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 Bioactive Compounds
Against SARS-CoV-2

Coronaviruses can cause illness, severe disease, and death; for example, Middle East respiratory syndrome (MERS),
severe acute respiratory syndrome (SARS), and COVID-19. Vaccine development and antiviral drugs are challenged by the
propensity for viral mutations allowing these viruses to evade such therapies. There are a number of bioactive compounds
from natural sources, which can exert health benefits and act as antiviral therapies such as anti-inflammation, antioxidative
stress, and immune regulation. This book summarizes research on the potential efficacy and underlying mechanisms of
bioactive compounds and traditional medicines against SARS-CoV-2.

Key Features

• Summarizes the potential benefits of bioactive therapies for coronaviruses

• Focuses on COVID-19, but also covers MERS and SARS
• Provides alternatives to vaccines and other antiviral drugs whose efficacy is reduced by viral mutations
• Relevant for clinicians and public health officials
Taylor & Francis
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Bioactive Compounds
Against SARS-CoV-2

Edited by
Jen-Tsung Chen
Designed cover image: Dotted Yeti, Shutterstock ID 1649506930

First edition published 2024

by CRC Press
6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742

and by CRC Press

4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN

CRC Press is an imprint of Taylor & Francis Group, LLC

© 2024 Taylor & Francis Group, LLC

Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity
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Library of Congress Cataloging‑in‑Publication Data

Names: Chen, Jen-Tsung, editor.

Title: Bioactive compounds against SARS-CoV-2 / edited by Jen-Tsung Chen.
Description: First edition. | Boca Raton : CRC Press, 2023. | Includes bibliographical references and index.
Identifiers: LCCN 2023001389 (print) | LCCN 2023001390 (ebook) | ISBN 9781032347998 (hardback) |
ISBN 9781032347967 (paperback) | ISBN 9781003323884 (ebook)
Subjects: MESH: SARS-CoV-2--drug effects | COVID-19 Vaccines | Antiviral Agents | Drug Resistance,
Viral | Mutation--drug effects | COVID-19 Drug Treatment
Classification: LCC RA644.C67 (print) |
LCC RA644.C67 (ebook) | NLM QW 168.5.C8 | DDC 614.5/924144--dc23/eng/20230621
LC record available at https://lccn.loc.gov/2023001389
LC ebook record available at https://lccn.loc.gov/2023001390

ISBN: 9781032347998 (hbk)

ISBN: 9781032347967 (pbk)
ISBN: 9781003323884 (ebk)

DOI: 10.1201/9781003323884

Typeset in Times
by KnowledgeWorks Global Ltd.
Contents
Preface...................................................................................................................................................................................xi
Editor.................................................................................................................................................................................. xiii
Contributors.......................................................................................................................................................................... xv

Chapter 1 Omics-Guided Intervention Strategies in Combatting SARS-CoV-2:

Recent Advancements and Challenges������������������������������������������������������������������������������������������������������������1
Somrita Padma, Nabarun Chandra Das, Ritwik Patra, Pritha Chakraborty, Abhijit Dey,
Suprabhat Mukherjee
1.1 Introduction............................................................................................................................................ 1
1.2 Origin of SARS-CoV-2...........................................................................................................................2
1.3 Pathophysiology of COVID-19............................................................................................................... 3
1.4 Omics-Guided Therapeutic Strategies................................................................................................... 4
1.5 Conclusions and Future Directives......................................................................................................... 7
Acknowledgments............................................................................................................................................. 8
References......................................................................................................................................................... 8

Chapter 2 The Application of Computer-Aided Drug Design Methods for Developing Natural
Compound-Based Therapeutics Against SARS-CoV-2��������������������������������������������������������������������������������� 12
Priya Godara, Dhaneswar Prusty
2.1 Introduction.......................................................................................................................................... 12
2.2 What Is a Computer-Aided Drug Design Approach (CADD)? Types? Related Software?.................. 13
2.3 How Has CADD Elevated the Entire Drug Development Pipeline?.................................................... 15
2.4 Natural Compound Libraries Used for CADD Against SARS-CoV-2................................................ 16
2.5 Remarkable Successes of CADD in Designing Natural Compound-Based
Therapeutics for COVID-19 Treatment................................................................................................ 16
2.6 The Challenges and Scope Associated with CADD............................................................................ 18
2.7 Conclusions........................................................................................................................................... 19
Acknowledgments........................................................................................................................................... 19
References....................................................................................................................................................... 19

Chapter 3 Therapeutic Targets of Host Factors for Potential COVID-19 Treatments���������������������������������������������������� 23

Sabarinath Neerukonda
3.1 Introduction.......................................................................................................................................... 23
3.2 General Replication Cycle of SARS-CoV-2......................................................................................... 23
3.3 High-Content Screening of Host Factors.............................................................................................25
References.......................................................................................................................................................34

Chapter 4 Phytocompounds: A Paradigm Shift as Therapeutics for COVID-19����������������������������������������������������������� 39

Vijeta Prakash, Paridhi Bhatia, Shweta Dang, Pammi Gauba, Reema Gabrani
4.1 Introduction.......................................................................................................................................... 39
4.2 Alkaloids.............................................................................................................................................. 39
4.3 Phenolics...............................................................................................................................................46
4.4 Terpenes................................................................................................................................................ 47
4.5 Organosulfur Compounds.................................................................................................................... 47
4.6 Conclusions........................................................................................................................................... 48
Acknowledgments........................................................................................................................................... 48
References....................................................................................................................................................... 48

v
vi Contents

Chapter 5 Flavonoids as Potential Therapeutic Agents Against the SARS-CoV-2 Infection: An Overview����������������� 53
Anand Kumar Pandey, Shalja Verma
5.1 Introduction.......................................................................................................................................... 53
5.2 Flavonoids and Their Classification..................................................................................................... 53
5.3 SARS-CoV-2, Related Pathologies and Lead Drug Targets................................................................. 55
5.4 Flavonoids as Therapeutics Against SARS-CoV-2.............................................................................. 59
5.5 Conclusions........................................................................................................................................... 62
References....................................................................................................................................................... 62

Chapter 6 Antiviral Activities of Flavonoids Against COVID-19 and Other Virus-Causing Ailments������������������������ 68
Arthi Boro, Biju Reji Souparnika, Srinivasan Atchaya, Shanmugam Ramya, Natchiappan Senthilkumar,
Shanmugam Velayuthaprabhu, Rengasamy Lakshminarayanan Rengarajan, Arumugam
Vijaya Anand
6.1 Introduction.......................................................................................................................................... 68
6.2 Types of Flavonoids.............................................................................................................................. 69
6.3 Respiratory Virus................................................................................................................................. 71
6.4 Effects on Different Organs................................................................................................................. 73
6.5 Effects of Flavonoids............................................................................................................................ 74
6.6 Conclusions........................................................................................................................................... 77
References....................................................................................................................................................... 77

Chapter 7 Potential Therapeutic Effects of Flavonoids Against the COVID-19 Infection: The Mode of Action���������� 82
Aswani R.B., Honey Pavithran, Ranjith Kumavath
7.1 Introduction.......................................................................................................................................... 82
7.2 Biological and Structural Organization of SARS-CoV-2..................................................................... 83
7.3 Druggability of SARS-CoV-2 and Treatment Opportunities...............................................................84
7.4 Discussion on the Potential Antiviral Properties of Flavonoids.......................................................... 86
7.5 Prospects of Using Flavonoids as Potential Anti-SARS-CoV Virus Agents....................................... 87
7.6 Conclusions........................................................................................................................................... 89
References.......................................................................................................................................................90

Chapter 8 Terpenes and Terpenoids: Potent Antiviral Agents Against SARS-CoV-2����������������������������������������������������94

Dwaipayan Sinha, Moumita Chatterjee, Srijonee Choudhury, Sanchita Seal, Tapas Das,
Shilpi Sharma, Swastika Banerjee, Shahana Chowdhury
8.1 Background...........................................................................................................................................94
8.2 Initial Backdrop of the Disease............................................................................................................94
8.3. Initial Therapeutic Options in Coronavirus Treatment........................................................................ 95
8.4 Possible Disadvantages of Synthetic Medicines and Vaccines............................................................97
8.5 Natural Products as a Therapeutic Option Against SARS-CoV-2....................................................... 98
8.6 Terpene-A Natural Product.................................................................................................................. 98
8.7 The Action of Terpenes Against SARS-CoV-2 (Selected Studies/Examples).....................................99
8.8 Terpenes from Marine Organisms: Potential Anti-SARS-CoV-2 Agents.......................................... 101
8.9 Present Status of Use of Terpenes in Pharmaceutical Industries to Counter
SARS-CoV-2 and Allied Viruses....................................................................................................... 102
8.10 Mechanism of Action of Terpenes and In Silico Approaches Against SARS-CoV-2........................ 102
8.11 Prospects and Conclusive Remarks.................................................................................................... 103
References..................................................................................................................................................... 104
Contents vii

Chapter 9 Phytonutrients and Secondary Metabolites to Cease SARS-CoV-2 Loop��������������������������������������������������� 111

Ajith Sivasangar Latha, Poornima Prasad Anita, Nihala Sidhic, Elizabeth James,
Mohandass Kaviya, Kathirvel Bharathi, Balasubramanian Balamuralikrishnan,
Arumugam Vijaya Anand
9.1 Introduction........................................................................................................................................ 111
9.2 Phytochemicals................................................................................................................................... 112
9.3 Phytonutrients and Secondary Metabolites........................................................................................ 112
9.4 Sources and Functions of Phytonutrients and Secondary Metabolites
with Antiviral Activity....................................................................................................................... 113
9.5 Antiviral Activity of Phytonutrients and Secondary Metabolites Against SARS-CoV-2.................. 116
9.6 Conclusions......................................................................................................................................... 118
References..................................................................................................................................................... 120

Chapter 10 Treatment Strategies Against COVID-19 Using Bioactive Compounds and Plant
Secondary Metabolites��������������������������������������������������������������������������������������������������������������������������������� 125
Rabia Javed, Syeda Aroosa Mohsin, Humaira Fatima
10.1 Introduction........................................................................................................................................ 125
10.2 Genomic Organization of SARS-CoV-2............................................................................................ 126
10.3 Life Cycle of SARS-CoV-2................................................................................................................ 126
10.4 Pathogenesis of SARS-CoV-2............................................................................................................. 127
10.5 Treatment Strategies of SARS-CoV-2................................................................................................ 127
10.6 Ethnobotanically Important Plants Presenting Potent Antiviral Activity.......................................... 130
10.7 Natural Composites Accustomed to Controlling SARS-CoV-2......................................................... 131
10.8 Biotechnological Approaches to Enhance the Production of Secondary Metabolites....................... 131
10.9 Conclusions and Future Recommendations....................................................................................... 134
Compliance with Ethical Standards.............................................................................................................. 135
References..................................................................................................................................................... 136

Chapter 11 The Potential Contribution of Vitamin K as a Nutraceutical to Scale Down the Mortality
Rate of COVID-19���������������������������������������������������������������������������������������������������������������������������������������� 140
Shanmugam Ramya, Arthi Boro, Irudhayaraj Peatrise Geofferina, Arumugam Jananisri,
Ganesh Vishalini, Balasubramanian Balamuralikrishnan, Meyyazhagan Arun, Arumugam
Vijaya Anand
11.1 Introduction........................................................................................................................................ 140
11.2 Vitamin K........................................................................................................................................... 140
11.3 The Deficiency of Vitamin K And COVID-19................................................................................... 143
11.4 Plausible Mechanisms for Vitamin K Against SARS-CoV-2............................................................ 143
11.5 Vitamin K Antagonist........................................................................................................................ 146
11.6 Biomarkers to Detect Vitamin K Deficiency..................................................................................... 147
11.7 Vitamin K is a Potent Nutraceutical................................................................................................... 148
11.8 Possible Potentials of Vitamin K as an Anti-COVID-19 Agent......................................................... 149
11.9 Conclusions......................................................................................................................................... 155
References..................................................................................................................................................... 155

Chapter 12 Gastrointestinal Symptoms in COVID-19 Patients: Prevention and Treatment������������������������������������������ 160

Mohamed Bouhrim, Noureddine Bencheikh, Nour Elhouda Daoudi, Amine Elbouzidi,
Hayat Ouassou, Loubna Kharchoufa, Doha Berraaouan, Jen-Tsung Chen, Christophe Hano,
Mohamed Addi, Mostafa Elachouri, Mohamed Bnouham
12.1 Introduction........................................................................................................................................ 160
12.2 Gastrointestinal Symptom Manifestations......................................................................................... 160
viii Contents

12.3 Gastrointestinal Symptoms Management........................................................................................... 161

12.4 Medicinal Plants are Commonly Used in Morocco to Treat Gastrointestinal Disorders.................. 163
12.5 Major Issues Remain Largely Unexplored......................................................................................... 163
12.6 Recommendations to Manage the Manifestation of Gastrointestinal
Symptoms Induced by COVID-19..................................................................................................... 167
References..................................................................................................................................................... 168

Chapter 13 Mechanisms and Management Options of Liver Injury in COVID-19: A Review�������������������������������������� 172
Nour Elhouda Daoudi, Mohamed Bouhrim, Omar Bouziane, Samira Mamri,
Mohamed Marghich, Hayat Ouassou, Mohamed Addi, Christophe Hano,
Jen-Tsung Chen, Mohamed Bnouham
13.1 Introduction........................................................................................................................................ 172
13.2 Action Mechanisms............................................................................................................................ 172
13.3 Management and Prevention.............................................................................................................. 174
13.4 Conclusions......................................................................................................................................... 176
References..................................................................................................................................................... 177

Chapter 14 Marine Species-Derived Bioactives Against Coronavirus Disease������������������������������������������������������������� 180

Augustus Akshaya Rani, Samsudeen Malik Basha, Syed Saadullah Ashfaq, Roy Apoorva,
Jana Sudipta, Shunmugiah Karutha Pandian
14.1 Introduction........................................................................................................................................ 180
14.2 The Entry of SARS-CoV-2................................................................................................................. 180
14.3 Druggable Targets.............................................................................................................................. 181
14.4 Marine Bioactive Compounds............................................................................................................ 183
14.5 Conclusions......................................................................................................................................... 195
14.6 Limitations......................................................................................................................................... 195
References..................................................................................................................................................... 195

Chapter 15 Potential Bioactive Compounds of Indian Flora Against SARS-CoV-2������������������������������������������������������ 203

Deepyaman Das, Joydeep Chakraborty, Harshita Shand, Rittick Mondal, Soumita Podder,
Suvankar Ghorai, Amit Kumar Mandal
15.1 Introduction........................................................................................................................................ 203
15.2 Phenolic Compounds.......................................................................................................................... 203
15.3 Terpenoids..........................................................................................................................................204
15.4 Alkaloids............................................................................................................................................207
15.5 Conclusions.........................................................................................................................................207
References.....................................................................................................................................................207

Chapter 16 Natural Product-Derived Active Compounds for the Management of COVID-19:

A Summary from Indochina������������������������������������������������������������������������������������������������������������������������ 211
Sora Yasri, Viroj Wiwanitkit
16.1 Introduction........................................................................................................................................ 211
16.2 Natural Sources and Natural Products for COVID-19 Management................................................. 212
16.3 Current Research and Perspectives in Southeast Asia....................................................................... 217
16.4 Conclusions......................................................................................................................................... 218
References..................................................................................................................................................... 218

Chapter 17 Novel Therapeutic Strategy Based on Nanomedicine Targeting Thrombosis for SARS-CoV-2����������������� 221
Xingping Quan, Xiaofeng Gong, Yonghua Zhao
17.1 Introduction........................................................................................................................................ 221
17.2 Thrombus Formation in COVID-19 Patients..................................................................................... 222
Contents ix

17.3 Antithrombotic Necessity and Strategy for COVID-19 Patients........................................................ 222

17.4 Nanomedicine as a Novel Therapeutic Strategy for COVID-19........................................................ 223
17.5 Vaccine-Induced Immune Thrombotic of COVID-19....................................................................... 225
17.6 Conclusions and Future Perspectives................................................................................................. 226
Acknowledgments......................................................................................................................................... 226
References..................................................................................................................................................... 227

Chapter 18 Nanomaterials in Combating Human Coronavirus Infections��������������������������������������������������������������������� 230

Mohanapriya Chelladuraia, Renganathan Sahadevana, Sekar Vijayakumarb
18.1 Introduction........................................................................................................................................ 230
18.2 Coronavirus........................................................................................................................................ 230
18.3 Challenges Faced with Antiviral Therapy.......................................................................................... 233
18.4 Nanotechnology.................................................................................................................................. 233
18.5 Nanoparticles in the Viral Arena....................................................................................................... 234
18.6 Conventional Nanoparticles Strategically Adopted for Treating SARS-CoV-2................................. 234
18.7 Other Possible Bio-Inspired Nanoparticles Strategically Adopted for the Treatment
of SARS-CoV-2.................................................................................................................................. 236
18.8 Point-of-Care Devices........................................................................................................................ 238
18.9 Affordability of Nanoparticle-Based Medicines................................................................................ 238
18.10 Conclusions......................................................................................................................................... 238
References..................................................................................................................................................... 239

Chapter 19 Structural Insights into Drug Targets from MERS, SARS-CoV, and SARS-CoV-2 and
Scope for Phytochemicals as Broad-Spectrum Antivirals and Immunomodulators����������������������������������� 243
Nikhil Singh, Rafiq Mohamed, U.V. Babu, Raghavendra P Rao, Prachi Singh
19.1 Introduction........................................................................................................................................ 243
19.2 Article Search and Methodology........................................................................................................244
19.3 Key Viral Proteins of Hcovs and Their Role in the Life Cycle of the Virus.....................................244
19.4 Conservations of Key Viral Proteins among HCoVS........................................................................ 245
19.5 Conclusions......................................................................................................................................... 250
References..................................................................................................................................................... 252

Chapter 20 Precision Phytochemicals for COVID-19-Induced Olfactory Dysfunction������������������������������������������������� 257

Sachiko Koyama, Vonnie D.C. Shields, Thomas Heinbockel
20.1 Introduction........................................................................................................................................ 257
20.2 Symptoms of COVID-19.................................................................................................................... 258
20.3 Chemosensory Dysfunction Caused by COVID-19........................................................................... 258
20.4 Phytochemicals with Anti-Inflammatory Effects.............................................................................. 259
20.5 Phytochemicals with Binding Affinity with SARS-CoV-2................................................................ 259
20.6 Signaling Pathways............................................................................................................................. 261
20.7 Future Directions for Clinical Treatment Options with Precision Phytochemicals........................... 263
References.....................................................................................................................................................264
Index����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������269
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Preface
Throughout the history of humankind the global economy research field attracting the attention of cross-discipline
and public health, and even globalization of the modern researchers. There is a widespread demand for reviewing
world, can be damaged greatly and repeatedly by cer- and refining related literature and disseminating the infor-
tain viruses that become pandemics. Among these, the mation not only to benefit the academic world but also the
Coronaviridae family (coronaviruses, CoV), which includes general public.
RNA viruses, has led to illnesses ranging from mild to This book aims to present the current research achieve-
severe diseases. In the past two decades, emerging varieties ments on the efficacy and underlying mechanisms of bio-
of CoV have contributed to lethal diseases, including severe active compounds from natural sources and traditional
acute respiratory syndrome (SARS), Middle East respira- medicines against SARS-CoV-2. This is an edited collec-
tory syndrome (MERS), and more recently coronavirus tion of a series of chapters in cross-disciplinary research
­disease 2019 (COVID-19) caused by severe acute respira- fields including bioinformatics, ethnopharmacology, drug
tory syndrome coronavirus 2 (SARS-CoV-2), which has design and discovery, nanotechnology, natural products,
caused the most severe public health crisis in modern times. molecular biology, omics, pathogenesis, pharmacology,
It can be a huge challenge to develop vaccines or anti- systems pharmacology, traditional medicines, and virology.
viral drugs to prevent or treat coronavirus infections in To ensure a broad source of knowledge and transparency,
humans due to the limited time period, and these viruses this book project is open for submission on the web page
keep mutating and gaining abilities to escape immunity. of ResearchGate. Also, over 500 invitations were sent to
Fortunately, it is well established that a wide range of bioac- potential contributors with high-quality publications in this
tive compounds from natural sources exert health benefits, field in recent years. Finally, 20 chapters have been included
and many have the therapeutic capacity of antiviruses and and this is just a snapshot of the great achievements in this
other related efficacies such as anti-inflammatory and anti- critical research field. As the editor, I truly hope this book
oxidant properties, as well as immune regulation against can enrich our understanding of the effects and potential
diseases caused by viruses. Hence, theoretically, bioactive applications of natural compounds on the management
compounds may have a high potential based on a paradigm of COVID-19 infection. It will be a critical reference and
shift for preventing and treating infections of coronaviruses suitable textbook for researchers, students, teachers, and
including SARS-CoV-2, and certainly have the poten- experts in a wide range of fields that are involved in the
tial to provide therapeutic interventions for the post-acute fight against SARS-CoV-2 and new variants in the future.
COVID-19 syndrome. Facing this emergence of COVID- In the end, I would like to thank all the authors for their
19, in recent years scientists have explored intensively the intelligence, time, and effort in organizing chapters, and the
effects and implications of a range of natural compounds assistance and instructions from the editorial office of the
for the treatment of this disease. It has become an emerging publisher are much appreciated.

xi
Taylor & Francis
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Editor
Jen-Tsung Chen is currently a Professor at the National medicinal plants, phytochemicals, plant physiology, and
University of Kaohsiung in Taiwan. He teaches cell biology, plant biotechnology. He has published over 90 scientific
genomics, proteomics, medicinal plant biotechnology, and papers and serves as an editorial board member for the
plant tissue culture. His research interests include bioactive journals Plant Methods and Plant Nano Biology.
compounds, chromatography techniques, in vitro culture,

xiii
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Contributors
Mohamed Addi Kathirvel Bharathi
Université Mohammed Premier Bharathiar University
Oujda, Morocco Coimbatore, India

Arumugam Vijaya Anand Paridhi Bhatia

Bharathiar University Centre for Emerging Diseases
Nadu, India Department of Biotechnology
Jaypee Institute of Information Technology
Poornima Prasad Anita Noida, Uttar Pradesh, India
Bharathiar University
Coimbatore, India Mohamed Bnouham
Mohammed First University
Roy Apoorva Oujda, Morocco
Alagappa University
Karaikudi, India Arthi Boro
Bharathiar University
Meyyazhagan Arun Nadu, India
CHRIST Deemed to Be University
Bengaluru, India Mohamed Bouhrim
Mohammed First University
Syed Saadullah Ashfaq Oujda, Morocco
Alagappa University
Karaikudi, India Joydeep Chakraborty
Raiganj University
Srinivasan Atchaya Raiganj, India
Bharathiar University
Nadu, India Pritha Chakraborty
Nazrul University
U.V. Babu West Bengal, India
Himalaya Wellness Company
Makali, Bangalore Moumita Chatterjee
V. Sivaram Research Foundation
Balasubramanian Balamuralikrishna Bangalore, India
Sejong University
Seoul, South Korea Mohanapriya Chelladurai
Anna University
Swastika Banerjee Chennai, India
Kairali College of +3 Science
Champua, India Jen-Tsung Chen
National University of Kaohsiung
Samsudeen Malik Basha Kaohsiung, Taiwan
Alagappa University
Karaikudi, India Srijonee Choudhury
University of Kalyani
Noureddine Bencheikh Kalyani, India
Mohammed First University
Oujda, Morocco Shahana Chowdhury
German University
Doha Berraaouan Gazipur, Bangladesh
University Mohamed Premier
Oujda, Morocco

xv
xvi Contributors

Shweta Dang Suvankar Ghorai

Centre for Emerging Diseases Raiganj University
Department of Biotechnology Raiganj, India
Jaypee Institute of Information Technology
Noida, Uttar Pradesh, India Priya Godara
Central University of Rajasthan
Nour Elhouda Daoudi Ajmer, India
Mohammed First University
Oujda, Morocco Xiaofeng Gong
Fudan University
Deepyaman Das Shanghai, China
Raiganj University
Raiganj, India Christophe Hano
University of Orleans
Nabarun Chandra Das Orléans, France
Nazrul University
West Bengal, India Ouassou Hayat
University Mohamed First
Tapas Das Oujda, Morocco
University of Kalyani
Kalyani, India Thomas Heinbockel
Howard University
Abhijit Dey Washington, DC (USA)
Presidency University
West Bengal, India Elizabeth James
Bharathiar University
Mostafa Elachouri Coimbatore, India
Mohammed First University
Oujda, Morocco Arumugam Jananisri
Bharathiar University
Amine Elbouzidi Coimbatore, India
Université Mohammed Premier
Oujda, Morocco Rabia Javed
Memorial University of Newfoundland and Labrador
Daoudi Nour Elhouda Corner Brook, Canada
University Mohamed First
Oujda, Morocco Mohandass Kaviya
Bharathiar University
Humaira Fatima Coimbatore, India
Quaid-i-Azam University
Islamabad, Pakistan Loubna Kharchoufa
Mohammed First University
Reema Gabrani Oujda, Morocco
Centre for Emerging Diseases
Department of Biotechnology Sachiko Koyama
Jaypee Institute of Information Technology Indiana University
Noida, Uttar Pradesh, India Indianapolis, IN (USA)

Pammi Gauba Ranjith Kumavath

Centre for Emerging Diseases Central University of Kerala
Department of Biotechnology Kasaragod, India
Jaypee Institute of Information Technology Pondicherry University
Noida, Uttar Pradesh, India Puducherry, India

Irudhayaraj Peatrise Geofferina Ajith Sivasangar Latha

Bharathiar University Bharathiar University
Coimbatore, India Coimbatore, India
Contributors xvii

Amit Kumar Mandal Ritwik Patra

Raiganj University Nazrul University
Raiganj, India West Bengal, India

Bnouham Mohamed Honey Pavithran

University Mohamed First Central University of Kerala
Oujda, Morocco Kasaragod, India

Bouhrim Mohamed Soumita Podder

University Mohamed First Raiganj University
Oujda, Morocco Raiganj, India

Marghich Mohamed Vijeta Prakash

University Mohamed First Centre for Emerging Diseases
Oujda, Morocco Department of Biotechnology
Jaypee Institute of Information Technology
Rafiq Mohamed Noida, Uttar Pradesh, India
Himalaya Wellness Company
Makali, Bangalore Dhaneswar Prusty
Central University of Rajasthan
Syeda Aroosa Mohsin Ajmer, India
Quaid-i-Azam University
Islamabad, Pakistan Xingping Quan
University of Macau
Rittick Mondal Macao, China
Raiganj University
Raiganj, India Aswani R.B.
Central University of Kerala
Suprabhat Mukherjee Kasaragod, India
Nazrul University
West Bengal, India Shanmugam Ramya
Bharathiar University
Sabarinath Neerukonda Nadu, India
U.S. Department of Health and Human Services,
Silver Spring, MD, USA Augustus Akshaya Rani
Alagappa University
Bouziane Omar Karaikudi, India
University Mohamed First
Oujda, Morocco Raghavendra P Rao
Himalaya Wellness Company
Hayat Ouassou Makali, Bangalore
Mohammed First University
Oujda, Morocco Rengasamy Lakshminarayanan Rengarajan
Madurai Kamaraj University
Somrita Padma Tamil Nadu, India
Nazrul University
West Bengal, India Renganathan Sahadevan
Anna University
Anand Kumar Pandey Chennai, India
Bundelkhand University
Uttar Pradesh, India Mamri Samira
University Mohamed First
Shunmugiah Karutha Pandian Oujda, Morocco
Alagappa University
Karaikudi, India
xviii Contributors

Sanchita Seal Biju Reji Souparnika

Polba Mahavidyalaya (Affiliated to Bharathiar University
University of Burdwan) Nadu, India
Polba, Hooghly, India
Jana Sudipta
Natchiappan Senthilkumar Alagappa University
Institute of Forest Genetics and Tree Karaikudi, India
Breeding (IFGTB-ICFRE)
Tamil Nadu, India Shanmugam Velayuthaprabhu
Bharathiar University
Harshita Shand Tamil Nadu, India
Raiganj University
Raiganj, India Shalja Verma
Bundelkhand University
Shilpi Sharma Uttar Pradesh, India
University of Delhi and
Delhi, India Indian Institute of Technology
Uttarakhand, India
Vonnie D.C. Shields
Towson University Sekar Vijayakumar
Towson, MD Shandong University
Weihai, P.R. China
Nihala Sidhic
Bharathiar University Ganesh Vishalini
Coimbatore, India Bharathiar University
Coimbatore, India
Nikhil Singh
Himalaya Wellness Company Viroj Wiwanitkit
Makali, Bangalore Parasitology Research Unit
Nakornratchasrima, Thailand
Prachi Singh
Himalaya Wellness Company Sora Yasri
Makali, Bangalore KM Center
Bangkok Thailand
Dwaipayan Sinha
Government General Degree College Yonghua Zhao
West Bengal, India University of Macau
Macao, China
 1 Omics-Guided Intervention Strategies
in Combatting SARS-CoV-2
Recent Advancements and Challenges
Somrita Padma, Nabarun Chandra Das, Ritwik Patra, Pritha Chakraborty,
Abhijit Dey, Suprabhat Mukherjee

1.1 INTRODUCTION (M), and nucleocapsid (N), which are required for viral rep-
lication and pathogenesis (Gordon et al. 2020; Kim et al.
Declared a pandemic year, 2019 brought havoc to the global 2020). Current evidence has reported a close association
population. Widespread severe acute respiratory syndrome between immunological patterns with disease progres-
coronavirus 2 (SARS-CoV-2), an infectious virus-related sion. Choudhury and Mukherjee (2020) have reported the
disease, resulted in approximately 3 million deaths and involvement of toll-like receptor 4 (TLR4) in recognizing
1 million recoveries among 180 million confirmed cases the viral spike protein and eliciting the immune response
(Tiwari et al. 2021). It has been known to cause severe mor- against SARS-CoV-2. Interaction of viral spike proteins
bidity and mortality, and destructively impact the world with pathogen recognition receptors (PRRs) through
economy (Yang et al. 2020; Mukherjee 2022). Studies have myeloid differentiation factor 88 (MyD88)-dependent
revealed that SARS-CoV-2 belongs to the Coronaviridae or -independent pathways activates downstream signaling
family and is classified into four genera: α-coronavirus via NF-κB transcription factors to release proinflammatory
(α-CoV), β-coronavirus (β-CoV), γ-coronavirus (γ-CoV), cytokines (Mukherjee 2022). Thus, TLR4 has been a target
and δ-coronavirus (δ-CoV) (Woo et al. 2010; Singh et al. for developing strategies because it has augmented protec-
2021). Of the four genera, α-CoV and β-CoV tend to infect tion against several pathogens. It has the potential to provide
mammals; however, β-CoV has affected the human popula- a new dimension in designing vaccines and omics-based
tion and caused present-day COVID-19. In most countries, therapeutics against viral infections including SARS-CoV-2
COVID-19 cases have followed an exponential growth (Dowling and Mansell 2016). Choudhury et al. (2022) have
trajectory during the early and peak stages of the disease developed “AbhiSCoVac” as a single vaccine candidate
outbreak, although variations in the trajectory are due to against virulent strains of SARS-CoV-2 to elicit an immu-
variations in disease testing, population size, healthcare sta- nogenic response by producing cytokines, antibodies, and
tus, and environmental and social parameters (Singh et al. interleukins. Apart from vaccines, in this post-genomic era,
2021). Severe clinical manifestations have been reported genomic studies have greatly contributed to understanding
including fever with dry cough, shortness of breath, pneu- the viral origin, and their dynamics and integrating omics-
monia, low oxygen saturation, and severe acute respiratory based technologies in the development of new diagnostics,
distress syndrome (ARDS) with an urgent need for hospi- vaccines, and therapeutics. Omics-based studies have been
talization (some in critical care) (Guan et al. 2020; Yang explained as a collective technology of genomics, transcrip-
et al. 2020). However, critical conditions are more prevalent tomics, metabolomics, and proteomics (Singh et al. 2021).
among individuals with underlying comorbidities such as These omics-based approaches have opened new areas of
diabetes, malignant tumors, cardiovascular disease, kidney research in the field of cancer biology, finding a novel target
disorders, and/or compromised immune systems (Li et al. for various inflammatory and infectious diseases, and gen-
2020a; Chen et al. 2021a). Evidence has suggested SARS- erating vaccine candidates against those diseases (Das et al.
CoV-2 transmission occurs via inhalation and exhalation of 2020, 2021; Patra et al. 2021d). This technology follows the
droplets between infected patients and a normal individual principle of detecting and verifying different gene and pro-
as well as fomite-mediated transmission among the com- tein products of a given biological sample and, hence, can
munity (Andersen et al. 2020; Singh et al. 2021) assist in defining gene and/or protein expression profiles
Structurally, SARS-CoV-2 is a single-stranded, envel- associated with SARS-CoV-2 (Chen et al. 2020a; Gordon
oped, positive-sense RNA virus with a genome size of et al. 2020). In this chapter, we will be discussing different
~30 kb (Kim et al. 2020). The viral genome encodes for 16 omics-based strategies used to explore the pathophysiology
nonstructural proteins (Nsps) and four structural proteins and to counteract this pandemic effectively.
including spike glycoprotein (S), envelope (E), membrane

DOI: 10.1201/9781003323884-1 1
2 Bioactive Compounds Against SARS-CoV-2

1.2 ORIGIN OF SARS-CoV-2 originates from bats and transfers to humans through
dromedary camels/camelids (Cui et al. 2019; Nova 2021).
Since the outbreak of this novel pneumonia (coronavirus Investigations also revealed that Chinese horseshoe bats
disease 2019 [COVID-19]) virus in the city of Wuhan in and palm civets are responsible for the transmission of
China’s Hubei province, there have been critical allegations SARS-CoV in humans (Guan et al. 2003; Poon et al. 2005;
and political speculation that SARS-CoV-2 is not from a Hu et al. 2015). Genomic analysis and several lines of sero-
natural source and that it is a laboratory-borne pathogenic logical reports claimed that both SARS-CoV and SARS-
virus (Andersen et al. 2020; Wu et al. 2020; Zhou et al. CoV-2 belong to the subgenus Sarbecovirus, which falls
2020b). Many studies have been done to stop these allega- under the genus Betacoronavirus (Hu et al. 2015; Holmes
tions against China’s Wuhan Virology Laboratory as well et al. 2021). Furthermore, comparative studies at the nucle-
as to find out the cause of this pandemic (Abdelrahman otide level exposed 79% similarity between SARS-CoV
et al. 2020; Zhang and Holmes 2020). It is very important and SARS-CoV-2 across the whole genome and about 72%
now to track the chain of evolution of coronaviruses to pre- similarity in spike glycoprotein (Zhang and Holmes 2020;
dict the next spillovers and be prepared against them. Zhou et al. 2020b); whereas the proteomics data explained
All previous six human coronaviruses (SARS-CoV, that the structural proteins (except spike) of SARS-CoV-2
MERS-CoV, HKU1, NL63, OC43, and 229E) d­ ocumented have more than 90% sequence identity with amino acids of
over the past 20 years have zoonotic origins (Ye et al. 2020; SARS-CoV (Lu et al. 2020; Zhou et al. 2020b). In addition,
Holmes et al. 2021). Evidence from phylogenetic analysis structural studies explained that the S protein of SARS-
suggests that HCoV-OC43 and HKU1 may derive from CoV-2 is stereochemically less active than SARS-CoV
viruses associated with rodents, whereas the remaining in binding hACE2 (Shang et al. 2020; Wan et al. 2020).
four are of bat origin (Su et al. 2016; Forni et al. 2017; Cui Collectively, these findings initially help to determine that
et al. 2019). It has also been discovered that intermediate SARS-CoV-2 is not a deliberately manipulated laboratory
hosts like cattle and/or swine (in the case of HCoV-OC43) construct, but that it arose from a common ancestor from
actually transmit the human coronaviruses to humans. which SARS-CoV evolved (Figure 1.1A).
Interspecies transmission of HCoV-229E and MERS-CoV

FIGURE 1.1 Phylogenetic map depicting the relation of human coronaviruses with bat and Pangolin coronaviruses. Rectangular
(A) and unrooted map (B) was generated utilizing the NCBI taxonomy database and a phylogenetic tree generator (phyloT).
Omics-Guided Intervention Strategies in Combatting SARS-CoV-2 3

Taking a clue from studies like Rahalkar and Bahulikar of RBD responsible for strong affinity toward hACE2 were
did in 2020, scientists started their investigations to find the found to be similar in both Pangolin-CoV and SARS-CoV-2
primary reservoir host of the SARS-CoV-2 virus. A study by (Lam et al. 2020; Li et al. 2020b). In support, an in silico
Rahalkar and Bahulikar (2020) reported that the six pneu- docking study performed by Choudhury and Mukherjee
monia cases from the Mojiang copper mine in 2012 had a (2020) exhibited a similar affinity of both Pangolin-CoV
resemblance to COVID-19 and were due to the involvement and SARS-CoV-2 viruses against hACE2. With several
of SARS-like CoVs from horseshoe bats. In this context, a contradictions, these findings suggested pangolin species
whole genome analysis by Zhou et al. (2020a) revealed that also are a natural reservoir of CoVs that resemble SARS-
SARS-CoV-2 shares a 96.1% resemblance with nucleotides CoV-2. Indeed, the absence of the furin cleavage site from
of bat SARSr-CoV RaTG13, whereas the study by Lu et al. both RaTG13 and Pangolin-CoV solidifies the prediction
(2020) noticed 87% and 88% nucleotide sequence similarity of the natural emergence of SARS-CoV-2 (Segreto and
to bat SARSr-CoV ZC45 and ZXC21. In addition, genomic Deigin 2021; Chan and Zhan 2022). Taking into account
and evolutionary evidence also explored the RmYN02 virus all the theory and experimental evidence, Ye et al. (2020)
from Rhinolophus bats from China’s Yunnan province and proposed in their review that SARS-CoV-2 could be trans-
found more than 96% sequence identity with genomic mitted to humans through direct transmission from bats or
regions 1ab, 3a, E, 6, 7a, N, and 10, which are ­responsible through intermediate host pangolins. The study also clari-
for encoding structural and Nsps (Zhou et al. 2020a). fied that more investigation is needed to solve the puzzle of
However, the S1/S2 region, which is six nucleotides shorter the origin of this zoonotic disease (Ye et al. 2020).
when compared with RaTG13, and has a theoretically According to the World Health Organization (WHO),
approved inability to bind with hACE2, increased the phy- more than 617 million confirmed COVID-19 cases and
logenetic distance of RmYN02 from SARS-CoV-2 (Zhou 6.5 million deaths were recorded globally on October 7,
et al. 2020a; Deigin and Segreto 2021). These findings in 2022 (WHO Coronavirus 2022). An evolutionary study
the nucleotide sequences support the prediction that viruses revealed this global concern is due to the emergence of
with similarity to SARSr-CoV RaTG13 may be the ances- two specific variants of SARS-CoV-2, variants of interest
tors of SARS-CoV-2 viruses (Shahhosseini et al. 2021). (VOIs) and variants of concern (VOCs) (Tao et al. 2021;
Moreover, at the protein level, RaTG13 shares 97.45% Chakraborty et al. 2022). Among the concern variants,
homology with the S region of SARS-CoV-2 (Jaimes et al. Omicron is currently threatening the whole world with
2020). Furthermore, the 1000 nucleotide difference from its high transmissibility (Araf et al. 2022; WHO Omicron
SARS-CoV-2, and decreased efficiency of targeting hACE2 2022). Weekly epidemiological data published by WHO spe-
due to mutations in four of five of the most significant amino cifically denotes the BA.5 descendent lineage of Omicron
acids from the RBD region, prove that the allegations raised dominating over other variants due to its high mutation rates
against designing SARS-CoV-2 via targeted mutagenesis of (WHO COVID-19 2022). However, Alpha, Beta, Gamma,
SARSr-CoV RaTG13 are untrue (Shang et al. 2020; Zhang Delta, and Epsilon, which were previously known as VOC
et al. 2020; Hakim 2021). However, the study of Choudhury for their toll on mankind, are currently considered variants
and Mukherjee (2020) proved a different story. According being monitored (VBMs) (CDC SARS-CoV-2 2022).
to them, SARSr-CoV RaTG13 is more efficient than SARS-
CoV-2 in targeting hACE2, but both SARSr-CoV RaTG13
1.3 PATHOPHYSIOLOGY OF COVID-19
and SARS-CoV-2 share a monophyletic group. Collectively
all evidence of the direct zoonotic origin of SARS-CoV-2 is The immune pathology of the coronavirus begins when the
the intermediate horseshoe bat (Rhinolophus affinis), which viral spike protein binds with the human ACE2 receptor. It
is mostly found in the Yunnan Province of China (Ruiz- results in the disruption of the renin-angiotensin receptor
Medina et al. 2022) (Figure 1.1B). leading to an alteration in blood pressure and electrolyte
Emerging reports of SARS-CoV from Foshan and balance (Patra et al. 2021c). Upon binding, the virus repli-
Guangzhou in China’s Guangdong province in 2002 and cates and releases virus particles that induce pyroptosis and
2003 clearly illustrated that these outbreaks were due to the release damage-associated molecular patterns (DAMPs)
sale of live animals such as civets and raccoon dogs (Guan and/or pathogen-associated molecular patterns (PAMPs).
et al. 2003; Xu et al. 2004). Scientists surmised that selling The PRRs are a class of receptor that binds to the PAMPs
of such live animals at the Wuhan market in 2019 might and/or DAMPs to elicit immune responses (Mukherjee
be the reason for the century’s deadliest outbreak, SARS- et al. 2016). Herein, DAMPs release upon binding with the
CoV-2 (Freuling et al. 2020; Xiao et al. 2021). Whole ACE2 receptor, which is recognized by the host epithe-
genome analysis identified about 91.02% homology between lial and endothelial macrophages of the alveoli to initiate
Pangolin-CoVs and SARS-CoV-2, which is significantly the production of inflammatory cytokines including tumor
less compared with SARSr-CoV RaTG13 and SARS-CoV-2 necrosis factor (TNF)-α; interleukin (IL)-1β, IL-6, IL-2,
(Zhang et al. 2020; Makarenkov et al. 2021). In contrast, IL-7, IL-8, IL-9, and IL-17; granulocyte-colony stimulat-
sequencing reports also explored whether Pangolin-CoV ing factor; and several chemokines like chemokine ligand
had a closer resemblance to the S1 region of SARS-CoV-2 2 (CCL2), CCL3, and CCL4 resulting in the huge efflux
than RaTG13 (Wang et al. 2020). All five key amino acids of the cytokines causing a cytokine storm (Bonam et al.
4 Bioactive Compounds Against SARS-CoV-2

2020; Patra et al. 2021c). This cytokine storm is the prin- The application of BlueDot, Metabiota, and HealthMap in
cipal contributor to the pathophysiology of COVID-19. It early detection, disease tracking, and disease monitoring
results in the translocation of monocytes, basophils, and helped determine the disease impact and real-time disease
CD4+ and CD8+ T cells at the site of infection followed outbreak (Patra et al. 2021b). Moreover, the advancement
by a surge in the blood neutrophil count (Bonam et al. in disease detection by standardizing the automated AI
2020; Cao 2020). These provide feedback to regulate the algorithm-based reverse transcription-polymerase chain
T-cell activation and natural killer (NK) cell production. reaction (RT-PCR) computed tomography (CT) scan and
Herein, the CD4+ T cell induces the B cells for the pro- magnetic resonance imaging (MRI) easily and rapidly
duction of viral-specific antibodies, whereas the CD8+ detected and stratified the COVID-19 patient based on
T cells directly kill the virus-infected cell through cyto- the disease severity without the physical intervention of
toxic activity (Bonam et al. 2020; Cao 2020). TLRs are ­healthcare personnel (Patra et al. 2021a, b). The worldwide
the major class of PRRs acting as innate immune sensors development of an omics-based database for the submis-
recognizing the PAMPs, thereby activating the proinflam- sion of the sequence and structure of the SARS-CoV-2
matory cytokine to elicit the immune responses against ­proteins helped scientists all over the world to easily access
the invading pathogen (Mukherjee et al. 2016, 2019). The the available information on the mutational changes, patho-
dysfunction of the TLR signaling pathways is associated genesis, and development of new drugs and therapeutic
with various inflammatory and infectious diseases includ- strategies (Patra et al. 2021b). The global database GISAID
ing inflammatory bowel disease, filariasis, cardiac dis- (https://gisaid.org/) is an initiative that rapidly shares all the
ease, obesity, diabetes, asthma, and many others (Mifsud data available worldwide regarding the genetic sequence,
et al. 2014; Achek et al. 2016; Mukherjee et al. 2016). epidemiological data, mutational changes, and phyloge-
Interestingly, the TLRs are also targeted for the therapeutic netic relationships among the different clades (Shu and
intervention of these diseases. Therefore, situation-specific McCauley 2017). The initiative of Google’s DeepMind and
strategic switching of the role of TLR might help in eluci- EMBL-EBI collaboratively initiated the structural database
dating the inflammatory responses caused by COVID-19. based on deep-learning and AI-based algorithms to predict
A maiden report by Choudhury and Mukherjee (2020) and construct the structure of the different proteins associ-
illustrated the role of TLR4 as a receptor for the immu- ated with SARS-CoV-2 (Jumper et al. 2021). Collectively,
nopathogenesis of SARS-CoV-2. The bioinformatics study all these omics-guided techniques and databases made it
based on the molecular docking score reveals positive easy for researchers and healthcare providers all over the
binding of the spike protein with the binding pockets of world to minimize the consequences and fight against the
TLR4. The TLR4 present across the alveolar macrophages worldwide pandemic. The different omics-based servers
senses the viral antigens to induce an immune response and databases for the research related to COVID-19 and
by producing a huge amount of type 1 interferon (IFN) development of therapeutic interventions are discussed in
and proinflammatory cytokines (Patra et al. 2020, 2021c; Table 1.1.
Mukherjee 2022). This type 1 IFN functions in the initia-
tion of early innate responses to h­ inder the pathogenesis by 1.4.1 TLRs as a Potential Target for Therapeutic
upregulating antiviral effectors like RNAaseL to restrict
Intervention Against SARS-CoV-2
the replication and feedback on the production of IFN
inducible genes. Intriguingly, the intracellular TLRs (viz., The dual role of the TLRs in both the initiation of immune
TLR3, TLR7, TLR8, and TLR9) can sense the viral RNA responses and the generation of anti-­ inflammatory
across the cytoplasm by the CARD domain containing the responses led several researchers to target TLRs for thera-
RNA helicases (Frieman et al. 2008). In this connection, peutic intervention against COVID-19. The study by Patra
the study by Choudhury et al. (2021b) revealed the com- et al. (2020) revealed that the early activation of the TLR
parative sensing of TLR3, TLR7, TLR8, and TLR9 with signaling pathways using several TLR agonists might
the mRNA of NSP10, S2, and E proteins of SARS-CoV-2 trigger the immune response against the invading SARS-
to elicit the immune responses and further target for thera- CoV-2 pathogen. As discussed in an earlier section, the
peutic interventions. putative role of TLR4 in COVID-19 pathogenesis and
evidence from the molecular docking study illustrates
1.4 OMICS-GUIDED THERAPEUTIC the highest binding affinity in the spike protein–TLR4
complex, which shows hydrogen bonds and hydrophilic
STRATEGIES
interactions (Choudhury and Mukherjee 2020). Thus,
Since the beginning of the COVID-19 global pandemic, therapeutic targeting of this complex and the TLR signal-
omics-guided techniques have proven to be efficient in fight- ing pathway proves to be an appropriate strategy. Studies
ing against this deadly disease. The role of artificial intel- have shown that dying cells and/or activated immune cells
ligence (AI) and the Internet of Things (IoT) in the early in response to SARS-CoV-2 form complexes with DNA,
prediction of disease and developing a disease prediction RNA, and DAMPs followed by binding with RAGE trans-
model helped various countries generate an early warning located to the lungs’ endolysosomal system and triggering
against the vulnerability of the disease (Patra et al. 2021a). TLR activation, especially TLR4 (Bonam et al. 2020; Cao
Omics-Guided Intervention Strategies in Combatting SARS-CoV-2 5

TABLE 1.1
Application of Various Omics-Based Servers and Databases Useful in the Discovery and Therapeutic
Intervention of COVID-19
Sl. Function Tools and Database Web Link Reference
No
1. Genetic sequence, epidemic data GISAID (Global initiative on https://www.gisaid.org/ (Bogner et al. 2006)
sharing all influenza data)
2. Genetic sequence GenBank https://www.ncbi.nlm.nih.gov/ (Benson et al. 2012)
genbank/
3. Genetic sequence, chip data, methylation, GEO (Gene Expression Omnibus) https://www.ncbi.nlm.nih.gov/ (Edgar et al. 2002)
chromatin structure, genome protein gds/?term=covid-19
interaction
4. Genomics data visualization tool UCSC SARS-CoV-2 Genome https://genome.ucsc.edu/covid19. (Fernandes et al. 2020)
Browser html
5. Archiving and distribution of genetic and BioGRID (Biological General https://thebiogrid.org/ (Oughtred et al. 2021)
protein interaction Repository for Interaction Datasets)
6. Gene variation, protein sequence, CNGBdb (China National https://db.cngb.org/gisaid/ (Chen et al. 2020b)
experiment and genome sequence data GeneBank DataBase)
7. Protein interaction data for drug transfer PROMISCUOUS 2.0 http://bioinformatics.charite.de/ (Gallo et al. 2021)
promiscuous2
8. RNA-sequence and transcriptomics data CovidExpress https://stjudecab.github.io/ (Singh et al. 2021)
visualization tool covidexpress/
9. Drug targeting DockCoV2 https://covirus.cc/drugs/ (Chen et al. 2021c)
10. Proteomics PRIDE (IDEntification Database) https://www.ebi.ac.uk/pride/ (Ternent et al. 2014)
11. Immune and genetic analysis COVID profiler http://genomics.lshtm.ac.uk/ (Ward et al. 2021)
12. Homology analysis UniProt https://sparql.uniprot.org/ (Apweiler et al., 2004)
13. Protein structure Protein Data http://www.rcsb.org/pdb/ (Kouranov et al. 2006)
Bank
14. 3D structure of protein and predict domain CATH https://www.cathdb.info/ (Sillitoe et al. 2021)
15. K-mer mapping UniqueKMER https://github.com/OpenGene/ (Chen et al. 2021b)
UniqueKMER
16. SARS-CoV-2 identification tool FASTV https://github.com/OpenGene/fastv (Chen et al. 2021b)
17. Detect and monitor metagenomic pathogen IDseq https://idseq.net (Kalantar et al. 2020)
18. Streamlined inactivation, amplification, and SHINE (Streamlined Highlighting https://github.com/broadinstitute/ (Arizti-Sanz et al.
detection of SARS-CoV-2 of Infections to Navigate Handlens 2020)
Epidemics)
19. Assessment of viral genetic diversity from V-pipe https://cbg-ethz.github.io/V-pipe/ (Posada-Céspedes
next-generation sequencing data et al. 2021)
20. Detection, annotation and classification of VIRify https://github.com/EBI- (Mitchell et al. 2020)
viruses Metagenomics/emg-viral-pipeline
21. Validation and annotation of virus sequence
VADR (Viral Annotation DefineR) https://github.com/nawrockie/vadr (Schäffer et al. 2020)
22. Analyzing genome (VBRC)Viral Bioinformatics https://www.4virology.net (Ehlers et al. 2002)
Research Center
23. Annotation and monitoring of SARS-CoV-2 Coronapp http://giorgilab.unibo.it/ (Mercatelli et al. 2021)
mutations coronannotator/
24. SARS-CoV-2 genome functional annotation CorGAT https://hub.docker.com/r/ (Singh et al. 2021)
laniakeacloud/galaxy_corgat
25. Analysis of variant IPD 2.0 (Infectious pathogen https://www.actrec.gov.in/ (Robinson et al. 2013)
detector) pi-webpages/AmitDutt/IPD/IPD.
html
26. Identification of novel mutations MicroGMT (Microbial genomics https://github.com/qunfengdong/ (Katoh and Standley
mutation tracker) MicroGMT 2013)
27. Analysis the relativeness of virus on the VirusDIP (Virus Data https://db.cngb.org/virusdip/ (Singh et al. 2021)
evolutionary tree Integration Platform)
28. Predict and analyze protein interaction VirHostNet http://virhostnet.prabi.fr/ (Sansone et al. 2019)
between viral proteins and targeted drugs
6 Bioactive Compounds Against SARS-CoV-2

2020). In this condition, Andersson et al. (2020) demon- against SARS-CoV-2 (Choudhury and Mukherjee 2020).
strated that inhibiting the RAGE-HMGB1-mediated acti- Similarly, many other repurposed drugs were determined
vation of the TLR4 signaling cascade shows therapeutic using bioinformatics-based approaches. In addition, several
results. Acetylcholine, heparin, statins, dexmedetomidine, phytochemicals are also efficient in reducing the pathogen-
ketamine, and other TLR4 antagonists like eritoran and esis of COVID-19. An in silico study using the computa-
aryl quinolinyl hydrazone derivatives inhibit the activa- tional approaches by Das et al. (2022a) screened more than
tion. From an immunological point of view, the main point 30 bioactive phytochemicals with previously documented
of therapy against COVID-19 is to suppress the cytokine antiviral activities to determine the binding affinity with the
storm. In this regard, the inactivation of the production of spike glycoprotein using molecular docking and molecular
proinflammatory cytokines like IL-1β and IL-6 reduces dynamic simulation followed by an ADMET study. The
the disease severity. Studies have shown that IL-37 acts results signify cajaninstilbene acid and papaverine targeted
on the mTOR, thereby increasing AMP kinase activity the TLR4, thereby mitigating the SARS-CoV-2-induced
and inflammatory response by suppressing the prolifera- lethal ­proinflammatory responses (Das et al. 2022a).
tion of IL-1β, TNF, CCL2, and IL-6 (Conti et al. 2020).
Moreover, as previously disclosed, the intracellular TLRs
(TLR3, TLR7, TLR8, TLR9) sense the mRNA of the 1.4.3 Omics-Guided Postulation of Monoclonal
NSP10, S2, and E protein of SARS-CoV-2, thus, targeting
Antibodies and Vaccine Candidate
these complexes shows a defensive role against coronavi-
rus disease. The application of a TLR agonist, especially Against SARS-CoV-2
a TLR3 agonist like poly IC:LC, inhibits the replication The milestone finding of the positive correlation and bind-
of coronavirus and serves as an inhibitor to IFN signaling ing of TLR4 with the SARS-CoV-2 spike protein directed
(Kumaki et al. 2017; Patra et al. 2021c). us to develop various therapeutic strategies. The recent
method for the therapeutic management of COVID-19
1.4.2 Omics-Guided Development of Drugs uses the application of monoclonal antibodies (mAbs) gen-
erated against SARS-CoV-2. In this regard, several mAbs,
and Phytochemicals as Therapeutics
including bamlanivimab, regdanvimab, tixagevimab,
Since the beginning of the worldwide pandemic, research- cilgavimab, etesevimab, casirivimab, imdevimab, and
ers focused on quickly discovering the key drugs to fight sotrovimab, were potentially used as therapeutics and
against the pathological consequences of COVID-19. Along are currently under clinical trials for final validations
with the discovery of new drugs, repurposing ­previously (Chen et al. 2021a; Das et al. 2022b). Herein, the multi-
approved drugs against other pathogenic infections to target omics-based technique on the different mAbs generated
the spike protein were also being studied. In this regard, against the Alpha (B.1.1.7) and Delta (B.1.617.2) lin-
the application of computational-based approaches in eages of SARS-CoV-2 by Das et al. (2022b) shows that
drug discovery and drug delivery was the prime method tixagevimab, regdanvimab, and cilgavimab effectually
used for the rapid and cost-effective technique of thera- neutralize the SARS-CoV-2 Alpha strains, whereas tixa-
peutic intervention. The 3D structure of the viral protein gevimab, bamlanivimab, and sotrovimab formed a stable
available across the various databases provided a path for complex with Delta variants. The bioinformatics-based
repurposing various drugs against pathogenesis. The other approach to conjugate the DRH3 of regdanvimab with a
multi-omics tools for modeling the protein based on the sotrovimab framework shows the most stable and effective
sequence homology included the Swiss-model (https://swiss- strategy for mAb-mediated neutralization of coronavirus
model.expasy.org/), iTESSAR (https://zhanggroup.org/I- disease (Das et al. 2022b).
TASSER/), and Modeller (https://salilab.org/modeller/) The most important and pathbreaking application of
to help resolve the limitation of structural databases. The the multi-omics-guided application for therapeutic inter-
DrugBank (https://go.drugbank.com/) and PubChem vention of COVID-19 includes the designing of a multi-
(https://pubchem.ncbi.nlm.nih.gov/) libraries provided all epitope-based peptide vaccine against the coronavirus
the relevant information about the previously approved disease. It reduces the cost of vaccine production and lim-
drugs in the treatment of various infectious and inflamma- its the challenges linked with potency, productivity, and
tory diseases. Applications of these omics-based servers and time barriers. Herein, targeting the TLR signaling path-
databases help to develop computer-aided drug discovery. ways for the formulation of effective vaccine candidates
In this regard, dozens of research papers are still being pub- provides clues to various researchers. The development of
lished targeting the spike protein. The study of Choudhury a multi-epitope-based vaccine using the three major pro-
et al. (2021a) demonstrated the efficacy of ivermectin homo- teins of SARS-CoV-2 (viz., nucleocapsid protein, mem-
logs, hydroxychloroquine, and remdesivir against the spike brane glycoprotein, and spike glycoprotein) to determine
trimer, viral replicase, protease and human TMPRSS2 the epitopes for both T and B cells shows both humoral and
using molecular docking, and in the molecular simulation cell-mediated immune responses (Kalita et al. 2020). This
study. It highlights ivermectin as a potential drug candidate vaccine candidate was found to interact effectively with
Omics-Guided Intervention Strategies in Combatting SARS-CoV-2 7

TLR3 and cascade the TLR3 signaling pathway for thera- 1.5 CONCLUSIONS AND FUTURE DIRECTIVES
peutic amelioration of COVID-19. Similarly, various other
bioinformatics-based studies targeted other TLRs includ- Since the beginning of the worldwide COVID-19 pandemic,
ing TLR4 and TLR5 (Rahman et al. 2020). The chimeric researchers and healthcare providers have been continuously
vaccine named “Cov-RMEN” consisting of six pathogenic and rigorously working to develop different therapeutic
peptides of SARS-CoV-2 shows effective binding and interventions to predict, track, diagnose, and ameliorate the
immunogenicity with TLR3 and TLR4, which leads to immunopathogenesis and consequences of the COVID-19
activation of dendritic cells for antigen processing and pre- crisis. In this regard, the application of multiple omics-
sentation (Rahman et al. 2020). However, the unremitting based approaches including AI and IoT proved to provide
changes in the genomic sequence and the imminent pro- advancement and benefit to various infrastructural, eco-
duction of different strains of the COVID-19 virus created nomical, pharmaceutical, and healthcare sectors. This chap-
challenges for producing an effective vaccine candidate. ter focused on the various multi-omics-based approaches in
Interestingly, the development of AbhiSCoVac, a single disease prediction, diagnosis, understanding the molecular
vaccine candidate formulated to target all the different immunopathogenesis, development of therapeutic interven-
strains of SARS-CoV-2, brings a ray of hope (Choudhury tion using repurposed drugs, phytochemicals, mAbs, and
et al. 2022). The vaccine construct shows strong interaction finally, multi-epitope-based peptide vaccines. The use of
with several vital immune sensors including TLR2, TLR4, these computational approaches fast-tracks the research in
MHC-I, and MHC-II proteins eliciting immune responses the field of COVID-19 and provides different intervention
for the generation of immunoglobins, anti-inflammatory strategies. The worldwide availability of open-access data-
cytokine, and various interleukins to fight against all coro- bases provides all the information about COVID-19 with-
navirus variants. Collectively, all the multi-omics-based out any limitations or boundaries. However, there are still
approaches to elucidate the immune response and thera- various lacunas in the knowledge of the evolution and fast-
peutic intervention of COVID-19 generated a huge pool of changing variants of SARS-CoV-2. With the emergence of
knowledge and strategies to overcome the consequences the new strain and continuous mutations, the application and
and fight against the life-­taking coronavirus disease. The development of new omics-based intervention techniques
application of various omics-based approaches in the is the most important requirement. The developments and
whole process from discovery to targeting COVID-19 is advancements in multi-omics-based research bridge these
illustrated in Figure 1.2. gaps and provide a better future in COVID-19 research.

FIGURE 1.2 Schematic representation of the application of various multi-omics-guided approaches in the immunopathogenesis and
therapeutic intervention of COVID-19.
8 Bioactive Compounds Against SARS-CoV-2

ACKNOWLEDGMENTS Chen J, Gao K, Wang R, Wei G-W (2021a) Revealing the threat
of emerging SARS-CoV-2 mutations to antibody therapies.
SM acknowledges UGC-STRIDE (KNU/R/STRIDE/1077/ bioRxiv 2021.04.12.439473. https://doi.org/10.1101/2021.
21:F.2-12/2019 [STRIDE-1]) and DST-SERB (CRG/2021/ 04.12.439473
002605) for supporting his research activities and labo- Chen S, He C, Li Y, et al (2021b) A computational toolset for rapid
ratory through research awards. RP acknowledges DST- identification of SARS-CoV-2, other viruses and microor-
ganisms from sequencing data. Brief Bioinform 22:924–
SERB for the award of Junior Research Fellowship (JRF).
935. https://doi.org/10.1093/bib/bbaa231
SP acknowledges SVMCM for her fellowship. PC acknowl- Chen T-F, Chang Y-C, Hsiao Y, et al (2021c) DockCoV2: A
edges DST-INSPIRE for her fellowship (JRF). drug database against SARS-CoV-2. Nucleic Acids Res
49:D1152–D1159. https://doi.org/10.1093/nar/gkaa861
Choudhury A, Das NC, Patra R, et al (2021a) Exploring the
binding efficacy of ivermectin against the key proteins of
REFERENCES
SARS-CoV-2 pathogenesis: An in silico approach. Future
Abdelrahman Z, Li M, Wang X (2020) Comparative review of Virol 16:277–291. https://doi.org/10.2217/fvl-2020-0342
SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A Choudhury A, Das NC, Patra R, Mukherjee S (2021b) In silico
respiratory viruses. Front Immunol 11:552909. https://doi. analyses on the comparative sensing of SARS-CoV-2
org/10.3389/fimmu.2020.552909 mRNA by the intracellular TLRs of humans. J Med Virol
Achek A, Yesudhas D, Choi S (2016) Toll-like receptors: 93(4):2476–2486. https://doi.org/10.1002/jmv.26776
Promising therapeutic targets for inflammatory diseases. Choudhury A, Mukherjee S (2020) In silico studies on the com-
Arch Pharm Res 39:1032–1049. https://doi.org/10.1007/ parative characterization of the interactions of SARS-
s12272-016-0806-9 CoV-2 spike glycoprotein with ACE-2 receptor homologs
Andersen KG, Rambaut A, Lipkin WI, et al (2020) The proximal and human TLRs. J Med Virol 92:2105–2113. https://doi.
origin of SARS-CoV-2. Nat Med 26:450–452. https://doi. org/10.1002/jmv.25987
org/10.1038/s41591-020-0820-9 Choudhury A, Sen Gupta PS, Panda SK, et al (2022) Designing
Andersson U, Ottestad W, Tracey KJ (2020) Extracellular AbhiSCoVac – a single potential vaccine for all ‘corona
HMGB1: A therapeutic target in severe pulmonary inflam- culprits’: Immunoinformatics and immune simulation
mation including COVID-19? Mol Med 26:42. https://doi. approaches. J Mol Liq 351:118633. https://doi.org/10.1016/j.
org/10.1186/s10020-020-00172-4 molliq.2022.118633
Araf Y, Akter F, Tang Y-D, et al (2022) Omicron variant of Conti P, Ronconi G, Caraffa AL, et al (2020) Induction of pro-
SARS-CoV-2: Genomics, transmissibility, and responses inflammatory cytokines (IL-1 and IL-6) and lung inflam-
to current COVID-19 vaccines. J Med Virol 94:1825–1832. mation by Coronavirus-19 (COVI-19 or SARS-CoV-2):
https://doi.org/10.1002/jmv.27588 Anti-inflammatory strategies. J Biol Regul Homeost Agents
Arizti-Sanz J, Freije CA, Stanton AC, et al (2020) Streamlined 34:327–331.
inactivation, amplification, and Cas13-based ­detection of Cui J, Li F, Shi Z-L (2019) Origin and evolution of pathogenic
SARS-CoV-2. Nat Commun 11:5921. https://doi.org/10.1038/ coronaviruses. Nat Rev Microbiol 17:181–192. https://doi.
s41467-020-19097-x org/10.1038/s41579-018-0118-9
Benson DA, Cavanaugh M, Clark K, et al (2012) GenBank. Das CN, Labala KR, Patra R, et al (2022a) In silico identification
Nucleic Acids Res 41:D36–D42. of new anti-SARS-CoV-2 agents from bioactive phytocom-
Bogner P, Capua I, Lipman DJ, et al (2006) A global initiative pounds targeting the viral spike glycoprotein and human
on sharing avian flu data. Nature 442:981. https://doi.org/ TLR4. Let. Drug Des Discov 19:175–191.
10.1038/442981a Das NC, Chakraborty P, Bayry J, Mukherjee S (2022b) In
Bonam SR, Kaveri SV, Sakuntabhai A, et al (2020) Adjunct immu- silico analyses on the comparative potential of therapeu-
notherapies for the management of severely ill COVID-19 tic human monoclonal antibodies against newly emerged
patients. Cell Rep Med 1(2):100016. SARS-CoV-2 variants bearing mutant spike protein. Front
Cao X (2020) COVID-19: Immunopathology and its implica- Immunol 12. https://doi.org/10.3389/fimmu.2021.782506
tions for therapy. Nat Rev Immunol 20:269–270. https://doi. Das NC, Patra R, PS Sen Gupta, et al (2020) Designing of a novel
org/10.1038/s41577-020-0308-3 multi-epitope peptide based vaccine against Brugia malayi:
CDC SARS-CoV-2 (2022) SARS-CoV-2 Variant Classifications An in silico approach. Infect Genet Evol 87:104633. https://
and Definitions. https://www.cdc.gov/coronavirus/2019- doi.org/10.1016/j.meegid.2020.104633
ncov/variants/variant-classifications.html Das NC, Sen Gupta PS, Biswal S, et al (2021) In-silico evidences
Chakraborty C, Bhattacharya M, Sharma AR, et al (2022) Continent- on filarial cystatin as a putative ligand of human TLR4. J
wide evolutionary trends of emerging SARS-CoV-2 variants: Biomol Struct Dyn 40(19):8808–8824. https://doi.org/10.
Dynamic profiles from Alpha to Omicron. GeroScience 1080/07391102.2021.1918252
44:2371–2392.https://doi.org/10.1007/s11357-022-00619-y Deigin Y, Segreto R (2021) SARS-CoV-2’s claimed natural origin
Chan YA, Zhan SH (2022) The emergence of the spike furin is undermined by issues with genome sequences of its rela-
cleavage site in SARS-CoV-2. Mol Biol Evol 39:msab327. tive strains. BioEssays 43:2100015. https://doi.org/10.1002/
https://doi.org/10.1093/molbev/msab327 bies.202100015
Chen B, Garmire L, Calvisi DF, et al (2020a) Harnessing big Dowling JK, Mansell A (2016) Toll-like receptors: The Swiss
“omics” data and AI for drug discovery in hepatocellular army knife of immunity and vaccine development. Clin
carcinoma. Nat Rev Gastroenterol Hepatol 17:238–251. Transl Immunol 5:e85. https://doi.org/10.1038/cti.2016.22
https://doi.org/10.1038/s41575-019-0240-9 Edgar R, Domrachev M, Lash AE (2002) Gene expression omni-
Chen FZ, You LJ, Yang F, et al (2020b) CNGBdb: China National bus: NCBI gene expression and hybridization array data
GeneBank database. Yi Chuan = Hered 42:799–809. https:// repository. Nucleic Acids Res 30:207–210. https://doi.
doi.org/10.16288/j.yczz.20-080 org/10.1093/nar/30.1.207
Omics-Guided Intervention Strategies in Combatting SARS-CoV-2 9

Ehlers A, Osborne J, Slack S, et al (2002) Poxvirus orthologous Kumaki Y, Salazar AM, Wandersee MK, Barnard DL (2017)
clusters (POCs). Bioinformatics 18:1544–1545. https://doi. Prophylactic and therapeutic intranasal administration with
org/10.1093/bioinformatics/18.11.1544 an immunomodulator, Hiltonol® (Poly IC: LC), in a lethal
Fernandes JD, Hinrichs AS, Clawson H, et al (2020) The UCSC SARS-CoV-infected BALB/c mouse model. Antiviral Res
SARS-CoV-2 genome browser. Nat Genet 52:991–998. 139:1–12.
https://doi.org/10.1038/s41588-020-0700-8 Lam TT-Y, Jia N, Zhang Y-W, et al (2020) Identifying SARS-
Forni D, Cagliani R, Clerici M, Sironi M (2017) Molecular evo- CoV-2-related coronaviruses in Malayan pangolins. Nature
lution of human coronavirus genomes. Trends Microbiol 583:282–285. https://doi.org/10.1038/s41586-020-2169-0
25:35–48. https://doi.org/10.1016/j.tim.2016.09.001 Li R, Pei S, Chen B, et al (2020a) Substantial undocumented
Freuling CM, Breithaupt A, Müller T, et al (2020) Susceptibility infection facilitates the rapid dissemination of novel coro-
of raccoon dogs for experimental SARS-CoV-2 infection. navirus (SARS-CoV-2). Science 368:489–493. https://doi.
Emerg Infect Dis 26:2982–2985. https://doi.org/10.3201/ org/10.1126/science.abb3221
eid2612.203733 Apweiler, R., Bairoch, A., Wu, C. H., Barker, W. C., Boeckmann,
Frieman M, Heise M, Baric R (2008) SARS coronavirus and B., Ferro, S., Gasteiger, E., Huang, H., Lopez, R., Magrane,
innate immunity. Virus Res 133:101–112. M., Martin, M. J., Natale, D. A., O'Donovan, C., Redaschi,
Gallo K, Goede A, Eckert A, et al (2021) PROMISCUOUS N., & Yeh, L. S. (2004). UniProt: the Universal Protein
2.0: A resource for drug-repositioning. Nucleic Acids knowledgebase. Nucleic Acids Research, 32(Database issue),
Res 49:D1373–D1380. https://doi.org/10.1093/nar/gkaa D115–D119. https://doi.org/10.1093/nar/gkh131
1061 Li X, Giorgi EE, Marichannegowda MH, et al (2020b) Emergence
Gordon DE, Jang GM, Bouhaddou M, et al (2020) A SARS- of SARS-CoV-2 through recombination and strong purify-
CoV-2 protein interaction map reveals targets for drug ing selection. Sci Adv 6:198975. https://doi.org/10.1126/
repurposing. Nature 583:459–468. https://doi.org/10.1038/ sciadv.abb9153
s41586-020-2286-9 Lu R, Zhao X, Li J, et al (2020) Genomic characterisation and
Guan W, Ni Z, Hu Y, et al (2020) Clinical characteristics of coro- epidemiology of 2019 novel coronavirus: Implications for
navirus disease 2019 in China. N Engl J Med 382:1708– virus origins and receptor binding. Lancet 395:565–574.
1720. https://doi.org/10.1056/NEJMoa2002032 https://doi.org/10.1016/S0140-6736(20)30251-8
Guan Y, Zheng BJ, He YQ, et al (2003) Isolation and characteriza- Makarenkov V, Mazoure B, Rabusseau G, Legendre P (2021)
tion of viruses related to the SARS coronavirus from ani- Horizontal gene transfer and recombination analysis of
mals in Southern China. Science 302:276–278. https://doi. SARS-CoV-2 genes helps discover its close relatives and
org/10.1126/science.1087139 shed light on its origin. BMC Ecol Evol 21:5. https://doi.
Hakim MS (2021) SARS-CoV-2, Covid-19, and the debunking of org/10.1186/s12862-020-01732-2
conspiracy theories. Rev Med Virol 31:e2222. https://doi. Mercatelli D, Triboli L, Fornasari E, et al (2021) Coronapp: A web
org/10.1002/rmv.2222 application to annotate and monitor SARS-CoV-2 muta-
Holmes EC, Goldstein SA, Rasmussen AL, et al (2021) The ori- tions. J Med Virol 93:3238–3245. https://doi.org/10.1002/
gins of SARS-CoV-2: A critical review. Cell 184:4848– jmv.26678
4856. https://doi.org/10.1016/j.cell.2021.08.017 Mifsud EJ, Tan AC-L, Jackson DC (2014) TLR agonists as
Hu B, Ge X, Wang L-F, Shi Z (2015) Bat origin of human modulators of the innate immune response and their poten-
coronaviruses. Virol J 12:221. https://doi.org/10.1186/ tial as agents against infectious disease. Front Immunol
s12985-015-0422-1 5:79.
Jaimes JA, André NM, Chappie JS, et al (2020) Phylogenetic Mitchell AL, Almeida A, Beracochea M, et al (2020) MGnify:
analysis and structural modeling of SARS-CoV-2 spike The microbiome analysis resource in 2020. Nucleic
protein reveals an evolutionary distinct and proteolytically Acids Res 48:D570–D578. https://doi.org/10.1093/nar/
sensitive activation loop. J Mol Biol 432:3309–3325. https:// gkz1035
doi.org/10.1016/j.jmb.2020.04.009 Mukherjee S (2022) Toll-like receptor 4 in COVID-19: Friend
Jumper J, Evans R, Pritzel A, et al (2021) Highly accurate protein or foe? Future Virol 17:415–417. https://doi.org/10.2217/
structure prediction with AlphaFold. Nature 596:583–589. fvl-2021-0249
https://doi.org/10.1038/s41586-021-03819-2 Mukherjee S, Huda S, Sinha Babu SP (2019) Toll-like receptor
Kalantar KL, Carvalho T, de Bourcy CFA, et al (2020) IDseq- polymorphism in host immune response to infectious dis-
an open source cloud-based pipeline and analysis ser- eases: A review. Scand J Immunol 90:e12771. https://doi.
vice for metagenomic pathogen detection and monitoring. org/10.1111/sji.12771
Gigascience 9:giaa111. https://doi.org/10.1093/gigascience/ Mukherjee S, Karmakar S, Babu SPS (2016) TLR2 and TLR4
giaa111 mediated host immune responses in major infectious dis-
Kalita P, Padhi A, Zhang KYJ, Tripathi T (2020) Design of a eases: A review. Brazilian J Infect Dis 20:193–204.
peptide-based subunit vaccine against novel coronavirus Nova N (2021) Cross-species transmission of coronaviruses in
SARS-CoV-2. Microb Pathog 145:104236. humans and domestic mammals, what are the ecological
Katoh K, Standley DM (2013) MAFFT multiple sequence align- mechanisms driving transmission, spillover, and disease
ment software version 7: Improvements in performance and emergence? Front Public Heal 9. https://doi.org/10.3389/
usability. Mol Biol Evol 30:772–780. https://doi.org/10.1093/ fpubh.2021.717941
molbev/mst010 Oughtred R, Rust J, Chang C, et al (2021) The BioGRID database:
Kim D, Lee J-Y, Yang J-S, et al (2020) The architecture of SARS- A comprehensive biomedical resource of curated protein,
CoV-2 transcriptome. Cell 181:914–921.e10. https://doi. genetic, and chemical interactions. Protein Sci 30:187–200.
org/10.1016/j.cell.2020.04.011 https://doi.org/10.1002/pro.3978
Kouranov A, Xie L, de la Cruz J, et al (2006) The RCSB PDB Patra R, Bhattacharya M, Mukherjee S (2021a) IoT-Based
information portal for structural genomics. Nucleic Acids Computational Frameworks in Disease Prediction and
Res 34:D302–D305. Healthcare Management: Strategies, Challenges, and
10 Bioactive Compounds Against SARS-CoV-2

Potential. In: Marques G, Bhoi AK, Albuquerque VHC Shang J, Ye G, Shi K, et al (2020) Structural basis of receptor
de, K.S. H (eds). IoT in Healthcare and Ambient Assisted recognition by SARS-CoV-2. Nature 581:221–224. https://
Living. Springer Singapore, Singapore, pp 17–41. doi.org/10.1038/s41586-020-2179-y
Patra R, Chandra Das N, Mukherjee S (2020) Targeting human Shu Y, McCauley J (2017) GISAID: Global initiative on sharing
TLRs to combat COVID-19: A solution? J Med Virol all influenza data–from vision to reality. Eurosurveillance
93:615–617. https://doi.org/10.1002/jmv.26387 22:30494.
Patra R, Das NC, Bhattacharya M, et al (2021b) Applications Sillitoe I, Bordin N, Dawson N, et al (2021) CATH: Increased
of Artificial Intelligence (AI) Protecting from COVID-19 structural coverage of functional space. Nucleic Acids Res
Pandemic: A Clinical and Socioeconomic Perspective. 49:D266–D273. https://doi.org/10.1093/nar/gkaa1079
In: Kautish S, Peng S-L, Obaid AJ (eds). Computational Singh R, Singh PK, Kumar R, et al (2021) Multi-omics approach
Intelligence Techniques for Combating COVID-19. in the identification of potential therapeutic biomolecule
Springer International Publishing, Cham, pp 45–60. for COVID-19. Front Pharmacol 12:652335. https://doi.
Patra R, Das NC, Mukherjee S (2021c) Toll-Like Receptors org/10.3389/fphar.2021.652335
(TLRs) as Therapeutic Targets for Treating SARS-CoV-2: Su S, Wong G, Shi W, et al (2016) Epidemiology, genetic
An Immunobiological Perspective. In: Asea, AAA, Kaur, recombination, and pathogenesis of coronaviruses. Trends
P (eds). Coronavirus Therapeutics – Volume I. Advances Microbiol 24:490–502. https://doi.org/10.1016/j.tim.2016.
in Experimental Medicine and Biology, vol 1352. Springer 03.003
International Publishing, Cham, pp 87–109. https://doi. Tao K, Tzou PL, Nouhin J, et al (2021) The biological and clini-
org/10.1007/978-3-030-85109-5_6 cal significance of emerging SARS-CoV-2 ­variants. Nat
Patra R, Mukherjee S, Das NC (2021d) Exploring the differen- Rev Genet 22:757–773. https://doi.org/10.1038/s41576-021-
tial expression and prognostic significance of the COL11A1 00408-x
gene in human colorectal carcinoma: An integrated bio- Ternent T, Csordas A, Qi D, et al (2014) How to submit MS pro-
informatics approach. Front Genet 12:88. https://doi.org/ teomics data to ProteomeXchange via the PRIDE data-
10.3389/fgene.2021.608313 base. Proteomics 14:2233–2241. https://doi.org/10.1002/
Poon LLM, Chu DKW, Chan KH, et al (2005) Identification of a pmic.201400120
novel coronavirus in bats. J Virol 79:2001–2009. https://doi. Tiwari V, Kumar M, Tiwari A, et al (2021) Current trends in
org/10.1128/JVI.79.4.2001-2009.2005 diagnosis and treatment strategies of COVID-19 infection.
Posada-Céspedes S, Seifert D, Topolsky I, et al (2021) V-pipe: A Environ Sci Pollut Res Int 28:64987–65013. https://doi.
computational pipeline for assessing viral genetic diversity org/10.1007/s11356-021-16715-z
from high-throughput data. Bioinformatics 37:1673–1680. UniProt Consortium (2021) UniProt: The universal protein
https://doi.org/10.1093/bioinformatics/btab015 knowledgebase in 2021. Nucleic Acids Res 49:D480–D489.
Rahalkar MC, Bahulikar RA (2020) Lethal pneumonia cases in https://doi.org/10.1093/nar/gkaa1100
Mojiang miners (2012) and the mineshaft could provide Wan Y, Shang J, Graham R, et al (2020) Receptor recognition
important clues to the origin of SARS-CoV-2. Front Public by the novel coronavirus from Wuhan: An Analysis based
Heal 8. https://doi.org/10.3389/fpubh.2020.581569 on decade-long structural studies of SARS coronavirus. J
Rahman MS, Hoque MN, Islam MR, et al (2020) Epitope- Virol 94. https://doi.org/10.1128/JVI.00127-20
based chimeric peptide vaccine design against S, M and E Wang M-Y, Zhao R, Gao L-J, et al (2020) SARS-CoV-2: Structure,
proteins of SARS-CoV-2 etiologic agent of global pandemic biology, and structure-based therapeutics development.
COVID-19: An in silico approach. bioRxiv 2020.03.30. Front Cell Infect Microbiol 10. https://doi.org/10.3389/
015164. https://doi.org/10.1101/2020.03.30.015164 fcimb.2020.587269
Robinson J, Halliwell JA, McWilliam H, et al (2013) IPD–the Ward D, Higgins M, Phelan JE, et al (2021) An integrated in silico
immuno polymorphism database. Nucleic Acids Res immuno-genetic analytical platform provides insights into
41:D1234–D1240. https://doi.org/10.1093/nar/gks1140 COVID-19 serological and vaccine targets. Genome Med
Ruiz-Medina BE, Varela-Ramirez A, Kirken RA, Robles- 13:4. https://doi.org/10.1186/s13073-020-00822-6
Escajeda E (2022) The SARS-CoV-2 origin dilemma: WHO Coronavirus (2022) WHO Coronavirus (COVID-19)
Zoonotic transfer or laboratory leak? BioEssays 44:2100189. Dashboard | WHO Coronavirus (COVID-19) Dashboard
https://doi.org/10.1002/bies.202100189 with Vaccination Data. https://covid19.who.int/
Sansone S-A, McQuilton P, Rocca-Serra P, et al (2019) WHO COVID-19 (2022) Weekly epidemiological update on
FAIRsharing as a community approach to standards, repos- COVID-19 - 5 October 2022. https://www.who.int/publica­
itories and policies. Nat Biotechnol 37:358–367. tions/m/item/weekly-epidemiological-update-on-covid-
Schäffer AA, Hatcher EL, Yankie L, et al (2020) VADR: 19---5-october-2022
Validation and annotation of virus sequence submissions WHO Omicron (2022) Update on Omicron. https://www.who.int/
to GenBank. BMC Bioinformatics 21:211. https://doi.org/ news/item/28-11-2021-update-on-omicron
10.1186/s12859-020-3537-3 Woo PCY, Huang Y, Lau SKP, Yuen K-Y (2010) Coronavirus
Segreto R, Deigin Y (2021) The genetic structure of SARS-CoV-2 genomics and bioinformatics analysis. Viruses 2:1804–
does not rule out a laboratory origin: SARS-COV-2 chime- 1820. https://doi.org/10.3390/v2081803
ric structure and furin cleavage site might be the result of Wu F, Zhao S, Yu B, et al (2020) A new coronavirus associated
genetic manipulation. Bioessays 43:e2000240. https://doi. with human respiratory disease in China. Nature 579:265–
org/10.1002/bies.202000240 269. https://doi.org/10.1038/s41586-020-2008-3
Shahhosseini N, Wong G, Kobinger GP, Chinikar S (2021) Xiao X, Newman C, Buesching CD, et al (2021) Animal sales
SARS-CoV-2 spillover transmission due to recombination from Wuhan wet markets immediately prior to the
event. Gene Reports 23:101045. https://doi.org/10.1016/j. COVID-19 pandemic. Sci Rep 11:11898. https://doi.org/10.
genrep.2021.101045 1038/s41598-021-91470-2
Omics-Guided Intervention Strategies in Combatting SARS-CoV-2 11

Xu R-H, He J-F, Evans MR, et al (2004) Epidemiologic clues to Zhang Y-Z, Holmes EC (2020) A genomic perspective on the
SARS origin in China. Emerg Infect Dis 10:1030–1037. origin and emergence of SARS-CoV-2. Cell 181:223–227.
https://doi.org/10.3201/eid1006.030852 https://doi.org/10.1016/j.cell.2020.03.035
Yang X, Yu Y, Xu J, et al (2020) Clinical course and outcomes Zhou H, Chen X, Hu T, et al (2020a) A novel bat coronavirus
of critically ill patients with SARS-CoV-2 pneumonia in closely related to SARS-CoV-2 contains natural inser-
Wuhan, China: A single-centered, retrospective, obser- tions at the S1/S2 cleavage site of the spike protein. Curr
vational study. Lancet Respir Med 8:475–481. https://doi. Biol 30:2196–2203.e3. https://doi.org/10.1016/j.cub.2020.
org/10.1016/S2213-2600(20)30079-5 05.023
Ye Z-W, Yuan S, Yuen K-S, et al (2020) Zoonotic origins of Zhou P, Yang X-L, Wang X-G, et al (2020b) A pneumonia out-
human coronaviruses. Int J Biol Sci 16:1686–1697. https:// break associated with a new coronavirus of probable
doi.org/10.7150/ijbs.45472 bat origin. Nature 579:270–273. https://doi.org/10.1038/
Zhang T, Wu Q, Zhang Z (2020) Probable pangolin origin of s41586-020-2012-7
SARS-CoV-2 associated with the COVID-19 outbreak.
Curr Biol 30:1578. https://doi.org/10.1016/j.cub.2020.03.063
 2 The Application of Computer-Aided
Drug Design Methods for Developing
Natural Compound-Based
Therapeutics Against SARS-CoV-2
Priya Godara, Dhaneswar Prusty

2.1 INTRODUCTION found inhibitory effects of medicinal plants and aromatic

herbal-based compounds against COVID.7
The Coronaviridae family consisting of the novel coronavi- Identifying and validating an appropriate therapeutic
rus led to the recent coronavirus disease 2019 (COVID-19) target is the initial stage of the drug development pipeline.
outbreak.1 In December 2019, the initial severe acute respi- The other steps include hit-to-lead discovery and lead mol-
ratory syndrome coronavirus 2 (SARS-CoV-2)-related cases ecule optimization, which precedes further by preclinical
were identified in Wuhan, China. The virus metastasized and clinical trials.8 The classical approach to drug design
globally in just a couple of months despite previous knowl- is an extensive process that takes nearly 10–15 years, with
edge about its transmission. According to the World Health around 2.558 billion USD spent on bringing the drug to
Organization (WHO), there were 611,421,786 reported market.9,10 Despite the massive investment of time and
cases, including around 6,512,438 confirmed deaths, as of resources, the success rate is merely 13%.11 The lack of
September 26, 2022 (https://covid19.who.int). Scientists, optimal pharmacokinetic properties is the main reason for
as well as global health specialists from all around the drug failure (40–60%) during the later stages.1 In contrast,
world, have tirelessly worked and collaborated to create using bioinformatics techniques has relieved a great deal
and execute extremely effective vaccines and novel thera- of pressure on the price of performing experiments and
peutic approaches to combat COVID-19.2 The search for the animal sacrifices during the experiments.13 The com-
efficient therapeutics was complicated by the unavailability puter-aided drug designing (CADD) approach may greatly
of s­ pecific therapeutics. speed up the process of drug discovery, minimize uncer-
Moreover, the rising incidences of resistance in the tainty, predict drug-like properties, and significantly lower
pathogens caused a pressing urgency to identify and research and development (R&D) costs.14 Therefore, the
develop novel substances to combat it. The bioavailability importance of in silico studies was particularly highlighted
and specificity of these compounds need to be extremely during the COVID-19 pandemic. Although the biology of
high with low toxicity. Among the various therapeutic com- coronaviruses had been studied previously, the computa-
pounds, natural compound molecules easily fit into these tional tools developed primarily for SARS-CoV-2 have just
criteria because a majority of the substances have ideal lately reached their full potential due to the quick detection,
drug-like properties along with pharmacokinetic character- comprehension, and prophylaxis of COVID-19.15
istics, making them likely candidates for use as lead com- The pre-built web servers, including Phyre2 and
pounds against different SARS-CoV-2 targets.3 They are I-TASSER, proved extremely useful for the protein struc-
distinguished by their vast array of different scaffolds and ture homology modeling even before the assessment of
complex structural makeup, acting as a reservoir for poten- crystallographic structures.16,17 Exploring appropriate target
tial drug candidates. Natural products (NPs) have a long sequences for the design of vaccines was substantially facil-
history as principal resources for developing new drugs to itated by using VaxiJen, B-cell, and T-cell epitope identifi-
treat various infectious diseases.4 The lengthy history of cation in silico methods.18
using conventional medications offers insights into their The in silico methods are incredibly useful for testing
safety along with efficiency.5 The vast range of compounds potential therapeutics against molecular targets, enabling
in plant-based extracts include alkaloids, terpenes, lignans, the most promising ones to be chosen for additional in vitro
flavonoids, phenolic acids, proteins, and coumarins. Recent and in vivo testing.19 Although CADD is based on some
research has concentrated on the possible antiviral activity predictions and approximations, it significantly speeds up
of plant-derived compounds against viruses like influenza, the entire drug development pipeline. The various suc-
herpes simplex, coronavirus, and dengue.6 Boukhatem et al. cessful attempts of CADD in SARS-CoV-2 drug discovery

12 DOI: 10.1201/9781003323884-2
The Application of Computer-Aided Drug Design Methods 13

FIGURE 2.1 Classification of the computer-aided drug design approach with its related tools.

during the pandemic point to a great scope of possibilities research is based on two major approaches: ligand-based
in the future.20 drug designing (LBDD) and structure-based drug design-
Due to the pre-availability of the pharmacodynamic and ing (SBDD) (Figure 2.1).
pharmacokinetic-related data of the broad-spectrum anti-
viral drug compounds, the potential repurposing of these
2.2.1 Structure-Based Drug Designing (SBDD)
drugs utilizing the CADD-based approaches has proved
to be one of the promising strategies to expedite the drug The SBDD approach relies on the three-dimensional (3D)
­discovery against SARS-CoV-2.21 structure of the target receptor, including its active site, to
Overall, this chapter intends to provide a rapid and con- better understand its interaction with the respective ligand.
cise overview of several CADD techniques that have been It is carried out through virtual screening, molecular dock-
deployed on a large scale for developing natural compound- ing, and by the molecular dynamics simulation (MDS)
based therapeutics for SARS-CoV-2, which can help deal approach.23 The various steps in SBDD involve target struc-
with future pandemic situations. ture preparation, identification/prediction of the active bind-
ing site, finding/preparation of a suitable compound library,
2.2 WHAT IS A COMPUTER-AIDED DRUG molecular docking, and finally, the MDS. The 3D s­ tructures
DESIGN APPROACH (CADD)? TYPES? of the target receptors can be directly obtained from the
protein data bank (PDB). In case of non-availability of the
RELATED SOFTWARE?
crystal structure of the respective protein, the 3D structure
CADD is the in silico approach that makes use of computer can be deciphered through the threading, homology mod-
algorithms to calculate the combinations of simulated com- eling, and ab initio modeling approaches, among which
pounds and their targets. CADD has become more precise homology modeling is the most widely used. It gives a more
due to the continuous advancements in computer technol- accurate prediction of the 3D structure of the protein from
ogy. Additionally, the network pharmacology technology its amino acid sequence utilizing a suitable template in the
advancements have made it possible to quickly unravel the PDB.24 The various tools for homology modeling include
intricate connections between compounds and their various the SWISS-MODEL server, Phyre, Phyre2, RaptorX,
targets.22 The CADD approach applied during COVID-19 HHpred, ESyPred3D, etc. Ab initio structure prediction is
14 Bioactive Compounds Against SARS-CoV-2

made when there is the absence of any template structure include Schrödinger, AutoDock, GOLD, AutoDock Vina,
in the database; it also recognizes a global optimization CDOCKER, FlexX, DOCK6, SwissDock, Surflex, etc. The
problem for obtaining the values of the dihedral angle for top best-docked complexes are carried out through molec-
protein stability using the tools I-TASSER, ROBETTA, and ular dynamics simulations that predict the stability of the
trRosetta.25 The protein threading approach is applied when complexes in the dynamic environment. The commonly
the target protein shares less than 25% sequence similarity used bioinformatics tools for MDSs include Desmond,
with the rest proteins in the PDB. It considers the structural GROMACS, NAMD (Nanoscale Molecular Dynamics),
information of the template, including solvent accessibility WebGro, etc.
and secondary structure for predicting the structure, and
uses tools like HHpred, RaptorX, IntFOLD, etc.26
2.2.2 Ligand-Based Drug Designing (LBDD)
Further, the ligand-binding sites on the target receptor
are essential for a specific type of docking, which can be LBDD is carried out when the 3D structure of the target
obtained from the previously published literature. In case receptor is unavailable.28 LBDD depends on the informa-
of the unavailability of information regarding the active tion regarding the ligand and target receptor interactions.
site, active sites can be predicted, or else blind docking LBDD is carried forward through pharmacophore model-
can be performed.27 The various tools for active site pre- ing, quantitative structure-activity relationship (QSAR), and
diction include MetaPocket, MSPocket, DoGSite Scorer, artificial intelligence (AI). Catalyst, DISCO, LigandScout,
Q-SiteFinder, CASTp, NSiteMatch, and the DEPTH server. PharmMapper, PharmaGist, Pharmer, and ZINCPharmer
Natural compound libraries can be selected from the vari- are the various tools used for pharmacophore modeling
ous available databases (Table 2.1). Molecular docking is studies. QSAR is widely used for predicting the biologi-
used to study the binding affinity between the ligands and cal property of lead compounds.29 QikProp, CODESSA
the target receptor, which is computed in the form of bind- PRO, PreADMET, E-DRAGON, DRAGON 7.0, PaDEL
ing energy scores. The various tools for molecular docking Descriptor, and ADMET Predictor are the various programs

TABLE 2.1
List of the Natural Compound Libraries/Databases Used Against SARS-CoV-2
S. No Natural Compounds Sources Website Link References – PMID/DOI Google Scholar
Citations
1. NPASS (Natural Product Activity http://bidd.group/NPASS/ 32470577 137
and Species Source Database)
2. CMNPD (Comprehensive Marine https://www.cmnpd.org 34772509 43
Natural Products Database)
3. ZINC natural products catalog https://zinc12.docking.org/browse/catalogs/ DOI: 10.34172/PS.2021.11 2388
natural-products
4. MarinLit database https://marinlit.rsc.org/ 34226782 499
5. ZINC database http://zinc15.docking.org 33963942 4127
6. Selleckchem.com https://www.selleckchem.com/ 33200697, 33134310 64
7. IBS Database (InterBioScreen) https://www.ibscreen.com/ 34395160 31
8. Sigma-Aldrich plant profiler https://www.sigmaaldrich.com/life-science/nutrition- 32897138 45
chemical library research/learning-center/plant-profiler.html
9. Dr. Duke’s Phytochemical and https://www.nal.usda.gov/dr-dukes-database 35337962 262
Ethnobotanical Databases
10. Phytochemical Interactions http://www.genome.jp/db/pcidb 35337962 245
Database
11. Super Natural III database https://bioinf-applied.charite.de/supernatural_new/ 33330376 259
12. Natural Compound and Natural- www.molport.com 33330376 4
Like Compound Database
13. FooDB Version 1.0 https://foodb.ca 33330376 20
14. COVID-19 Docking Server https://ncov.schanglab.org.cn/ 32692801 142
15. PubChem database https://pubchem.ncbi.nlm.nih.gov/ 33888980 3567
16. SWEETLEAD database https://simtk.org/home/sweetlead DOI: 10.22037/smsj. 109
v2i3.31997
17. South African Natural Compound https://sancdb.rubi.ru.ac.za/ DOI: 10.22037/smsj. 93
Database (SANCDB) v2i3.31997
18. SymMap database http://www.symmap.org/ DOI: 10.22037/smsj. 207
v2i3.31997
The Application of Computer-Aided Drug Design Methods 15

used for building the QSAR models. AI applications in drug estimate the consequences of mutation on the drug binding
discovery are numerous, including the prediction of toxicity with SARS-CoV-2 receptors.37
and the drug molecules’ biological properties.30 Machine As a result, CADD can significantly accelerate the drug
learning and deep learning are the two significant meth- development process. Drug development is typically a chal-
ods for rational drug designing.31 Random forest, support lenging task accomplished in mainly six phases: (1) iden-
vector machine, and naive Bayesian are the widely used tification of target and validation, (2) lead discovery and
machine learning algorithms in drug discovery.32–34 The optimization, (3) preclinical trials, (4) clinical trials, (5)
convolutional neural network, recurrent neural network, approval, and (6) post-marketing surveillance (Figure 2.2).
deep neural network, and restricted Boltzmann machine are Applying the CADD approach can significantly enhance
the various deep learning methods used in drug discovery.11 the quality and accuracy to prevent potential late-stage
failure.23 The target identification and validation step
2.3 HOW HAS CADD ELEVATED THE ENTIRE includes finding a potential molecular target and classify-
ing and determining whether it is suited for drug discov-
DRUG DEVELOPMENT PIPELINE?
ery, like predicting protein structure through 3D structure
The classical drug development pipeline generally takes modeling, reverse docking, protein structural elucidation
10–12 years, with just around 9.6–13.8% of overall drugs through nuclear magnetic resonance (NMR) spectroscopy,
passing FDA standards.35 Further, it is also quite expensive, and X-ray crystallography.38 The second step comprises the
challenging, and time-consuming. In the interim, the bio- discovery of lead compounds and optimization using vari-
informatics approach emerged as one of the cornerstones ous approaches, including high-throughput virtual screen-
in the SARS-CoV-2 drug development, which elevated ing (HTVS), QSAR, and molecular docking. These hit
the entire drug development pipeline. With the consistent molecules are then assessed and modified to enhance their
increase in SARS-CoV-2 infection-related deaths, CADD, activity.39 The next step includes performing the preclini-
which reduces time and lowers the costs of generating cal studies/trials conducted before human clinical trials to
therapeutic compounds, has become a quick and reliable impart detailed information on the toxicity parameter and
strategy in pharmaceutical research.36 Although the SARS- the appropriate dosage of the drugs.40 The findings of the
CoV-2 genome’s inherent mutability impedes disease pre- preclinical studies determine whether the drug is suitable for
vention and treatment, CADD can be used effectively to testing on humans.40 The last step in the drug development

FIGURE 2.2 Comparison between the computer-aided-drug design (CADD) approach and traditional drug development pipeline.
16 Bioactive Compounds Against SARS-CoV-2

phase includes clinical trials. Following the successful com- Naik et al. successfully conducted HTVS of nearly 3963
pletion of clinical trials, the following drug gets approval natural compounds from the NPASS Database on endori-
from U.S. Food and Drug Administration (FDA), allowing bonuclease, 3C-like proteinase, exoribonuclease, helicase,
it to be sold for clinical use.41 Incorporating computational methyltransferase, and RNA-dependent RNA polymerase
studies into drug discovery has significantly increased the targets of SARS-CoV-2. The study revealed three natural
knowledge of structural, chemical, and biological data in compounds, NPC52382, NPC214620, and NPC270578, as
the phases mentioned earlier.42 The lack of optimal phar- potent multitargeting natural compounds for SARS-CoV-2
macokinetic properties (ADMET) has been reported in drug discovery.49
40–60% of cases, leading to the failure of drugs at the final This increasing interest in NPs and their use led to an
stages of development.12 The use of CADD techniques in enormous increase in the virtual screening of the natu-
preliminary investigations by pharmaceutical corporations ral compound libraries of various databases during the
and academic research teams has greatly aided in the accel- pandemic (Table 2.1). In 2020, Kong et al. introduced the
eration of the drug development process while lowering COVID-19 Docking Server (https://ncov.schanglab.org.
overall costs and reducing failures in the final phases.14 cn/) in their study; it was designed especially for docking
the peptides, antibodies, and small molecules against the
2.4 NATURAL COMPOUND LIBRARIES USED various therapeutic targets of COVID-19. The users need
to upload the small molecule/peptide/antibody file as input,
FOR CADD AGAINST SARS-CoV-2
and the targets in the virus can be selected from the list
Natural compound-based products have been the focus provided on server.50
of the research group’s attention for the last few decades,
and interest related to these keeps rising steadily. The dis-
tinct chemical diversity that NPs have acquired through- 2.5 REMARKABLE SUCCESSES OF
out a long period of evolution has resulted in variances in CADD IN DESIGNING NATURAL
their biological activity and drug-likeness properties. Even COMPOUND-BASED THERAPEUTICS
prior to the advent of modern chemical pharmacology, NPs
FOR COVID-19 TREATMENT
were already employed for millennia as constituents of tra-
ditional medications, particularly as active ingredients in When the SARS-CoV-2 genomic data became available
herbal cures.43 weeks after the outbreak, the CADD approach was a piv-
Due to financial constraints, religious tenets, or the otal part of COVID-19 research.51 The computational tools
difficulty in obtaining pharmaceuticals, some traditional helped rapidly predict the promising inhibitory compounds
medical techniques, such as Indian Ayurveda, African against the numerous SARS-CoV-2 targets, contributing to
herbal remedies, and traditional Chinese medicine (TCM), the disease pathogenesis. The successful application of bio-
continue to be the first choice of therapy for many people informatics techniques against SARS-CoV-2 in the current
today.44 NPs are now established as one of the essential context is a remarkable accomplishment for the scientific
sources for creating novel lead compounds as well as scaf- community. In silico studies like genome-wide association
folds in modern pharmacology.45,46 Additionally, scientific studies, next-generation sequencing, and CADD not only
studies detailing the beneficial effects of NPs on the recov- led to the sequencing of the genome of SARS-CoV-2 but
ery process of numerous human and animal ailments are also predicted and interpreted the genetic variations, phy-
published weekly in peer-reviewed journals. Surprisingly, logenetic relationships, sequencing errors, and promising
NPs extracted from bacteria serve as the foundation for therapeutics against SARS-CoV-2 in a short span. From the
major classes of antibiotics and antifungals. NPs and their initial stages of identification and characterization of viral
derivatives are frequently utilized to treat many malignan- samples to the final stages of drug design, computational
cies, cardiovascular disorders, diabetes, and other ailments. approaches have been extensively used to discover signifi-
For example, from 1981 to 2014, NPs accounted for more cant insights regarding SARS-CoV-2 genomic content and
than half of all newly discovered medications.45 structure, variability, and species diversity, and ultimately
However, most of these tested NP extracts are without to anticipate potential drugs/vaccines.13
structural information. In this regard, the NP library pro- A structure-based virtual screening approach was
vides a collection of structural and experimental data on attempted by Jayaraj et al. to screen nearly 3112 home-
the different compounds, which are then used for HTVS opathy and 14,682 Kabasura Kudineer compounds against
against various targets.47 Regarding this, various libraries NSP-13, NSP-15, spike proteins, NSP-12, NSP-14, and main
from different databases have multiplied quickly over the protease targets of SARS-CoV-2. It was found that a total
last 20 years as generalist or specialized sources for NP of five Kabasura Kudineer compounds (88583189, 250395,
information. 44259583, 129677029, and 44259584) and five homeopa-
The NP Activity & Species Source (NPASS) Database thy compounds (14590080, 3802778, 5315832, 320361, and
(https://bidd.group/NPASS/) is a free database that addi- 74029795) showed higher docking scores. The molecular
tionally provides the literature-reported experimental activ- dynamics simulations confirmed the complexes’ stable
ity like MIC, Ki, GI50, IC50, and EC50 values of compounds.48 interactions during the simulations.52 Zhang et al. attempted
The Application of Computer-Aided Drug Design Methods 17

to screen 1871 natural compounds against the receptor- cyanidin, curcumin, catechin, B-carotene, vanillin, thymol,
binding domain (RBD) of the spike glycoprotein, thereby resveratrol, astaxanthin, gingerol, and eugenol against the
preventing it from binding with the host’s angiotensin-con- Mpro and RdRp receptors of SARS-CoV-2 using the COVID-
verting enzyme 2 (ACE2) receptor. It was predicted that a 19 Docking Server. It was predicted that riboflavin, daidzein,
total of six compounds, including 20(S)-ginsenoside Rg3, phycocyanobilin, cyanidin, and genistein showed effective
isochlorogenic A, isobavachalcone, 20(R)-ginsenoside Rg3, binding to the receptors.61 Xu et al. conducted a novel study
epigallocatechin gallate (EGCG), and bakuchiol (Bkc) in which a total of 2030 flavonoid inhibitor compounds were
showed a higher docking score. Among these, isoliensinine virtually screened against the COVID-19 3CL protein recep-
(Isl), Ibvc, salvianolic acid A (SalA), and EGCG were found tor. According to their research, the compound rutin outper-
to be potent against SARS-CoV-2.53 forms the other candidate compounds.62
So far, there have been nearly 16 marine FDA-approved Majumder et al. screened compounds from the plant
drugs; dozens are in clinical development, with the vast profiler chemical library of Sigma-Aldrich (https://www.
majority aimed at cancer therapeutics.54 Ara A® is the sigmaaldrich.com/life-science/nutrition-research/­learning-
only marine-obtained antiviral medication against herpes center/plant-profiler.html) against the Mpro receptor of
simplex. Both of the previously recognized anticancer the corona­ virus, and among these the compound rutin
medications (Bryostatin® and Griffithsin) from red algae obtained from Allium sativum (garlic) showed the ­highest
are currently in clinical trials for the human immunodefi- binding score. Following that, the SwissSimilarity web
ciency virus (HIV).55 PharmaMar recently announced that tool (http://www.­swisssimilarity.ch/) was used to search
phase II clinical trials for the treatment of COVID-19 had for r­utin-like compounds, and nearly 40 ­compounds were
begun, with the anti-tumor drug Aplidin® (derived from docked to the Mpro receptor. Finally, molecular ­docking
soil-derived actinomycete) showing a higher effect for and MDS studies suggest kaempferol 3-O-rutinoside,
coronavirus treatment than ivermectin. In an attempt to find ­quercetin-3-O-α-L-arabinopyranoside, peonidin 3-O-
alternatives for COVID-19 medications from the marine glucoside, and 4-(3,4-dihydroxyphenyl)-7-methoxy-5-[(6-
ecosystem, researchers are refocusing their work on this O-β-D-xylopyranosyl-β-D-glucopyranosyl)oxy]-2H-1-ben-
domain by employing SBDD approaches.56 zopyran-2-one as the potent lead compounds against
Gentile et al. proposed 17 lead-like main protease (Mpro) COVID-19.63 In their recent study, Moradi et al. studied
inhibitors by screening a Marine Natural Product (MNP) the ability of plant-based inhibitors of protease to inhibit
library containing 14,064 compounds obtained from the papain-like protease activity of SARS-CoV-2. The molecu-
brown algae using the SBDD approach.57 Khan et al. pre- lar docking and molecular dynamics study confirmed the
dicted the compound (11R)-11-epi-Fistularin-3 obtained suppressive role of VcTI (2PLX) obtained from Veronica
from the Aplysinidae sponge as the potent inhibitor of hederifolia against SARS-CoV-2.64
SARS-CoV-2 Mpro through the MDS and molecular docking A molecular docking approach was used by Nouadi
study.58 Several studies have been published regarding the et al. when docking nearly 54 compounds extracted from
application of LBDD methodologies for finding inhibitors the medicinal Moroccan plants against 3CLpro and ACE2,
against SARS-CoV-2. Ghosh et al. proposed 13 NP com- and among them, rutin, luteolin-glucoside, taxol, and gen-
pounds as potent Mpro inhibitors consisting of 1 pentacyclic kwanine were predicted as anti-SARS-CoV-2 compounds
triterpenoid, 11 flavonoids, and 1 lignan.59 A CADD method based on their high-affinity scores.65
comprising both structure- and ligand-based approaches was Nikunj et al. predicted 9-dodecenoic acid, n-decanoic
carried out using 11,162 MNPs from the Reaxys® database, acid, and methyl ester as effective against SARS-CoV-2 by
7 MNPs derived by a team from marine-derived actinomy- screening the red algae-derived phytochemicals against the
cetes, and 14 MNPs from the clinical trials. Among these, RBD domain.66 In their study, Joseph et al. concluded that
494 MNPs sorted by the QSAR method of ligand-based green tea extracts is one of the promising remedies against
CADD were carried out for the molecular docking approach coronaviruses.67
of structure-based CADD. A total of five compounds con- A virtual screening study was carried out by dock-
sisting of notoamide I, two bromoindole derivatives, benzo[f] ing nearly 6570 herbal compounds from SWEETLEAD
pyrano[4,3-b]chromene, and emindole SB beta-mannoside Database, the South African natural compound database
were predicted as potent inhibitors of SARS-CoV-2 Mpro.56 (SANCDB), and SymMap database, which predicted cyclo-
An SBDD approach was carried out by screening FDA- mulberrin and Sodwanone B as the lead compounds against
approved drugs of the LOPAC library against the S-RBD of the Mpro target of SARS-CoV-2.68
SARS-CoV-2 and the ACE-2 receptor. The molecular dock- A molecular docking approach was carried out by
ing and MDS study showed that five compounds, GNF-5, Prasanth et al. using nearly 48 compounds derived from
TNP, eptifibatide acetate, RS504393, and GR 127935 hydro- cinnamon through the PubMed database. The MDS study
chloride hydrate showed an effective binding with the ACE2 predicted pavetannin C1 and tenufolin as potent hits against
receptor. Moreover, BMS195614, GSK1838705A, KT203, the spike and Mpro receptors of SARS-CoV-2.69
and KT185 showed a strong binding with viral S-protein.60 The CADD approach has greatly helped to develop
Pendyala et al. attempted to screen the bioactive compounds, drugs against SARS-CoV-2 like Veklury (remdesivir),
including riboflavin, daidzein, phycocyanobilin, genistein, Olumiant (baricitinib), and galidesivir, which are currently
18 Bioactive Compounds Against SARS-CoV-2

in clinical trials or even approved by FDA (https://clinical- lack precision; thus, they are required to be validated fur-
trials.gov/). Eweas et al. screened the repurposed drugs like ther through the various Web lab approaches.78 Most CADD
hydroxychloroquine, remdesivir, chloroquine, favipiravir, studies on the natural compounds’ antiviral properties and
and ivermectin against the spike protein and membrane TCMs are still preliminary, so in-depth in vivo studies are
proteins receptor-like nucleoproteins, nsp14, RdRp, and required to elucidate the fundamental cellular and molecu-
viral proteases using the SBDD approaches. Finally, remde- lar processes.79
sivir and ivermectin were predicted as the most promising LBDD and SBDD are the two types of CADD
drugs based on molecular docking, molecular mechanics approaches that play a significant part in the quicker
Poisson–Boltzmann surface area (MM-PBSA), and molec- and more affordable design and characterization of drug
ular dynamics studies.70 An SBDD approach was carried molecules. Given the likelihood of SARS-CoV-2 devel-
out by docking five FDA-approved drugs against the Ebola oping mutations as well as the advent of drug resistance
virus, hepatitis C virus, HIV, and 13 compounds in the clin- concerns, it is imperative to go further and think about
ical development pipeline against hepatitis C virus against pursuing polypharmacological drugs that would be more
the RNA-dependent RNA polymerase of SARS-CoV-2. efficacious and possibly aid in overcoming the issues of
The study suggested tenofovir, remdesivir, galidesivir, drug resistance.23 Russo et al. employed the in silico plat-
ribavirin, and sofosbuvir as effective drugs against SARS- form, the Immune System Simulator (ISS), to investigate
CoV-2.71 Among these, galidesivir is currently undergoing the COVID-19 vaccine trials, which in turn proved to be
phase 1 clinical trials. a successful method for developing SARS-CoV-2 vac-
Similarly, an in silico study was carried out by docking cines. These cutting-edge in silico technology platforms
baricitinib, chloroquine, ruxolitinib, azithromycin, hydroxy- may be incredibly beneficial in the pace of various other
chloroquine, and quinacrine against the Mpro receptor of future pandemics and may aid in developing prompt and
SARS-CoV-2. The results predicted quinacrine, baricitinib, affordable therapies.80 A few in silico methods have also
and azithromycin as potent inhibitors, among which bar- been employed to forecast the long-term consequences of
icitinib is approved by the FDA.72. Bisindolylmaleimide IX viruses on general health.81,82
was predicted as the effective inhibitor against the 3CLpro Concerning SARS-CoV-2, the in silico methods helped
receptor of SARS-CoV-2 through the HTVS and MDS to analyze the structures, find the drug targets, predict the
study, which was further validated through the in vitro potent lead compounds, and evaluate the effectiveness
assays.73 Similarly, it was concluded that usnic acid was an of possible natural compound components in preventing
effective lead compound against SARS-CoV-2 based on the SARS-CoV-2 infection as well as drug repurposing. The
in silico and in vitro approaches.74 In their in silico and in expansion of AI and machine learning algorithms has
vitro studies, Kumar et al. found (2S,3S)-3-amino-1-(4-(4- largely facilitated categorizing the previous data and their
(tert-butyl)benzyl)piperazin-1-yl)-4-phenylbutan-2-ol as an application and making precise predictions and plans for
effective inhibitor against the uridylate-specific endoribo- future pandemics.83
nuclease target of SARS-CoV-2.75 Even though more and more in silico tools are made
Finally, computational approaches have made it easier continuously for predicting molecular structures, inter-
to comprehend SARS-CoV-2 biological targets underlying preting the proteomes and genomes, drug designing, and
mechanisms and information regarding the interactions molecular dynamics simulations, none of them is entirely
between receptor and therapeutic compounds at the molec- perfect.84 Accordingly, the search for better in silico tools
ular level.15 to address the problems will continue. Nevertheless,
future scientific studies will undoubtedly be heavily
2.6 THE CHALLENGES AND SCOPES influenced by the accessibility of databases, which may
be general or specialized. The opportunities in bioinfor-
ASSOCIATED WITH CADD
matics include understanding functional aspects of the
The application of computational biology techniques, such human genome, which may intensify and improve the
as molecular modeling, and computer-aided virtual screen- identification of drug targets and enable individualized
ing, has made significant strides in identifying new potent treatments. Therefore, bioinformatics and other science
compounds and comprehending the structural features of disciplines must work collectively to prosper for the ben-
numerous coronavirus molecular targets.76 Because the efit of humankind.85
safety and side effects of FDA-approved pharmaceuticals The cost of a laboratory trial, which demands financial
are already established, the CADD approach made it pos- and human resources, will be reduced by concentrating
sible to find novel drugs or repurpose existing ones. Given only on a few selected targets.19 Investigating the effective-
the severe outcomes of COVID-19 and the unavailability ness of compounds derived from NPs against COVID-19 is
of any significant FDA-approved therapeutics, this justifies one complementation of the in silico studies.86
the necessity for a potent robust form of therapy in a shorter The rate at which scientific data is created and commu-
duration.77 nicated today has never been faster. In this regard, the dis-
Despite the immense values of CADD methods, the cipline of bioinformatics, particularly involving the CADD
binding affinities through the computational techniques approach, is a rapidly evolving field in drug discovery.
The Application of Computer-Aided Drug Design Methods 19

2.7 CONCLUSION F.; Wink, M.; Wolfender, J.-L.; Xiao, J.; Yeung, A. W. K.;
Lizard, G.; Popp, M. A.; Heinrich, M.; Berindan-Neagoe, I.;
The recent pandemic due to COVID-19 has caused wide- Stadler, M.; Daglia, M.; Verpoorte, R. Supuran, C. T.; The
spread devastation. The process of discovering and devel- International Natural Product Sciences Taskforce; Supuran,
oping a novel medicine is typically resource intensive and C. T., Natural products in drug discovery: Advances and
tedious. With this consideration, computational techniques opportunities. Nature Reviews Drug Discovery 2021, 20
(3), 200–216.
have recently been increasingly used in pharmaceutical
6. Stan, D.; Enciu, A. M.; Mateescu, A. L.; Ion, A. C.;
firms and scientific disciplines to increase the efficacy and Brezeanu, A. C.; Stan, D.; Tanase, C., Natural compounds
effectiveness of drug development pipelines. From analyz- with antimicrobial and antiviral effect and nanocarriers
ing recent publications pertaining to the applications of in used for their transportation. Frontiers in Pharmacology
silico techniques to handling various COVID-19 aspects 2021, 12, 723233.
of infection, it may be concluded that CADD has greatly 7. Boukhatem, M. N.; Setzer, W. N., Aromatic herbs, medici-
helped in investigating unknown molecular structures, ana- nal plant-derived essential oils, and phytochemical extracts
lyzing the interaction of lead compounds with their viral as potential therapies for coronaviruses: Future perspec-
tives. Plants (Basel) 2020, 9 (6), 800.
targets, categorizing COVID-19 data using AI and deep 8. Vohora, D.; Singh, G., Pharmaceutical Medicine and
learning techniques, and developing new preventive and Translational Clinical Research. Academic Press: 2018.
therapeutic agents. 9. Song, C. M.; Lim, S. J.; Tong, J. C., Recent advances in
computer-aided drug design. Briefings in Bioinformatics
2009, 10 (5), 579–591.
ACKNOWLEDGMENTS 10. DiMasi, J. A.; Grabowski, H. G.; Hansen, R. W., Innovation
in the pharmaceutical industry: New estimates of R&D
PG is thankful to the Central University of Rajasthan for pro- costs. Journal of Health Economics 2016, 47, 20–33.
viding a University fellowship. DP is thankful to the Central 11. Zhong, F.; Xing, J.; Li, X.; Liu, X.; Fu, Z.; Xiong, Z.; Lu, D.;
University of Rajasthan for the computational facilities. Wu, X.; Zhao, J.; Tan, X.; Li, F.; Luo, X.; Li, Z.; Chen, K.;
Zheng, M.; Jiang, H., Artificial intelligence in drug design.
Conflict of Interest Statement Science China Life Sciences 2018, 61 (10), 1191–1204.
The authors declare that there are no conflicts of interest. 12. Hou, T.; Xu, X., Recent development and application of
virtual screening in drug discovery: An overview. Current
Pharmaceutical Design 2004, 10 (9), 1011–1033.
REFERENCES 13. Ray, M.; Sable, M. N.; Sarkar, S.; Hallur, V., Essential inter-
pretations of bioinformatics in COVID-19 pandemic. Meta
1. Pal, M.; Berhanu, G.; Desalegn, C.; Kandi, V., Severe Acute Gene 2021, 27, 100844.
Respiratory Syndrome Coronavirus-2 (SARS-CoV-2): An 14. Yu, W.; MacKerell, A. D. Jr., Computer-aided drug design
update. Cureus 2020, 12 (3), e7423. methods. Methods in Molecular Biology 2017, 1520,
2. Alam, M. A.; Parra-Saldivar, R.; Bilal, M.; Afroze, C. A.; 85–106.
Ahmed, M. N.; Iqbal, H. M. N.; Xu, J., Algae-derived bioac- 15. Hufsky, F.; Lamkiewicz, K.; Almeida, A.; Aouacheria, A.;
tive molecules for the potential treatment of SARS-CoV-2. Arighi, C.; Bateman, A.; Baumbach, J.; Beerenwinkel, N.;
Molecules 2021, 26 (8), 2134. Brandt, C.; Cacciabue, M.; Chuguransky, S.; Drechsel, O.;
3. Gopal, D.; Skariyachan, S., Recent Perspectives on COVID- Finn, R. D.; Fritz, A.; Fuchs, S.; Hattab, G.; Hauschild,
19 and Computer-Aided Virtual Screening of Natural A. C.; Heider, D.; Hoffmann, M.; Hölzer, M.; Hoops, S.;
Compounds for the Development of Therapeutic Agents Kaderali, L.; Kalvari, I.; von Kleist, M.; Kmiecinski, R.;
Towards SARS-CoV-2. In In Silico Modeling of Drugs Kühnert, D.; Lasso, G.; Libin, P.; List, M.; Löchel, H. F.;
Against Coronaviruses. Methods in Pharmacology and Martin, M. J.; Martin, R.; Matschinske, J.; McHardy,
Toxicology, Roy, K., Ed. Humana, New York, NY: 2020; A. C.; Mendes, P.; Mistry, J.; Navratil, V.; Nawrocki,
pp 433–471. https://doi.org/10.1007/7653_2020_44 E. P.; O’Toole, Á. N.; Ontiveros-Palacios, N.; Petrov, A. I.;
4. Lachance, H.; Wetzel, S.; Waldmann, H., Role of Natural Rangel-Pineros, G.; Redaschi, N.; Reimering, S.; Reinert,
Products. In Lead Generation Approaches in Drug Discovery, K.; Reyes, A.; Richardson, L.; Robertson, D. L.; Sadegh, S.;
Rankovic, Z., Morphy, R., Eds. Wiley: 2010, p 187. Singer, J. B.; Theys, K.; Upton, C.; Welzel, M.; Williams,
5. Atanasov, A. G.; Zotchev, S. B.; Dirsch, V. M.; Orhan, L.; Marz, M., Computational strategies to combat COVID-
I. E.; Banach, M.; Rollinger, J. M.; Barreca, D.; Weckwerth, 19: Useful tools to accelerate SARS-CoV-2 and coronavirus
W.; Bauer, R.; Bayer, E. A.; Majeed, M.; Bishayee, A.; research. Briefings in Bioinformatics 2021, 22 (2), 642–663.
Bochkov, V.; Bonn, G. K.; Braidy, N.; Bucar, F.; Cifuentes, 16. Zheng, W.; Zhang, C.; Li, Y.; Pearce, R.; Bell, E. W.; Zhang,
A.; D’Onofrio, G.; Bodkin, M.; Diederich, M.; Dinkova- Y., Folding non-homologous proteins by coupling deep-
Kostova, A. T.; Efferth, T.; El Bairi, K.; Arkells, N.; learning contact maps with I-TASSER assembly simula-
Fan, T.-P.; Fiebich, B. L.; Freissmuth, M.; Georgiev, tions. Cell Reports Methods 2021, 1 (3), 100014.
M. I.; Gibbons, S.; Godfrey, K. M.; Gruber, C. W.; Heer, 17. Kelley, L. A.; Mezulis, S.; Yates, C. M.; Wass, M. N.;
J.; Huber, L. A.; Ibanez, E.; Kijjoa, A.; Kiss, A. K.; Lu, Sternberg, M. J., The Phyre2 web portal for protein model-
A.; Macias, F. A.; Miller, M. J. S.; Mocan, A.; Müller, R.; ing, prediction and analysis. Nature Protocols 2015, 10 (6),
Nicoletti, F.; Perry, G.; Pittalà, V.; Rastrelli, L.; Ristow, 845–858.
M.; Russo, G. L.; Silva, A. S.; Schuster, D.; Sheridan, H.; 18. Sohail, M. S.; Ahmed, S. F.; Quadeer, A. A.; McKay,
Skalicka-Woźniak, K.; Skaltsounis, L.; Sobarzo-Sánchez, M. R., In silico T cell epitope identification for SARS-
E.; Bredt, D. S.; Stuppner, H.; Sureda, A.; Tzvetkov, CoV-2: Progress and perspectives. Advanced Drug Delivery
N. T.; Vacca, R. A.; Aggarwal, B. B.; Battino, M.; Giampieri, Reviews 2021, 171, 29–47.
20 Bioactive Compounds Against SARS-CoV-2

19. Sadeghi, M.; Miroliaei, M.; Fateminasab, F.; Moradi, 35. Chakravarti, R.; Singh, R.; Ghosh, A.; Dey, D.; Sharma, P.;
M., Screening cyclooxygenase-2 inhibitors from Allium Velayutham, R.; Roy, S.; Ghosh, D., A review on potential
sativum L. compounds: In silico approach. Journal of of natural products in the management of COVID-19. RSC
Molecular Modeling 2021, 28 (1), 24. Advances 2021, 11 (27), 16711–16735.
20. Maghsoudi, S.; Taghavi Shahraki, B.; Rameh, F.; Nazarabi, 36. Onawole, A. T.; Sulaiman, K. O.; Kolapo, T. U.; Akinde,
M.; Fatahi, Y.; Akhavan, O.; Rabiee, M.; Mostafavi, E.; F. O.; Adegoke, R. O., COVID-19: CADD to the rescue.
Lima, E. C.; Saeb, M. R.; Rabiee, N., A review on com- Virus Res 2020, 285, 198022.
puter-aided chemogenomics and drug repositioning for 37. Sharma, T.; Abohashrh, M.; Baig, M. H.; Dong, J. J.; Alam,
rational COVID-19 drug discovery. Chemical Biology & M. M.; Ahmad, I.; Irfan, S., Screening of drug databank
Drug Design 2022, 100 (5), 699–721. against WT and mutant main protease of SARS-CoV-2:
21. Pillaiyar, T.; Meenakshisundaram, S.; Manickam, M., Recent Towards finding potential compound for repurposing
discovery and development of inhibitors targeting coronavi- against COVID-19. Saudi Journal of Biological Science
ruses. Drug Discovery Today 2020, 25 (4), 668–688. 2021, 28 (5), 3152–3159.
22. Rubio-Perez, C.; Tamborero, D.; Schroeder, M. P.; Antolín, 38. Ain, Q. U.; Batool, M.; Choi, S., TLR4-targeting therapeu-
A. A.; Deu-Pons, J.; Perez-Llamas, C.; Mestres, J.; tics: Structural basis and computer-aided drug discovery
Gonzalez-Perez, A.; Lopez-Bigas, N., In silico prescription approaches. Molecules 2020, 25 (3), 627.
of anticancer drugs to cohorts of 28 tumor types reveals 39. Zhu, T.; Cao, S.; Su, P. C.; Patel, R.; Shah, D.; Chokshi,
targeting opportunities. Cancer Cell 2015, 27 (3), 382–396. H. B.; Szukala, R.; Johnson, M. E.; Hevener, K. E., Hit
23. Gurung, A. B.; Ali, M. A.; Lee, J.; Farah, M. A.; Al-Anazi, identification and optimization in virtual screening:
K. M., An updated review of computer-aided drug design Practical recommendations based on a critical literature
and its application to COVID-19. BioMed Research analysis. Journal of Medical Chemistry 2013, 56 (17),
International 2021, 2021, 8853056. 6560–6572.
24. Muhammed, M. T.; Aki-Yalcin, E., Homology modeling in 40. Steinmetz, K. L.; Spack, E. G., The basics of preclinical
drug discovery: Overview, current applications, and future drug development for neurodegenerative disease indica-
perspectives. Chemical Biology & Drug Design 2019, 93 tions. BMC Neurology 2009, 9 (Suppl 1), S2.
(1), 12–20. 41. Dhodapkar, M.; Zhang, A. D.; Puthumana, J.; Downing,
25. Yuan, X.; Shao, Y.; Bystroff, C., Ab initio protein struc- N. S.; Shah, N. D.; Ross, J. S., Characteristics of clinical
ture prediction using pathway models. Comparative and studies used for US Food and Drug Administration supple-
Functional Genomics 2003, 4 (4), 397–401. mental indication approvals of drugs and biologics, 2017 to
26. Xu, J.; Jiao, F.; Yu, L., Protein structure prediction using 2019. JAMA Network Open 2021, 4 (6), e2113224.
threading. Methods in Molecular Biology 2008, 413, 42. Batool, M.; Ahmad, B.; Choi, S., A structure-based drug
91–121. discovery paradigm. International Journal of Molecular
27. Hassan, N. M.; Alhossary, A. A.; Mu, Y.; Kwoh, C.-K., Sciences 2019, 20 (11), 2783.
Protein-ligand blind docking using QuickVina-W with 43. Cragg, G. M.; Newman, D. J., Natural products: A continu-
inter-process spatio-temporal integration. Scientific Reports ing source of novel drug leads. Biochimica et Biophysica
2017, 7 (1), 15451. Acta 2013, 1830 (6), 3670–3695.
28. Sharma, A.; Rajpoot, M.; Gupta, G., Drug designing in 44. Pan, S. Y.; Litscher, G.; Gao, S. H.; Zhou, S. F.; Yu, Z. L.;
novel drug discovery: Trends, scope and relevance. In Chen, H. Q.; Zhang, S. F.; Tang, M. K.; Sun, J. N.; Ko,
Chemical Drug Design. Girish Kumar Gupta and Vinod K. M., Historical perspective of traditional indigenous
Kumar Eds. Berlin: De Gruyter, 2016; pp 15–30. medical practices: The current renaissance and conserva-
29. Neves, B. J.; Braga, R. C.; Melo-Filho, C. C.; Moreira-Filho, tion of herbal resources. Evidence-based Complementary
J. T.; Muratov, E. N.; Andrade, C. H., QSAR-based virtual and Alternative Medicine 2014, 2014, 525340.
screening: Advances and applications in drug discovery. 45. Newman, D. J.; Cragg, G. M., Natural products as sources
Frontiers in Pharmacology 2018, 9, 1275. of new drugs over the nearly four decades from 01/1981
30. Patel, H. M.; Noolvi, M. N.; Sharma, P.; Jaiswal, V.; to 09/2019. Journal of Natural Products 2020, 83 (3),
Bansal, S.; Lohan, S.; Kumar, S. S.; Abbot, V.; Dhiman, 770–803.
S.; Bhardwaj, V., Quantitative structure–activity rela- 46. Khalifa, S. A. M.; Elias, N.; Farag, M. A.; Chen, L.; Saeed,
tionship (QSAR) studies as strategic approach in drug A.; Hegazy, M. F.; Moustafa, M. S.; Abd El-Wahed, A.;
discovery. Medicinal Chemistry Research 2014, 23 (12), Al-Mousawi, S. M.; Musharraf, S. G.; Chang, F. R.; Iwasaki,
4991–5007. A.; Suenaga, K.; Alajlani, M.; Göransson, U.; El-Seedi,
31. Patel, L.; Shukla, T.; Huang, X.; Ussery, D. W.; Wang, S., H. R., Marine natural products: A source of novel antican-
Machine learning methods in drug discovery. Molecules cer drugs. Marine Drugs 2019, 17 (9), 491.
2020, 25 (22), 5277. 47. Nahar, L.; Sarker, S. D., Chapter 5 – Application of
32. Qi, Y., Random Forest for Bioinformatics. Ensemble Computation in Building Dereplicated Phytochemical
Machine Learning: Methods and Applications. New York, Libraries. In Computational Phytochemistry, Sarker, S. D.;
NY: Springer: 2012. Nahar, L., Eds. Amsterdam: Elsevier: 2018; pp 141–163.
33. Li, F., The information content of forward-looking state- 48. Zeng, X.; Zhang, P.; He, W.; Qin, C.; Chen, S.; Tao, L.;
ments in corporate filings—A naïve Bayesian machine Wang, Y.; Tan, Y.; Gao, D.; Wang, B.; Chen, Z.; Chen, W.;
learning approach. Journal of Accounting Research 2010, Jiang, Y. Y.; Chen, Y. Z., NPASS: Natural product activity
48 (5), 1049–1102. and species source database for natural product research,
34. Somvanshi, M.; Chavan, P.; Tambade, S.; Shinde, S. A discovery and tool development. Nucleic Acids Research
review of machine learning techniques using decision tree 2017, 46 (D1), D1217–D1222.
and support vector machine. 2016 International Conference 49. Naik, B.; Gupta, N.; Ojha, R.; Singh, S.; Prajapati, V. K.;
on Computing Communication Control and Automation Prusty, D., High throughput virtual screening reveals
(ICCUBEA), IEEE: 2016; pp 1–7. SARS-CoV-2 multi-target binding natural compounds
The Application of Computer-Aided Drug Design Methods 21

to lead instant therapy for COVID-19 treatment. Inter­ 62. Xu, Z.; Yang, L.; Zhang, X.; Zhang, Q.; Yang, Z.; Liu, Y.; Wei,
national Journal of Biological Macromolecules 2020, S.; Liu, W., Discovery of potential flavonoid inhibitors against
160, 1–17. COVID-19 3CL proteinase based on virtual screening strat-
50. Kong, R.; Yang, G.; Xue, R.; Liu, M.; Wang, F.; Hu, J.; Guo, egy. Frontiers in Molecular Bioscience 2020, 7, 556481.
X.; Chang, S., COVID-19 docking server: A meta server for 63. Majumder, R.; Mandal, M., Screening of plant-based natu-
docking small molecules, peptides and antibodies against ral compounds as a potential COVID-19 main protease
potential targets of COVID-19. Bioinformatics 2020, 36 inhibitor: An in silico docking and molecular dynamics
(20), 5109–5111. simulation approach. Journal of Biomolecular Structure &
51. Chukwudozie, O. S.; Duru, V. C.; Ndiribe, C. C.; Aborode, Dynamics 2022, 40 (2), 696–711.
A. T.; Oyebanji, V. O.; Emikpe, B. O., The relevance 64. Moradi, M.; Golmohammadi, R.; Najafi, A.; Moosazadeh
of bioinformatics applications in the discovery of vac- Moghaddam, M.; Fasihi-Ramandi, M.; Mirnejad, R., In
cine candidates and potential drugs for COVID-19 treat- silico analysis of inhibiting papain-like protease from SARS-
ment. Bioinformatics and Biology Insights 2021, 15, CoV-2 by using plant-derived peptides. International Journal
11779322211002168. of Peptide Research and Therapeutics 2022, 28 (1), 24.
52. Jayaraj, J. M.; Jothimani, M.; Palanisamy, C. P.; Pentikäinen, 65. Nouadi, B.; Ezaouine, A.; El Messal, M.; Blaghen, M.;
O. T.; Pannipara, M.; Al-Sehemi, A. G.; Muthusamy, K.; Bennis, F.; Chegdani, F., Prediction of anti-COVID 19 ther-
Gopinath, K., Computational study on the inhibitory effect apeutic power of medicinal Moroccan plants using molecu-
of natural compounds against the SARS-CoV-2 proteins. lar docking. Bioinformatics and Biology Insights 2021, 15,
Bioinorganic Chemistry and Applications 2022, 2022, 11779322211009199.
8635054. 66. Patel, N.; Prajapati, N.; Patel, L.; Richa, P.; Kalasariya, H.,
53. Zhang, D.; Hamdoun, S.; Chen, R.; Yang, L.; Ip, C. K.; Qu, Red algae derived phycocompounds as an inhibitors of
Y.; Li, R.; Jiang, H.; Yang, Z.; Chung, S. K.; Liu, L.; Wong, SARS CoV-2. Journal of Interdisciplinary Cycle Research
V. K. W., Identification of natural compounds as SARS- 2020, 12, 662–681.
CoV-2 entry inhibitors by molecular docking-based virtual 67. Joseph, J.; Karthika, T.; Das, A. V. R.; Raj, S. V., The use of
screening with bio-layer interferometry. Pharmacological pseudotyped coronaviruses for the screening of entry inhib-
Research 2021, 172, 105820. itors: Green tea extract inhibits the entry of SARS-CoV-1,
54. Jimenez, P. C.; Wilke, D. V.; Branco, P. C.; Bauermeister, MERS-CoV, and SARS-CoV-2 by blocking receptor-spike
A.; Rezende-Teixeira, P.; Gaudêncio, S. P.; Costa-Lotufo, interaction. Current Pharmaceutical Biotechnology 2022,
L. V., Enriching cancer pharmacology with drugs of marine 23 (8), 1118–1129.
origin. British Journal of Pharmacology 2020, 177 (1), 68. Dowlati Beirami, A.; Hatamabadi, D.; Iranpanah, S.; Rezaei,
3–27. M.; Ziai, S. P. S. P. A., In silico identification of potentially
55. Riccio, G.; Ruocco, N.; Mutalipassi, M.; Costantini, M.; effective herbal inhibitors of SARS-Cov-2 main protease by
Zupo, V.; Coppola, D.; de Pascale, D.; Lauritano, C., Ten- virtual screening method: Potential anti-COVID-19 mol-
year research update review: Antiviral activities from ecules. School of Medicine Students’ Journal 2020, 2 (3),
marine organisms. Biomolecules 2020, 10 (7), 1007. 2–6.
56. Gaudêncio, S. P.; Pereira, F., A computer-aided drug design 69. Prasanth, D.; Murahari, M.; Chandramohan, V.; Panda, S. P.;
approach to predict marine drug-like leads for SARS- Atmakuri, L. R.; Guntupalli, C., In silico identification of
CoV-2 main protease inhibition. Marine Drugs 2020, 18 potential inhibitors from Cinnamon against main protease and
(12), 633. spike glycoprotein of SARS CoV-2. Journal of Biomolecular
57. Gentile, D.; Patamia, V.; Scala, A.; Sciortino, M. T.; Piperno, Structure & Dynamics 2021, 39 (13), 4618–4632.
A.; Rescifina, A., Putative inhibitors of SARS-CoV-2 main 70. Eweas, A. F.; Alhossary, A. A.; Abdel-Moneim, A. S.,
protease from a library of marine natural products: a virtual Molecular docking reveals ivermectin and remdesivir as
screening and molecular modeling study. Marine Drugs potential repurposed drugs against SARS-CoV-2. Frontiers
2020, 18 (4), 225. in Microbiology 2021, 11, 592908.
58. Khan, M. T.; Ali, A.; Wang, Q.; Irfan, M.; Khan, A.; Zeb, 71. Jorgensen, S. C. J.; Tse, C. L. Y.; Burry, L.; Dresser, L. D.,
M. T.; Zhang, Y. J.; Chinnasamy, S.; Wei, D. Q., Marine Baricitinib: A review of pharmacology, safety, and emerg-
natural compounds as potents inhibitors against the main ing clinical experience in COVID-19. Pharmacotherapy
protease of SARS-CoV-2-a molecular dynamic study. 2020, 40 (8), 843–856.
Journal of Biomolecular Structure and Dynamics 2021, 39 72. Marinho, E. M.; Batista de Andrade Neto, J.; Silva, J.;
(10), 3627–3637. Rocha da Silva, C.; Cavalcanti, B. C.; Marinho, E. S.; Nobre
59. Ghosh, K.; Amin, S. A.; Gayen, S.; Jha, T., Chemical- Júnior, H. V., Virtual screening based on molecular docking
informatics approach to COVID-19 drug discovery: of possible inhibitors of Covid-19 main protease. Microbial
Exploration of important fragments and data mining based Pathogenesis 2020, 148, 104365.
prediction of some hits from natural origins as main pro- 73. Gupta, Y.; Maciorowski, D.; Zak, S. E.; Jones, K. A.;
tease (Mpro) inhibitors. Journal of Molecular Structure Kathayat, R. S.; Azizi, S. A.; Mathur, R.; Pearce, C. M.;
2021, 1224, 129026. Ilc, D. J.; Husein, H.; Herbert, A. S.; Bharti, A.; Rathi,
60. Choudhary, S.; Malik, Y. S.; Tomar, S., Identification of B.; Durvasula, R.; Becker, D. P.; Dickinson, B. C.; Dye,
SARS-CoV-2 cell entry inhibitors by drug repurposing J. M.; Kempaiah, P., Bisindolylmaleimide IX: A novel anti-
using in silico structure-based virtual screening approach. SARS-CoV2 agent targeting viral main protease 3CLpro
Frontiers in Immunology 2020, 11, 1664. demonstrated by virtual screening pipeline and in-vitro
61. Pendyala, B.; Patras, A., In silico Screening of Food validation assays. Methods 2021, 195, 57–71.
Bioactive Compounds to Predict Potential Inhibitors of 74. Oh, E.; Wang, W.; Park, K. H.; Park, C.; Cho, Y.; Lee, J.;
COVID-19 Main protease (Mpro) and RNA-dependent RNA Kang, E.; Kang, H., (+)-Usnic acid and its salts, inhibitors
polymerase (RdRp). ChemRxiv. Cambridge: Cambridge of SARS-CoV-2, identified by using in silico methods and
Open Engage. 10.26434/chemrxiv.12051927.v2. 2020. in vitro assay. Scientific Reports 2022, 12 (1), 13118.
22 Bioactive Compounds Against SARS-CoV-2

75. Kumar, S.; Gupta, Y.; Zak, S. E.; Upadhyay, C.; Sharma, 81. Tetz, G.; Tetz, V., Prion-like domains in spike protein
N.; Herbert, A. S.; Durvasula, R.; Potemkin, V.; Dye, of SARS-CoV-2 differ across its variants and enable
J. M.; Poonam; Kempaiah, P.; Rathi, B., A novel compound changes in affinity to ACE2. Microorganisms 2022, 10 (2),
active against SARS-CoV-2 targeting uridylate-specific 280.
endoribonuclease (NendoU/NSP15): In silico and in vitro 82. Mohabatkar, H.; Behbahani, M.; Moradi, M., A concise in
investigations. RSC Medicinal Chemistry 2021, 12 (10), silico prediction report of a potential PRION-like domain
1757–1764. in SARS-COV-2 polyprotein. Journal of Microbiology,
76. Wang, J.; Zhang, Y.; Nie, W.; Luo, Y.; Deng, L., Computa­ Biotechnology and Food Sciences 2021, 11 (3), e4813.
tional anti-COVID-19 drug design: Progress and challenges. 83. Moradi, M.; Golmohammadi, R.; Najafi, A.; Moosazadeh
Briefings in Bioinformatics 2022, 23 (1), bbab484. Moghaddam, M.; Fasihi-Ramandi, M.; Mirnejad, R., A
77. Basak, S. C.; Kier, L. B., COVID-19 pandemic: How can contemporary review on the important role of in silico
computer-assisted methods help to rein in this global men- approaches for managing different aspects of COVID-19
ace? Current Computer-Aided Drug Design 2021, 17 (1), 1. crisis. Informatics in Medicine Unlocked 2022, 28,
78. Meng, X. Y.; Zhang, H. X.; Mezei, M.; Cui, M., Molecular 100862.
docking: A powerful approach for structure-based drug dis- 84. Sliwoski, G.; Kothiwale, S.; Meiler, J.; Lowe, E. W. Jr.,
covery. Current Computer-Aided Drug Design 2011, 7 (2), Computational methods in drug discovery. Pharmacological
146–157. Reviews 2014, 66 (1), 334–395.
79. Xian, Y.; Zhang, J.; Bian, Z.; Zhou, H.; Zhang, Z.; Lin, Z.; 85. Mehmood, M., Use of bioinformatics tools in differ-
Xu, H., Bioactive natural compounds against human coro- ent spheres of life sciences. Journal of Data Mining in
naviruses: A review and perspective. Acta Pharmaceutica Genomics & Proteomics 2014, 5, 1000158.
Sinica B 2020, 10 (7), 1163–1174. 86. Sattari, A.; Ramazani, A.; Aghahosseini, H., Repositioning
80. Russo, G.; Pennisi, M.; Fichera, E.; Motta, S.; Raciti, G.; therapeutics for COVID-19: Virtual screening of the potent
Viceconti, M.; Pappalardo, F., In silico trial to test COVID- synthetic and natural compounds as SARS-CoV-2 3CLpro
19 candidate vaccines: A case study with UISS platform. inhibitors. Journal of the Iranian Chemical Society 2021,
BMC Bioinformatics 2020, 21 (Suppl 17), 527. 18 (10), 2807–2827.
 3 Therapeutic Targets of
Host Factors for Potential
COVID-19 Treatments
Sabarinath Neerukonda

3.1 INTRODUCTION drug targets, host factor identification also facilitates a bet-
ter understanding of coronavirus biology.
Coronaviruses are positive-sense single-stranded (ss) After providing an overview of the coronavirus repli-
RNA viruses with the largest genomes (approximately 30 cation cycle, this chapter details genome-wide functional
kilobases [kb]). They are classified by the International genetic, proteomic, and chemical compound screening
Committee on Taxonomy of Viruses (ICTV) under the approaches that have been employed to identify host factors
order Nidovirales, suborder Coronavirinae and subfam- during the SARS-CoV-2 life cycle. Although only a handful
ily Orthocoronavirinae within the family Coronoviridae. of host factors have known functions at specific stages of
They infect a wide range of birds and mammals including the virus life cycle, the remaining host factors were dem-
humans, livestock, and domestic animals. Coronaviruses onstrated to have indispensable roles using knockout (KO),
are ­classified into four genera: Alphacoronavirus, Beta- small interfering RNA (siRNA) inhibition, and chemical
coronavirus, Gammacoronavirus, and Deltacoronavirus. compounds that are approved by the U.S. Food and Drug
Alpha, Beta, and Deltacoronaviruses largely cause respira- Administration (FDA) or in phase 2/3 clinical trials.
tory, hepatic, and intestinal infections in mammals, whereas
Gammacoronaviruses cause respiratory, intestinal, and
nephropathic infections in birds. Thus far, seven human 3.2 GENERAL REPLICATION CYCLE
coronaviruses (HCoVs) were identified and characterized.
OF SARS-CoV-2
Four HCoVs (229E, NL63, OC43, and HKU1) cause mild
respiratory infections and the remaining three HCoVs that The SARS-CoV-2 virion is an enveloped particle, sized
emerged in the 21st century (severe acute respiratory syn- around 104 nm in diameter with spherical or pleomorphic
drome coronavirus [SARS-CoV]-1, Middle East respiratory morphology.6–8 The virion envelope consists of spike (S)
syndrome coronavirus [MERS-CoV], and SARS-CoV-2) trimers, giving it crown-shaped morphology along with
can cause lethal respiratory disease. SARS-CoV-2 was ini- membrane (M) and envelope (E) proteins. The genome is
tially identified in late 2019 as a causative agent of a severe a 5′ capped positive-sense ssRNA with a 3′ polyadenylated
pneumonia outbreak in Wuhan, China.1–4 Several SARS- (polyA) tail and is packaged by a helical nucleoprotein (N)
CoV-2-related (SARS2r) coronaviruses have been identified into a nucleocapsid. SARS-CoV-2 entry is mediated by the
circulating in bats indicating bat origin, although whether binding of the S receptor-binding domain (RBD) located in
an intermediate host is involved in the human transmission the S1 subunit to the host receptor, angiotensin-­converting
is still unknown.1,5 Due to high transmissibility, asymptom- enzyme 2 (ACE2), on target cells.9 S glycoprotein is a
atic carriers, and infectivity, SARS-CoV-2 rapidly spread type I membrane glycoprotein which, upon binding to the
across continents causing a global pandemic and continue receptor, undergoes a conformational switch like influenza
to cause mortality and severe disruption to human and hemagglutinin (HA) to permit virus-cell membrane fusion;
economic activity. Although SARS-CoV-2 spike-targeting therefore, it is a class I fusion protein. Structurally, the S
vaccines based on messenger RNA (mRNA), adenovirus, glycoprotein in its prefusion conformation comprises four
and subunit platforms as well as therapeutic neutralizing domains in its S1 subunit: amino-terminal (N-terminal)
antibodies (nAbs) were rapidly deployed to curb the disease domain (NTD), RBD, and two carboxy-terminal domains
severity, several SARS-CoV-2 variants with the ability to (CTD1 and CTD2). A multibasic furin cleavage site pre-
undergo immune escape continue to emerge, challenging cedes the S2 subunit, which facilitates furin processing of
vaccine efficacy and nAb potency. Therefore, it is impera- S protein into S1/S2 subunits. The S2 subunit consists of
tive to identify novel therapeutic targets such as cellular host an upstream helix (UH), fusion peptide (FP), FP-proximity
factors that either facilitate (proviral) or antagonize (antivi- region (FPPR), heptad repeats 1 and 2 (HR1/2), central
ral) various aspects of coronavirus biology. Although both helix (CH), beta-hairpin (BH), connector domain (CD),
proviral and antiviral host factors can serve as potential stem helix (SH), transmembrane domain (TM), and a

DOI: 10.1201/9781003323884-3 23
24 Bioactive Compounds Against SARS-CoV-2

cytoplasmic tail. The pre-fusion conformation of the S tri- FP10 (Figure 3.1). Depending upon the availability of host
mer is maintained by the three S1 subunits sitting atop three cell proteases, the FP exposure event occurs at the plasma
S2 subunits to prevent premature triggering into the post- membrane or upon endocytosis.10 The FP located in the S2
fusion state. One or more RBDs in trimer access the cellular subunit inserts into the host cell membrane followed by the
ACE2 receptor in an “up” state, whereas the “down” state formation of a stable six-helix bundle that brings viral and
prevents RBD access to the receptor.8 In the up state, RBD cell membranes into proximity for fusion.11 Upon mem-
binding to the receptor results in conformational changes in brane fusion, nucleocapsid gets released into cytoplasm fol-
the S1 subunit, causing its “shedding” from the S2 subunit, lowed by the dissociation of N from genomic RNA, which
which then undergoes a series of conformational changes is ready for translation.
to form a stable elongated trimeric helix triggering fusion. Genomic RNA translation begins with the translation of
The binding step is followed by proteolytic cleavage of orf1a and orf1b encoded within two-thirds of the genome
S by the host cell proteases (transmembrane serine prote- at the 5′ end (Figure 3.1). Polyprotein (pp) 1a (pp1a) is
ase 2 [TMPRSS2] and cathepsin L [Cat L]) to expose the encoded by open reading frame (ORF)1a, whereas pp1ab

FIGURE 3.1 The general replication cycle of SARS-CoV-2. Upon spike recognition of ACE2, subsequent cleavage by TMPRSS2 at
the S2′site triggers fusion at the plasma membrane. Alternatively, in cells absent of TMPRSS2 expression, SARS-CoV-2 entry occurs
via endocytosis, with spike cleavage in the low pH endosomal environment by cathepsin L triggering fusion. Upon N protein disas-
sociation from the positive-sense (+) RNA genome, the translation-ready genome is immediately translated into polyproteins, pp1a and
pp1ab. Pp1a and pp1b are proteolytically processed into the Nsps 1–16, which assemble replication-transcription complexes (RTCs)
and remodel cellular membranes to form double-membrane vesicles (DMVs). DMVs establish an optimal environment for replicat-
ing and transcribing viral RNA. The first step in replication involves the synthesis of a negative-sense (−) complimentary copy of a
positive-sense genome, which serves as a template for the synthesis of nascent positive-sense RNA genomes, which may undergo
further rounds of translation or are packaged into budding virions. Discontinuous transcription of the positive-sense genomic RNA
yields negative-sense subgenomic RNAs, which serve as templates for the synthesis of positive-sense subgenomic RNAs that encodes
structural and accessory proteins. Newly synthesized viral RNAs leave DMVs through a double membrane-spanning transmembrane
pore to undergo further rounds of translation or virion assembly. Nascent positive genomic RNAs, encapsidated by N, as well as the
structural proteins S, M, and E, bud into the ERGIC to form new virions. Finally, progeny virions exit the host cells. (Figure created
by modifying template on Biorender.com.)
Therapeutic Targets of Host Factors for Potential COVID-19 Treatments 25

is translated as a single polyprotein of ORF1a and ORF1b 3.3 HIGH-CONTENT SCREENING

through a –1 programmed ribosomal frameshifting (PRF) OF HOST FACTORS
mechanism. PRF in coronaviruses is facilitated in part by
a conserved heptameric slippery sequence (U UUA AAC) 3.3.1 CRISPR-Based High-Content Screening
and a downstream pseudoknot secondary structure.12,13 for Host Factors
Several trans-acting host factors (e.g., SHFL, HNRNPH1,
HNRNPH2, ZAP-S, ELAVL1, and ZAP-L) are known to Large-scale Clustered Regularly Interspaced Short
regulate the PRF event.12,13 Both pp1a and pp1b are pro- Palindromic Repeats (CRISPR) screens are powerful tools
cessed into individual nonstructural proteins (Nsps) by that employ CRISPR-Cas9 genome editing for the discov-
viral proteases encoded within Nsp3 (papain-like pro- ery of genes, pathways, and host factors that play proviral
tease [PLpro]) and Nsp5 (3-chymotrypsin-like protease and antiviral roles during virus–host interactions. CRISPR
[3CLpro/Mpro]), producing 16 Nsps (Nsp1–Nsp16), which screens can be applied in pooled or arrayed screening for-
remodel host cellular environment to favor subsequent mats.23 In a pooled CRISPR screen, a library of CRISPR
steps in viral replication. Nsp1 inhibits host cell translation guide RNAs (gRNAs) and Cas9 proteins are typically
by interacting with the NXF1 of the mRNA export receptor introduced into target cells that are permissive to the virus
(NXF1/NXT1) and 40S ribosomal subunit to block cellu- of interest by lentiviral transduction at low multiplicity of
lar mRNA export into cytoplasm and mRNA entry tunnel, infection (MOI ~0.1). This is done so the cells do not receive
respectively.14,15 more than one gRNA to avoid interactions between different
The Nsp3, Nsp4, and Nsp6 subcomplex remodel endo- gRNAs and multiple gene perturbations in each cell.23,24 The
plasmic reticulum (ER) membranes to generate double-­ gRNA directs the Cas9 endonuclease to the complementary
membrane vesicles (DMVs), which shield replication products gene sequence, where Cas9-mediated double-stranded (ds)
from recognition by the host-pathogen sensors known as pat- DNA break undergoes error-prone repair to generate loss-
tern recognition receptors (PRRs), and serve as RNA replica- of-function (LOF) mutations, resulting in a pool of cells
tion factories.16 A double membrane spanning molecular pore with independent gene KOs.24 The gene KO cells are then
made of large multifunctional Nsp3 as its main component subjected to selection pressure, such as drug treatment, to
serves as an exit channel for RNA products from the interior retain and expand KO cell populations. The discovery of
of DMV into the cytosol.17 Nsp2-Nsp16 form the viral repli- proviral host factors is dependent upon virus-induced cell
cation-transcription complex (RTC), with Nsp12-16 in com- death or cytopathic effect (CPE), such that virus-suscepti-
plex with their cofactors carrying out various core enzymatic ble cells are rapidly cleared, and only virus-resistant cells
functions such as RNA-dependent RNA polymerase (Nsp12), remain. Alternatively, virus-resistant and virus-susceptible
helicase (Nsp13), exonuclease/N7-methyltransferase (Nsp14), cells can be enriched or sorted using fluorescent reporter
uridylate-specific endoribonuclease (NendoU/Nsp15), and virus or staining for virion protein on infected cells.24,25
S-adenosyl methionine-dependent 2′-O-methyltransferase The enrichment or depletion of each gRNA and respective
(Nsp16). The first step of RNA replication involves full- target gene is then determined by polymerase chain reac-
length negative-strand synthesis that is complementary to the tion (PCR) amplification of gRNAs and comparing gRNA
positive-sense genome. The negative-sense strands serve as representation in virus-infected cells and uninfected control
a template for the synthesis of positive-sense strands, which cells by sequencing.23–25 The enriched gRNAs in virus-
may undergo translation to yield more Nsps or are packaged resistant population compared with control cells represent
into newly budding virions. Furthermore, discontinuous tran- proviral host factors, because corresponding gene KO or
scription of the 3′ end of the positive-sense genome produces LOF prevents one or more steps of virus replication.
negative-strand subgenomic RNAs, which serve as templates Conversely, antiviral host factors can be identified by car-
for the synthesis of positive-strand subgenomic RNAs that rying out CRISPR-based gain-of-function (GOF) screens
encode structurally (S, E, M, and N) and accessory ORF3a, where the Cas9 protein is fused to activator domains such as
3b, 6, 7a, 7b, 8, and 9b proteins.18 Although accessory pro- the synergistic activation mediator transcriptional activators
teins are non-essential for replication, they carry out acces- (e.g., dCas9–VP64 + PP7-P65-HSF1), which are localized
sory functions such as innate immune evasion by interfering to target gene promoters by gRNAs to induce transactiva-
with PRR signaling cascades,19 as also demonstrated for Nsps tion.23,25–27 Upon viral infection, enriched gRNAs and their
and structural proteins M and N (reviewed in20). Nascent corresponding overexpressed gene hits in GOF screen are
RNA genomes complexed with N bud into the ER–Golgi antiviral restriction factors because their overexpression
intermediate compartment (ERGIC) to form enveloped par- prevents viral replication and CPE. Conversely, depleted
ticles containing S, M, and E proteins.21,22 Each step of the gRNAs are proviral. Regardless of approach, gRNAs tar-
coronavirus life cycle is tightly entwined by viral and host– geting genes essential for cell survival and proliferation can
protein interactions, and as described later, identification and result in the depletion of cells from the LOF or GOF cell
targeting host factors is a rational option due to higher genetic populations during the selection process; therefore, this can
barrier of the host compared with the virus, which undergoes lead to failure to identify the role of such depleted factors
frequent mutational escape to direct-acting antivirals (DAAs) in the virus life cycle. Furthermore, as a phenotypic selec-
as well as nAbs. tion of CRISPR screens is dependent on cells showing no
26 Bioactive Compounds Against SARS-CoV-2

CPE, and, therefore, no active viral replication, host factors phosphatidylserine receptor families, TIM146 and AXL,47
identified in such screens generally target earlier steps in which enhance viral entry by binding phosphatidylserine
the virus life cycle such as viral entry, genome translation, on the virion envelope, were described to serve as alter-
transcription, and RNA replication, but not final steps of native coreceptors. However, the role of lectins and phos-
assembly, budding, and viral egress.24,25 Compared with phatidylserine receptors in mediating SARS-CoV-2 entry
pooled screens, arrayed screens are more labor-intensive, is non-specific, as they do not s­upport infection in the
expensive, and limited in scale, as gene KO perturbations absence of ACE2.
are individually separated.23 But because each gene KO is Cellular heparan sulfate (HS) also serves as a SARS-
predefined in the study design, arrayed screen readouts gen- CoV-2 attachment factor and enhances infection efficiency
erally do not require sequencing.23 by possibly promoting “open” RBD conformation for ACE2
Several independent CRISPR-based LOF and GOF binding.48–50 In line with this, multiple screens identified
screens have been conducted on a genome-wide scale to dis- EXT1, a subunit of the copolymerase required for the syn-
cover SARS-CoV-2 proviral and antiviral host factors.26–37 thesis of the HS backbone. Target cells depleted for EXT1
Host factors discovered in all the screens thus far are based lacked binding of recombinant RBD and spike.34 Several
on cell viability or resistance to virus-induced CPE, and, other host factors that were known to markedly enhance
therefore, are likely to prevent detection of genes that are SARS-CoV-2 infection in ACE2-positive cells include
essential for cell survival or proliferation as well as genes neuropilin-1 (NRP1),51,52 Scavenger receptor B1 (­SR-B1),53
involved in the final stages of the virus life cycle, including basigin (CD147),54 low-density lipoprotein receptor class A
assembly, budding, and egress. Furthermore, experimental domain containing 3 (LDLRAD3),28 transmembrane pro-
parameters in the LOF and GOF screens such as the cell tein 30A (TMEM30A),26–28,34 transferrin receptor (TfR),55
type, cellular interferon (IFN) status, virus strain used, glucose regulatory protein 78 (GRP78),56 ASGPR1,
readout duration, and perturbation modality can influ- KREMEN1,57 and sialic acids.58–61 Coexpression of NRP1
ence host factor discovery.24 However, secondary screen- markedly enhanced SARS-CoV-2 infection.51,52 NRP1
ing of gene hits from published screens and the screen by binds to the C terminus of the S1 subunit generated upon
Rebendenne et al. in four different cell lines (A549-ACE2, furin cleavage whose amino acid sequence (682RRAR685)
Caco-2-ACE2, Huh7.5.1-ACE2, and Calu-3) confirmed that conforms to the “C-end rule” [R/K]xx[R/K] (“x” rep-
the hits identified in viability-based whole genome screens resents any amino acid) motif.51,52 LDLRAD3 interacts
are strongly cell-type specific.24,26 Although cells lining the with the spike NTD to enhance infection.28 SCARB1 is a
respiratory tract and lung are the primary sites of SARS- cell surface high-density lipoprotein (HDL) receptor that
CoV-2 replication, several cell lines of different origins facilitates the selective uptake of cholesteryl esters and
have been used for host factor discovery such as African other lipid components of receptor-bound HDL particles.
green monkey kidney-derived cells (Vero E6), human lung Through the putative cholesterol recognition amino acid
adenocarcinoma cells (A549-ACE2, Calu-3), human hepa- consensus motifs in NTD and RBD, the S1 subunit may
toma cells (Huh7, Huh7.5), and human colon adenocarci- bind cholesterol and HDL components, causing significant
noma (Caco-2) cells.26–37 uptake of HDL-bound viral particles by SR-B1.53 Although
an earlier report indicated a direct interaction between
3.3.2 Proviral Host Factors CD147 and spike RBD along with an alternate receptor
role of CD147 in ACE2-deficient cell types,54 a later report
3.3.2.1 Proviral Host Factors Involved in Entry showed no evidence of a direct interaction between the viral
As the primary receptor of SARS-CoV-2, ACE2 was iden- spike protein and two common isoforms of CD147, ques-
tified in multiple screens in different cell lines.9 ACE2 tioning the CD147 role in SARS-CoV-2 entry.62 ASGR1 and
is an 805-amino-acid carboxypeptidase consisting of an KREMEN1 were identified to facilitate SARS-CoV-2 entry
N-terminal metallopeptidase domain preceding collectrin in a receptome study, where greater interaction between
and TM domains in its C-terminal half.38 The primary RBD and ASGR1/KREMEN1 was found compared with
physiological function of ACE2 is to catalyze angioten- NTD and the S2 subunit.57
sin I and angiotensin II, generated by renin and ACE, TMEM30A encodes a subunit of the ATP8A2 flip-
into angiotensin-1–9 and angiotensin-1–7, respectively.38 pase complex that controls the distribution of phospholip-
Crystal structures of RBD-ACE2 complexes identified a ids across membrane leaflets.63 Although TMEM30A was
concave receptor-binding motif (RBM) structure with 17 shown to interact with a spike in a co-immunoprecipitation
RBM residues interacting with 20 ACE2 peptidase domain assay, and TMEM30A knockdown (KD) reduced SARS-
residues.38–41 Apart from ACE2, several cell surface mol- CoV-2 entry,28 whether TMEM30A plays a direct role in
ecules were described to serve as attachment factors attachment or an indirect role by maintaining phospholipid
or coreceptors for SARS-CoV-2. These include NTD- integrity is still unknown. TfR is an abundantly expressed
binding C-type lectin family members such as CLEC4G,28 glycoprotein on the plasma membrane of most human cells
DC-SIGN, and L-SIGN,42–45 whose primary function and promotes the entry of transferrin into cells for iron
is carbohydrate ­recognition of pathogen molecules and delivery. A direct interaction between RBD and TfR was
facilitating intercellular adhesion. In addition, members of shown in vitro along with SARS-CoV-2 infection of mice
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He had yet to discover that he was sensitive to an "atmosphere," but he was
already well on the road to that discovery. Hartley's room was big and
rather bare. He was athletic, and the wall-space was almost wholly given up
to a number of oars, and a dozen or so plainly-framed groups with a cricket
cap hanging from the corner of one. The exceptions were two other
photographs, one of the service on the sands at Eastbourne (in which Paul
had discovered Madeline) and one of the Cambridge University Missionary
Campaigners in some Midland town. The mantelshelf was overcrowded
with photographs of men, snapshots of children, and the cards of a variety
of chiefly religious societies and activities among which a Bump Supper
menu seemed out of place. The electric lights were naked; the window-
curtains commonplace; the tea had been homely. The room focussed
activities. It had made Paul feel instinctively "keen," as they said at the
Christian Union.

Manning, kneeling before the fire, was carefully pouring boiling water
into a Turkish coffee-pot of burnished copper. Delicate china coffee-cups
stood by a silver cigarette-box on an Indian lacquered table. A diffused light
filtered through silken lamp-shades, and two wall-sconces of candles lit the
pictures with a faint radiance. The corners and distances of the room were
heavy with shadows. Bronze chrysanthemums stood in a tall vase on an
otherwise bare overmantel. The chairs the big footstools, the lounge, the
carpet—all were soft, rich, heavy. The firelight glinted on the tooled leather
bindings of books in a case opposite him. The room made him feel
comfortable and introspective. Parker's Piece seemed to belong to a
different world.

He pulled himself together, and deliberately continued the conversation.

"But it is Christ Who matters, Manning," he said with real bravery.

The other replaced the kettle and set the coffee-pot on the table. He
selected a cigarette and lit it over the lamp. Then he settled himself
comfortably on the lounge. "Matters?" he queried. "Your technicalities are
new to me, Kestern."

But Paul was not going to shirk issues. "Yes," he said, "matters to your
soul, for life or death."
 "That, then," said Manning, "is my business. My soul is my own, at any
rate."

"No," said Paul, "it was bought with a price. 'Ye are not your own.'"

"I was not aware that I had put mine up for sale," retorted Manning, "and
if it was purchased, on your own showing, some nineteen hundred years
before it came into my possession at all, it seems to me that I don't get much
of a chance."

"Christ bought you to set you free," said Paul, and he mentally recalled a
favourite anecdote of his concerning a travelling Englishman and a freed
slave. For the first time, perhaps, he decided not to use it in this connection.

"Thanks," said Manning drily. "Then I claim my freedom."

"But," capped Paul, "you have to choose whom you will serve."

Manning flicked off his cigarette ash with a little gesture. "Look here,"
he said, "words are words. They serve a purpose, but they are not ends in
themselves. St. Paul used Jewish and Rabbinic phraseology, and appealed to
his day with metaphors. Thus—it's as old as the hills—if you talk of
purchase, you imply a seller. Did Christ buy me from Satan or from
Almighty God or from whom? So far as I am aware, the point is not even
yet settled. Nor is it meant to be settled. It implies a conception of the
universe generally that is outworn. Neither you nor I are ancient Jews. A
good deal of water has flowed under the bridges since Habakkuk."

Paul said nothing. In point of fact, he hardly understood. This was all
new to him.

"Well, I see God in art and beauty. He has given me a soul that finds Him
there, rather than in sacrifice of the fat of rams and the thunder of Sinai. I
take it, even you do not regard Him as tied to pitch-pine, corrugated iron
and Moody and Sankey's hymns. He is not to me a tribal deity, needing
propitiation and ordering the slaughter of women and children, flocks and
herds. 'Nothing but the Blood of Jesus,' sounded all right on Parker's Piece
and offers an emotional stimulus to uneducated people. But when you come
to definitions, the thought of the Old Testament leaves me rather cold. How
in the world can blood wash me clean?"

"But you believe in the Bible, surely?" queried Paul, puzzled and
honestly grieved.

"My dear fellow, what in the world do you mean by 'believe in'? I
believe in Browning. Personally, I believe in the present Government. As a
matter of fact, I believe in you."

Paul flushed. "But the Bible is the 'verbally inspired Word of God,'" he
ventured to quote.

"Which of the ten-score different versions?" queried Manning calmly.

"By the way, have you shown your verses to Tressor yet?"

If his visitor accepted the change of subject, it was because he was, for
the moment, clean bowled.

(4)

Paul had left a note asking Strether to breakfast, and he rather wondered
if, after the previous day's rag, his friend would come. But he came. To
mark the occasion, Paul had fish and an omelette sent up from the kitchens,
and over these burnt sacrifices he made his apology.

"Look here, Gussie," he said, "I'm sorry that rag ended as it did. I had no
idea the others had arranged it with old Sam like that, and I couldn't help
Donaldson kicking up all that row on the stairs. That was beastly, I admit.
I'm awfully sorry. Hope it won't make any difference to our friendship."

Strether growled in his throat. "Who bagged my boots?" he demanded,

with a sense of humour.

Paul laughed. "Let's rag Donaldson somehow," he suggested, "and I'll

give them back."
 Strether smiled. Then frowned. "Always talking about girls," he
muttered. Then, dropping the subject for good and all, "Come to The
Mikado this week," he invited.

"I've never been to the theatre," said Paul frankly.

The other nodded slowly in his meditative fashion. "So?" he queried.

"Yes," said Paul. "My people are against it. They say the stage is
immoral. I don't know...."

"Then so are newspapers," said Strether, "and so's Cambridge too for the
matter of that."

"That's different," objected Paul.

Strether laid down his knife and fork. "Going to the P.M., Sunday?" he
queried.

"Yes, I expect so," said Paul. "Why?"

"I'll come with you, if you'll come with me to The Mikado. I've never
been to a P.M. My people say prayer meetings make religion too
emotional."

Paul got up dubiously. He looked out of the window.

After all, there were, it seemed, many points of view in the world. Ought
he to see none other than his father's? And besides, if this would get Gus
Strether to a prayer meeting ...

"I'll go," he said. "I see that it is certainly foolish to condemn a thing you
haven't seen."

That night, over his fire in his own beloved room, he got out a secret and
personal diary which an evangelical missioner had urged him to keep, and
sat thoughtfully over it, pencil in hand. Then he wrote slowly: "Nov. 13. I
have decided to go to a theatre, since it is obviously unfair to condemn
anything unseen. I wish to be sure of the spirit in which I go and for what I
ought to look. Therefore I shall ask myself afterwards three questions, and I
write these down now to make certain that I do not forget:

1. If Christ came while I was there, should I mind?

2. Do I see anything bad in this play?
3. Has it helped my Christian life?"

Years later he turned up his old answers, written late on the Wednesday
night of the play, and smiled at their amazing and yet serious youthfulness.
"1. I should mind Christ's advent while I was in the theatre no more than I
should mind His coming while I was laughing over a humorous novel," so
ran the first answer.

"2. Honestly, I see nothing bad in the play. It was beautiful, the colour
and music bewitching, and the only fault, overmuch foolishness. But in the
bar and lounge, one felt that the men about were mostly of the sort who are
careless about their souls. Query: But what about a bump supper or a
smoking carriage?

"3. No, it has not helped my Christian life, but it has not, so far as I can
see, hindered it. Indirectly, it has perhaps helped me, just as exercise, music,
poetry and ordinary conversation, may be said to do.

"Note. Honestly, I have never enjoyed myself more in all my life."

Poor Paul!

(5)

But he was to enjoy himself still more that memorable term. Towards its
close, as a scholar, he received an invitation to the big college Feast of St.
Mary's, a commemoration to which some distinguished outsider was always
invited and which celebrated itself with the aid of a classic menu and some
historic music. Neither Strether nor Donaldson were asked, for neither had
achieved scholarship fame, and Manning was separated from the fresher by
an impassable gulf of table. Paul, in fact, sat between Judson and the wall
farthest from the High Table. Judson, cox of his boat, was a genial person,
but no particular friend of Paul's, and Judson, moreover, was frankly there
to eat and drink. Paul functioned merely automatically in regard to these. It
was the splendour, the glamour, that he feasted upon, and his imagination
saw to it that neither lacked. Even the sheer beauty of the shining plate, the
silver candelabra, the ancient hall and the glittering tables, touched, here
and there, with the orange and yellow and green and gold of the piled
dessert, was all but forgotten as he read his list of distinguished names,
caught the gleam of ribbons across this and that shirt-front, listened to the
clever short speeches, delighted in the historic music, shared, timidly, in the
ceremonies of toast and loving-cup. He saw a world worth entering. He was
intoxicated, though he drank no more than a shy glass of lemonade. If, in
the dark shadowed gallery away from the bustling waiters, there lingered
understanding spirits, as like as not Paul Kestern was the most entertaining
person present.

In the library, the great Tressor singled him out. "Well, Kestern," he said
smiling, "what did you think of it all?"

The boy looked at him gravely. "It was all rather wonderful to me, sir,"
he said.

"It was a good feast, certainly," said the other. "By the way, I fear I can't
get away from all this now, but I wanted to say a word to you about those
verses of yours. They are very distinctly good, I think. The shortest is the
best—The Spent Day. You'll do much better work, but in its own way, it's a
perfect poem."

Paul could hardly believe his ears. "It is awfully good of you to read
them," he managed to say.

"Oh not at all. I'm delighted. Look here, are you engaged to-morrow?
Come to luncheon, will you? You row, don't you? so you'll want to leave
early. I won't invite anybody else, and we can discuss them then. Good-
night."
 The big man, with the heavy eyebrows, slightly bowed shoulders and
kindly eyes, smiled, nodded, and passed on. Manning followed him up to
Paul. "What did he say?" he asked.

Paul hardly liked to tell him. It seemed fantastic as he said it.

Manning nodded. "I thought as much," he said, smiling. "Remember me,

Kestern, when you're a big man. I at any rate put one of your feet on the
ladder."

Paul mumbled something, and soon escaped. His fire was out in his
room, but it mattered little; he could not sit down to read or think quietly
after all this. Up and down he paced, repeating Tressor's words: "In its own
way, it's a perfect poem." A perfect poem! And Tressor had said it! Said it
after those songs, those speeches; said it in that company.

Then, as the boy passed and repassed, his eye fell on his text. He looked
at it critically: the frame and flowers and lettering were so extraordinarily
bad. A few weeks ago he had not remarked that. Still, it was the words that
mattered. What would the Master have thought of the college feast? Cana of
Galilee? Yes, but He would have been but a visitor. Could He have had a
real part in it?

Paul swung into a new train of thought. He considered the cost of it all.
Why, when he had refused the first cigar, Judson had said he never refused
a half-crown smoke. Half a crown for a cigar!—the thing was monstrous to
evangelical Paul. The smokes of the dinner alone would have kept a
catechist in India for a year! Probably the wines would have paid the annual
salary of a white missionary in China. And with every tick of the clock, a
heathen soul passed into eternity. How often he had said it! What, then, was
he doing among such things? What part had he in such extravagance? "One
is your Master, even Christ."

Paul sighed, and reached for his diary. "The feast was wonderful (he
wrote), extraordinarily beautiful I thought.... But..." Then he went to bed.

Wonder on wonders. The morning's post brought him a letter from the
editor of The Granta, accepting, magnanimously, a short story of an
imaginative nature that he had placed in Egypt with the aid of a Baedeker.
The editor asked, interestedly, if he had been there. He supposed that Paul
must have been, for the descriptions were so vivid.

Paul's porridge grew cold. He sat on with the letter in his hand.
Donaldson found him so, calling to go with him to a distant lecture.
"Hullo," he said, "not finished brekker? You're late again, four!"

"I say," said Paul, "The Granta's taken that yarn of mine about Egypt."

"By Jove, that's topping." Donaldson spoke enviously, staring at him.

"But I told you it was jolly good, didn't I?"

"You did," said Paul, "but I say, what do you think Tressor said last night
about my verse?"

"Can't say," said Donaldson.

Then Paul told him.

His friend whistled. "Damn it all, Paul," he said—"by the way, let me
call you 'Paul,' may I?—I should chuck all those preaching and praying
stunts of yours now."

"Why on earth——" began Paul, utterly surprised.

"Oh well, do as you think best. But it'll spoil you for literature. Didn't
Tressor tell you the other day that your essays were too like sermons? And
if you get in with Manning and all that set, Hartley and his crowd won't be
of any use."

Paul got up slowly and walked to the fire. He stood still awhile, gazing
into it. The other fidgeted. "Come on now, anyway," he said. "We shall be
late for that lekker."

"I shan't go this morning. I shall cut it."

"Right-o. Good-bye. I'm off," retorted the other, and departed, a little
huffed.
 Mrs. Roper came in to clear away. "Aren't you a-going to finish your
breakfuss, sir?" she asked.

"I've done, thanks," said Paul. "I don't want any more."

"Off 'is feed," said Mrs. Roper outside to her "help." "'Ad too much at
that there feast, I expect. 'Ere, you can 'ave them eggs."

As for Paul, he mounted his bicycle and rode out into the country. A
wintry sun lay on the bare woods and stubble fields, and it was all very
lovely. Even the close-cropped hedges were beautiful. The fallen beech-
leaves were a spread of old gold under the trees by Madingley.

CHAPTER III
CHRISTMAS CAROLS
... Doubt, which, like a ghost,
In the brain's darkness haunted me,
Was thus resolved: Him loved I most,
But her I loved most sensibly.
Lastly, my giddiest hope allow'd
No selfish thought, or earthly smirch;
And forth I went, in peace, and proud
To take my passion into Church;
Grateful and glad to think that all
Such doubts would seem entirely vain
To her whose nature's lighter fall
Made no divorce of heart from brain.
COVENTRY PATMORE: The Angel in the House.

(1)
 Paul, walking home from Claxted Station down Edward Street and past
Mr. Thornton's "Elite Photographic Studio," was puzzled. Some
bewildering spell had fallen upon Claxted in a couple of months. The
suburban station had a strange respectable air that sat ill on it, and whereas
a station may smell of dirt or smoke, it should not smell of stale paint.
Edward Street was horribly tidy, and gaped. The Town Hall and its
Libraries, once majestic centres of learning and authority, had been
cheapened. And the familiar road to his home appeared to have been newly
washed and to have shrunk in the process.

His father's house had only escaped the snare by a miracle, and Paul was
obsessed by a sense of that miracle. The case of stuffed birds in the hall, the
gilt presentation clock in the drawing-room, the old arm-chair in the dining-
room, the yards of commentaries and sermons in the study, with the
illuminated addresses above them, were miraculously pleasant. For days
after his return, he kept looking at them, and marvelling inwardly that they
were just the same. The furniture of Manning's and Mr. Tressor's rooms had
already made him feel that in his home recollections there must be some
mistake. But he knew now, staring about him, that there was not. And he
was still quite glad, and a little subdued.

"Oh, Paul," cried his mother, hurrying into the hall to meet him, "how
well you're looking! Are you glad to be back?"

"Very glad, mother darling," said Paul, kissing her. "Where's dad?"

"It's the Band of Hope night, dear, don't you remember? He's not back
yet. But he said he wouldn't be late for supper. Sit down over there where I
can see you, and tell me all about Cambridge."

Paul laughed. "That's a big order," he said. "I don't know where to
begin."

"Tell me about your children's service and the open air meetings, Paul,"
said his mother. "Is Mr. Hartley nice? Your father and I are so glad you've
made such friends."
 Paul thrust "The Literary Lounge," the College Feast, the Theatre,
Donaldson, Strether and Manning, into the back of his mind, and told her.

"And do you find the lectures hard?" she queried.

Paul laughed gaily. What a topsy-turvy notion of Cambridge his mother,

after all, must have!

His father's key grated in the door and Paul ran out into the hall. The
clergyman came in, followed by Mr. Derrick. "Ah, Paul," he said, "it is
good to see you home again. Come in, Mr. Derrick. Paul's just back. I'll get
you the books at once."

He entered the study, and Mr. Derrick held out his hand. Paul took in the
dapper little man, from his spotless tall linen collar to his neat black boots.
"How are you?" he said genially. "How goes things?"

"How do you do, Mr. Paul," said Mr. Derrick nervously. "We are all very
well, thank you. Have you had a good time at college? How short the terms
are! You seem scarcely to have gone away at all."

"Eh?" queried Paul, momentarily astonished. Then he recollected. "Yes,"

he confessed, "I suppose they do seem short. We read more in the vacs. than
in the terms, you know."

"I hope you will still be able to lend us a hand, however," said his visitor.

"Rather," said Paul. "Who's taking the children on Sunday?"

"I am, unless you'd rather."

"I put Paul down for the evening," said his father, returning. "I rather
hope he'll go to church with his mother in the morning. She'd enjoy having
him. You know what mothers are, Derrick."

"Yes, yes, to be sure," said the little man quickly. "I should have thought
of it. But I expect we shall see a good deal of you, Mr. Paul."

"Rather," said the young man again. "Are all the folk going strong?"
 "Yes. Mr. Vintner is secretary of the Missionary Committee in your
place. He's coming on well."

"Vintner!" exclaimed Paul. But he was ashamed of his instinctive

thought the next moment. "Splendid," he said.

Mr. Derrick nodded. "He gave a most helpful address on Henry Martyn
last week.... Thank you, Mr. Kestern. Are those the books? I'll go through
them to-night and let you have them on Sunday. I don't suppose it'll take me
long. Good-night. Good-night, Mr. Paul."

The clergyman thanked him and saw him out. "Capital fellow," he said,
entering the dining-room. "Wait till you're ordained, Paul, and you'll know
what such lay help means to a clergyman. Well, dear boy, and how are you?
Really I think you've grown. What do you think, mother?"

"I've been admiring his fancy waistcoat," said Mrs. Kestern. "Where did
you get it, Paul?"

(2)

Paul was soon aware that he was in for a delightful vacation. Not many
young men in their circle went to the University, and none at all, naturally,
from among "the workers." Paul was, therefore, lionised. It was impossible
for him not to be aware of it. He had always been a kind of natural leader,
but he was now something more. A glamour sat about him. It was possibly
Miss Ernest who made him aware of it first.

She was to play at the Mission Hall that first Sunday night, and Paul
called for her to take her down through the dark, slummy streets. She kept
him waiting some minutes, and when she came down, she was most
unusually resplendent even for her.

"How do you do?" she said, shaking hands and smiling. "Do you know, I
hardly dare call you Paul now?"
 "Why ever not?" he asked, closing the house door behind her.

"You're so much older," she said.

"Two months, Madeline," he protested, using her name deliberately.

"Is that all? It seems to me that you've been away ages."

Paul glanced at her. She was entirely demure, and did not look at him.

"Well," he confessed, "it seems a long time to me too. It's curious how
quickly Cambridge changes things. I hardly feel the same as I did two
months ago."

"I suppose you've met all sorts of ripping people."

"Rather. Do you know Mr. Tressor's at our college, and I've shown him
my verses. He said—he was awfully nice about them. And The Granta has
taken a story of mine."

"I'm not surprised," she said. "I always thought you had it in you."

Paul was a little piqued that she took it so easily, though on reflection he
perceived that this was a compliment. "It is impossible not to write at St.
Mary's," he said.

"Is it very lovely?" she asked softly.

"Oh, exquisite. You must see. Do you think you could come up in the
summer term? My rooms are small and high up you know, but perfect I
think. And the Hall and Chapel thrill me every time I see them. If you could
see the moonlight on our First Court!"

"Doesn't Claxted bore you after all that?"

Paul laughed. "It's rather quaint," he confessed. "It's really rather like
another world. Do you know, I've been to the theatre."

"Have you? Oh how splendid! I'd love to go."

 "Don't tell anyone," said Paul, cautiously.

"Of course not. What did you see?"

"The Mikado."

"Oh don't—I can't bear it. You make me so jealous. There you are,
leading your own life, and I'm tied down to this. You don't know how things
bore me at times."

Paul grew suddenly grave. "I think perhaps I am beginning to," he said,
and lapsed into silence.

A lay-reader took the service, and Paul, in cassock and surplice on the
platform of the little mission church, had leisure to observe. He had been
there a thousand times; very dear memories linked him to it; but not till now
had he looked about him critically. The place was an iron building of good
size, garishly lit with gas, and at one end was a platform which could be
screened off from the body of the hall. The curtains were drawn apart for
this service, and Paul from where he sat, stared sideways at the varnished
Table within the encircling wood railings; at the text above it; at the
harmonium opposite him, with the back of Miss Ernest visible, and the side
of her face, under its big hat, when she occasionally glanced at the lay-
reader who was taking the prayers and announcing the hymns. Below her
sat the choir of working men, and near them a couple of forms of girls who
"strengthened" their efforts. Paul scanned their faces surreptitiously with
amusement. There, against the wall, was old Miller who invariably started
each verse a word ahead of the rest, and got steadily more flat as the hymn
continued. Among the girls, he was surprised to see Miss Tillings. He
supposed she had been converted in his absence. In the front row was
Hodgson, a police-sergeant and a thoroughly good fellow. Next him,
McArthur, who played a cornet when he knew the tune. And then the
congregation, among them Mrs. Reynolds. If Edith Thornton were present,
he could not see her. But he looked.

The lay-reader was occasionally doubtful about his aspirates. He also

read an unduly large selection of collects. His voice, too, got on Paul's
nerves. He read for the hundredth time the short, staring gilt text above the
Table. "Till He Come." Except for the hymn notices, there was nothing else
to catch the attention. Oh yes, I.H.S. in a monogram under the text. Paul
wondered if the lay-reader knew what the letters meant. He wondered if any
of them knew what they meant. Then, as the reader began the prayer for
Parliament, if anyone knew what anything meant. Mrs. Reynolds, for
example. "That all things may be bordered and settled by their hendeavours,
upon the best and surest foundations...." "Amen"—very loudly from old
Miller. But he had heard that old Miller was a strong Conservative and
concerned with politics in his off hours. Curious; it struck Paul suddenly
that "the workers" never seemed to have politics. Oh, at last—Hymn 148.

Afterwards, they were all very kind. He shook hands with the departing
congregation, including Hilda Tillings. Hodgson was unfeignedly glad to
see him back. But outside, while Paul was smilingly making his way back
to the platform by which Madeline was standing drawing on her gloves, the
sergeant was rebuffed by old Miller.

"Good sermon, Miller," he said. "He's a fine young chap, and I'm glad
he's back."

"Eh, eh, sergeant, but I dunno as I 'olds with all this 'ere book-larning.
'E's got more grammar nor ever, and, seems ter me, less grace."

"Doesn't it all seem rather queer to you now?" asked Madeline, as they
walked home.

Paul shrugged his shoulders. "They're rattling good people," he said,

enigmatically.

"Yes, of course. By the way, do you remember that the Sale of Work is to
be this week. You will help me decorate our stall, won't you, Paul?"

"Rather. Is it this week? I'd forgotten. Do you want all that muslin stuff
tacked up again?"

"Yes. But we'll get you a step-ladder this year. The boxes collapsed last
time—remember?"
 He nodded, amused. "But why don't you try a new idea?" he suggested.
"Why always keep to the same old muslin?"

Madeline sighed. "We do always keep to the same old things, don't we?
But what could we do? Suggest something."

"Have a background of palms and cover the framework with ivy."

"That'd be lovely. But how could we get the ivy?"

"Leave that to me. I'll get it for you."

"Will you? Thanks so much. Could I help?"

Paul glanced at her carefully. She walked gracefully, but with her eyes
on the pavement. He admired her fair hair and her new hat, her trim figure.
After all, why not?

"Bicycle out with me on Friday and get some," he suggested. "There's

lots at Hursley."

Her voice was even as ever as she replied. "That would be delightful,"
she said. "Come in now and ask father, will you? Perhaps he'd come too.
And I say, do let me read your verses. I'd like to so much."

Paul was suddenly shy. "Oh they're nothing," he said.

She smiled. "Mr. Tressor did not think so," she retorted. "Paul, I wonder
if you're going to be a poet."

"I'm going to be a foreign missionary," he said.

"Well, you can be both. I expect abroad you would have no end of
inspiration. You're not likely to be sent among utter savages. You're more
likely to be made the head of some college or another, perhaps in India. You
could write too. I should think Calcutta or Delhi, or some place like that,
would be heavenly. And you'd go to the mountains in the summer. It makes
me envious to think of you. You'll have a glorious life."
 Paul grew grave. "I'd prefer to be among savages," he said.

"Why? Besides, do you think that's altogether right? God didn't give you
your gifts for you to waste them. And they want the other sort of missionary
just as much."

"I suppose they do," said Paul. "And if I lived that sort of life, I should
marry. One could. I've always doubted if a pioneer missionary ought to
marry."

Madeline nodded. "I think you're right. And besides, the wives of that
sort of missionary do get so awfully dowdy. I suppose it doesn't matter what
you wear among savages, and so they don't care any more about dressing.
I'm afraid I could never be so good as all that."

Paul laughed. "Honestly, I can't see you dowdy, Madeline," he said.

She smiled, but said nothing. He glanced at her shyly. "Summery frocks
and Indian Society would suit you," he said.

"Do you think so?" she said easily.

"Yes. By the way, you just must come up for the Mays. Will you
promise?"

"I'll come if I possibly can," she said, "and thank you ever so much for
asking me. Here we are. Now come in and settle with father about the ivy,
will you?"

(3)

The Annual Sale of Work was the parochial Feast of Claxted. A

distinguished visitor was always invited to open it; the stalls through which
one wandered, were so many courses, so to speak; and in the evening, there
were always songs, a few speeches, and light refreshments. So far as the
Mission Hall of the church was concerned, only the more superior members
were expected to put in an appearance, and these chiefly in the evening.
Thus Hodgson always came, but not old Miller. The Christian Endeavour
arranged little side-show concerts from six o'clock onwards, at half-hour
intervals, but even the Endeavourers were not seen in the afternoon. During
those sacred hours, the carriages drew up outside the Parish Room in
Edward Street, and there descended from them the elite and the wealthy of
the congregation. These, entering the half-empty room, caused a ripple of
comment to run through the stall-holders proportionate to their importance.
"Old Mrs. Wherry," Mrs. Ernest would whisper enthusiastically to
Madeline. "Oh, my dear, try to get her here at once. She always spends such
a lot, and she's so blind, she can't see what she buys. She just decides to
spend so much, I believe, and when it's spent, no one can get another penny
out of her. Do fetch her here."

"How can I, mother?" retorted Madeline, on this occasion.

"I'll try," said Paul, good-humouredly, and strolled off in her direction.

"Madeline, I saw you fastening that ivy with Paul," said Mrs. Ernest, as
he went.

"Well, mother?"

"My dear, anyone might have seen. I thought I saw Mrs. Cator watching.
And you know what she is likely to say."

Madeline tossed her pretty head. "I know what I am about, mother," she
said.

"I hope you do," sighed Mrs. Ernest. Her husband was a good man, but
without distinction, and truth to tell, she was tired of living on a curate's
stipend.

Paul came up with Mrs. Wherry. The old lady had been genuinely glad to
see him, and, since her own sons had been at Cambridge, she showed him
caustic good humour. "You want me to spend my money here, I suppose, do
you? Well, it doesn't much matter to me. Good afternoon, Mrs. Ernest. I see
you've adopted a system of pickets. Or is it Miss Ernest? Still everything's
fair in love and war, and certainly a Sale of Work is war. What have you? I
shall only buy things that I can send elsewhere."

Paul stood chatting with Madeline again while the old lady did her
shopping. A little hum of talk covered their conversation, which was broken
now and again as someone nodded and spoke to him, or he was sent off by
his father on some trivial errand. He was not as bored as usual, but drifted
back to the ivy-hung stall fairly regularly. At half past four he suggested tea.
"You can go, Madeline," said Mrs. Ernest. "I'll wait a little. Someone must
watch the stall."

"Come on then," said Paul, catching Madeline's eye, and she moved off
with him.

Formerly it had been hard to get Madeline for tea. Young men, who had
recently started going to the City, used to drop in about this time and take
her off. There were one or two about now, but she had no eyes for them. He
piloted her into a corner, and went to get the tea from the buffet which was
presided over by Mrs. Cator herself. She kept him chatting while a fresh pot
was made, and he was steering his way back to Madeline with the little tray
when he saw Edith.

It was early for her, for she arrived, as a rule, with the rest of the
Endeavourers. There she was, however, with her mother in a black dress
and a bead bonnet. Mrs. Thornton was well known in the congregation. She
aspired to rather a high estate, which was impossible for her, socially, with
her husband's shop in Edward Street.

Paul watched Edith bring her in. The girl was quiet and self-possessed,
and did not, apparently, see him. She steered her mother to a little table and
sat down by her. One of the Miss Cators, acting waitress, went up for the
order.

"Here's the tea," said Paul. "Sorry I was so long. You must want it."

"I do. Oh, and you've got eclairs! How delicious; I love them."

"I remembered that you did at the school-treat last August."

 "That terrible day! Do you remember how Mrs. Thornton would have
lunch at our table? Look—there she is. I do hope she doesn't see you. She's
sure to come over if she does. ''Ow do you do, Mr. Paul, and 'ow do you like
Cambridge? We're glad to see you back, I'm sure.'"

Paul sat down deliberately in such a position that he could see Edith.
"Don't, Madeline," he said. "She's a thoroughly good sort really, and means
well."

"Paul, you know perfectly well you used to laugh at her as much as any
of us."

"Did I? Then I was wrong. I'm beginning to see that the world is full of
queer sorts of people, and that the only real test is their sincerity."

"Well, then, some sincere people are impossible. You know they are. At
any rate I'm sincere enough to tell you that I think so."

Ethel Cator came up to them. She was a brunette, tall and thin, and in a
cap and apron she looked pretty. She was one of Madeline's friends. "Hullo,
Madeline," she said. "How are you two getting on? Have some more tea?"

"My dear, aren't you worn out with this tea business? Can't I give you a
hand? It's a slack time at the stall."

"Oh no. It's all right. But it's our busy time, of course. Have some more
eclairs. We're running a bit short, but I can get some for you."

"I couldn't. Really, I couldn't. Will you, Paul? I say, Ethel, are you going
to the school dance? Grace said yesterday she didn't know what you had
decided. Do come, my dear. I've said I'll go, and you must be there. I've
positively got a new frock for it."

"Look here," said Paul, laughing, "this is no place for me. I'm off. I'll tell
your mother to expect you in half an hour, Madeline. Good-bye. Good-bye,
Miss Cator. Your tea's topping. I'll send in everyone I see." And he walked
off.
 Madeline glanced quickly across the room; Mrs. Thornton and Edith
were making their way to the door; Paul caught them up as she watched.
She flushed slightly. Ethel Cator slipped into the empty place by her side,
and dropped her voice a little. "He's not keen on that girl, surely," she said.

Madeline shrugged her shoulders. "How should I know?" she asked,

with an assumption of indifference.

Ethel laughed. "Well, my dear, of course it's not my business, but I

thought you saw a good deal of him."

"Well, naturally, seeing what our fathers are."

"Has Cambridge changed him? I should have thought he'd have dropped
the Mission Hall now."

Ethel's tone was a little contemptuous, and it roused Madeline to the

defensive.

"My dear, you don't know Paul," she said coolly. "He doesn't play at
religion. He probably wants to speak to Miss Thornton about the Christian
Endeavour. It would take more than a term at Cambridge to make Paul
throw that over. And I like him for it."

Her friend got up. "I must go," she said. "I didn't mean to be a cat,
Madeline. Everybody knows Paul's a born parson, and of course he'll make
a good one."

"He wants to go to India," said Madeline, mollified and inconsequent,

and not realising that she lied. "He'll be a bishop one day, I expect."

Ethel looked envious, and rewarded her. "India!" she exclaimed, and sat
down again for a minute or two. The girls fell to discussing Simla with a
suburban imagination.

Mrs. Thornton had asked him "'Ow he liked Cambridge?" and Paul had
replied at length. But she had gone off at last, and left him with the tall girl
whose brown eyes had been alight with a flicker of amusement the while he
had talked to her mother. They were standing near the platform at the top of
the room, and a not yet opened "fishpond" with its appurtenances screened
them slightly. He was able to look her full in the face now and realise how
good she looked, though the little fur hat was slightly out of place there, and
her coat a little shabby.

"Mother's a dear," she said.

He nodded. "I know. Edith, I've longed to see you again. Why weren't
you at the Mission Hall on Sunday?"

"I couldn't go. I was ever so sorry."

"Really?"

She nodded. "I knew you were preaching. Mr. Derrick told us. But I had
to stay and help mother with the kiddies."

Paul saw a mental vision of the little rooms over the shop and the three
small Thornton children sprawling everywhere. Once or twice he had been
in on business for the Society, and he knew it well. Edith in that setting had
always puzzled him a little. She did not seem quite to belong to it, and yet
she moved about household jobs with a quiet dignity that did not in the least
suggest resentment or incongruity.

"You'll be here to-night?" he questioned.

She shook her head. "That's why I've come this afternoon."

"When are we going to meet then? I do so want to talk to you.

Cambridge is wonderful, Edith. There's heaps to say. I don't know why, but
I want to tell you things."

He couldn't know that she had to make a little effort to steady her voice.
"Do you, Paul," she said. "That's awfully good of you."

He studied her a minute, thinking rapidly. "Tell me what you're doing

this week," he demanded.