DRUG DESIGN (AN OVERVIEW)

APPAJI B MANDHARE, Ph.D. TORRENT RESEARCH CENTRE (Gandhinagar, India) [email protected]

Drug Design & Discovery: Introduction

Drugs:
Natural sources Synthetic sources

Targets:

Discovering and Developing the One Drug

Profile of Todays Pharmaceutical Business

Time to market: 10-12 years. By contrast, a chemist develops a new adhesive in 3 months! Why? (Biochemical, animal, human trials; scaleup; approvals from FDA, EPA, OSHA)

Pharmaceutical R&D
A Multi-Disciplinary Team
Administrative Support Analytical Chemistry Animal Health Anti-infective Disease Bacteriology

Behavioral Sciences Biochemistry Biology Biometrics Cardiology Cardiovascular Science Clinical Research
Communication Computer Science Cytogenetics Developmental Planning DNA Sequencing Diabetology Document Preparation Dosage Form Development Drug Absorption Drug Degradation Drug Delivery Electrical Engineering Electron Microscopy Electrophysiology Environmental Health & Safety Employee Resources

Endocrinology Enzymology Facilities Maintenance Fermentation Finance

Gastroenterology Graphic Design Histomorphology Intestinal Permeability Law Library Science

Mechanical Engineering Medicinal Chemistry Molecular Biology Molecular Genetics Molecular Models Natural Products Neurobiology Neurochemistry Neurology Neurophysiology Obesity

Oncology Organic Chemistry Pathology Peptide Chemistry Pharmacokinetics Pharmacology Photochemistry

Physical Chemistry Physiology Phytochemistry Planning Powder Flow Process Development Project Management Protein Chemistry Psychiatry Public Relations Pulmonary Physiology Radiochemistry Radiology Robotics Spectroscopy Statistics Sterile Manufacturing Tabletting Taxonomy Technical Information Toxicology Transdermal Drug Delivery Veterinary Science Virology X-ray Spectroscopy

Over 100 Different Disciplines Working Together

Formulation
Medical Services

Medicinal chemists today are facing a serious challenge because of the increased cost and enormous amount of time taken to discover a new drug, and also because of fierce competition amongst different drug companies

Drug Discovery & Development

Identify disease Drug Design - Molecular Modeling - Virtual Screening Find a drug effective against disease protein (2-5 years) Scale-up

Isolate protein involved in disease (2-5 years)

Preclinical testing (1-3 years)

Human clinical trials (2-10 years)

Formulation

FDA approval (2-3 years)

Technology is impacting this process

GENOMICS, PROTEOMICS & BIOPHARM.
Potentially producing many more targets and personalized targets

HIGH THROUGHPUT SCREENING

Identify disease Screening up to 100,000 compounds a day for activity against a target protein

VIRTUAL SCREENING
Isolate protein Using a computer to predict activity

COMBINATORIAL CHEMISTRY
Rapidly producing vast numbers of compounds Find drug

MOLECULAR MODELING
Computer graphics & models help improve activity

IN VITRO & IN SILICO ADME MODELS

Preclinical testing

Tissue and computer models begin to replace animal testing

History of Drug Discovery.

1909 - First rational drug design. Goal: safer syphilis treatment than Atoxyl. Paul Erhlich and Sacachiro Hata wanted to maximize toxicity to pathogen and minimize toxicity to human (therapeutic index). They found Salvarsan (which was replaced by penicillin in the 1940s)
HO O As O H 2N Atoxyl Salvarsan Na+ ClH.H 2N HO As As NH2 .HCl OH

1960 - First successful attempt to relate chemical structure to biological action quantitatively (QSAR = Quantitative structureactivity relationships). Hansch and Fujita

History of Drug Discovery

Mid to late 20th century - understand disease states, biological structures, processes, drug transport, distribution, metabolism. Medicinal chemists use this knowledge to modify chemical structure to influence a drugs activity, stability, etc. procaine = local anaesthetic; Procainamide = antirhythmic

O H 2N OCH2CH2N(C2H5 )2 Procaine H 2N

O NHCH2CH 2N(C 2H5) 2 Procainamide

Drug Discovery overview

Approaches to drug discovery:

Serendipity (luck)
Chemical Modification

Screening
Rational

Serendipity Chance favors the prepared mind

1928 Fleming studied Staph, but contamination of plates with airborne mold. Noticed bacteria were lysed in the area of mold. A mold product inhibited the growth of bacteria: the antibiotic penicillin

Chemical Modifications
(A) Homolog Approach: Homologs of a lead prepared
R-(CH2)n-N Homologs (n=2,3,4,5.....)

(B) Molecular Disconnection /Simplification

N CH3 H3C NCH2CH=C HO O MORPHINE PENTAZOCINE CH2OCONH2 C C3H7 CH2OCONH2

MEPROBAMATE

(C) Molecular Addition

N-CH2-CH=CH2

(D) Isosteric Replacements

H2N COOH P.A.B.A. H2N SO2NH2

HO

SULFANILAMIDE

NALORPHINE

Chemical Modification.
Traditional method. An analog of a known, active compound is synthesized with a minor modification, that will lead to improved Biological Activity.

Advantage and Limitation: End up with something very similar to what you start with.

Screening
Testing a random and large number of different molecules for biological activity reveals leads. Innovations have led to the automation of synthesis (combinatorial synthesis) and testing (high-throughput screening). Example: Prontosil is derived from a dye that exhibited antibacterial properties.

Irrational, based on serendipity & Intuition

Trial & error approach

Time consuming with low through output No de novo design, mostly Me Too Approach

Rational Drug Design

- Cimetadine (Tagamet)

Starts with a validated biological target and ends up with a drug that optimally interacts with the target and triggers the desired biological action.

Problem: histamine triggers release of stomach acid. Want a histamine antagonist to prevent stomach acid release by histamine = VALIDATED BIOLOGICAL TARGET.

Histamine analogs were synthesized with systematically varied structures (chemical modification), and SCREENED. N-guanyl-histamine showed some antagonist properties = LEAD compound.

Rational Drug Design - Cimetadine (Tagamet) - continued

a. Chemical modifications were made of the lead = LEAD OPTIMIZATION: b. More potent and orally active, but thiourea found to be toxic in clinical trials

c. Replacement of the group led to an effective and well-tolerated product:

d. Eventually replaced by Zantac with an improved safety profile

First generation Rational approach in Drug design

In 1970s the medicinal chemists considered molecules as topological entities in 2 dimension (2D) with associated chemical properties. QSAR concept became quite popular. It was implemented in computers and constituted first generation rational approach to drug design

2nd generation rational drug design

The acceptance by medicinal chemists of molecular modeling was favored by the fact that the QSAR was now supplemented by 3D visualization. The lock and key complementarity is actually supported by 3D model. Computer aided molecular design (CAMD) is expected to contribute to intelligent lead

MECHANISM BASED DRUG-DESIGN

Most rational approach employed today. Disease process is understood at molecular level & targets are well defined. Drug can then be designed to effectively bind these targets & disrupt the disease process

Very complex & intellectual approach & therefore requires detailed knowledge & information retrieval. (CADD Holds Great Future)
Drug Receptor Interaction is not merely a lock-key interaction but a dynamic & energetically favorable one

Evolutionary drug designing

Ancient times: Natural products with biological activities used as drugs. Chemical Era: Synthetic organic compounds Rationalizing design process: SAR & Computational Chemistry based Drugs Biochemical era: To elucidate biochemical pathways and macromolecular structures as target as well as drug.

DRUG DISCOVERY PROCESS

Target Identification and Validation

Lead Compounds

Evaluation
Chemical Libraries, Combichem, Natural Products

Clinical Trials

New Targets
Total Genome Druggable genes
Includes biological Space Small molecule Space New druggable space ? Existing drugs (450 Targets) targets)

~30,000

~3,000

HIGH-THROUGHPUT SCREENING
FUNCTIONAL INTEGRATION OF:
BIOLOGY CHEMISTRY SCREENING TECHNOLOGY INFORMATICS

BOTTLENECKS
Hundreds of Hits but NO Leads
Data mining Accurate profiling of molecules for further studies.

ALTERNATE STRATEGIES
Rational Design of Chemical Libraries Molecular Modeling Approach Virtual Screening Early ADME & Toxicity Profiling

Smart Drug Discovery platform

A view of Drug Discovery road map illustrating some key multidisciplinary technologies that enable the development of (a) Breakthrough medicines from promising candidates (b) LO & generation processes that are relative to novel ligands. Tomi K Sawyer, Nature Chemical Biology 2, (12) December 2006.

Molecular Modeling
NMR and X-ray structure determination QSAR/3D QSAR Structure-based drug design Rational drug design

QM, MM methods

Model construction Molecular mechanics Conformational searches Molecular dynamics

Homology modeling

Combinatorial chemistry Chemical similarity Chemical diversity

Bioinformatics Chemoinformatics

What is Molecular Modeling?

Molecular Graphics: Visual representation of molecules & their properties.

Computational Chemistry: Simulation of

atomic/molecular properties of compound through computer solvable equations.

( b-b0)[ V1cos] b (-0) [V1cos] Statistical Modeling: D-R, QSAR/3-D QSAR Molecular data Information Management: Organizational databases retrieval

/search & processing of properties of 1000 of compounds.

MM = Computation + Visualization + Statistical modeling + Molecular Data Management

COMPUTATIONAL TOOLS: QM/MM

(A) MOLECULAR MECHANICS (MM) (B) QUANTUM MECHANICS (QM)

COMPUTATIONAL TOOLS
Quantum Mechanics (QM)
Ab-initio and semi-empirical methods Considers electronic effect & electronic structure of the molecule Calculates charge distribution and orbital energies Can simulate bond breaking and formation Upper atom limit of about 100-120 atoms

COMPUTATIONAL TOOLS
Molecular Mechanics (MM)

Totally empirical technique applicable to both small and macromolecular systems a molecule is described as a series of charged points (atoms) linked by springs (bonds) The potential energy of molecule is described by a mathematical function called a FORCE FIELD

When Newton meets Schrdinger...

Sir Isaac Newton (1642 - 1727) Erwin Schrdinger (1887 - 1961)

F ma

When Quantum Chemistry Starts to Move...

Mixed QuantumClassical

Traditional QC Methods

Classical MD Simulations

First-Principles Car-Parrinello MD

Mixed Quantum-Classical in a complex environment - QM/MM

Main idea
Partitioning the system into
Classical MM

1. chemical active part treated by QM methods

2. Interface region 3. large environment that is modeled by a classical force field

interface

QM

Mixed Quantum-Classical in a complex environment - QM/MM

Main idea
Partitioning the system into
Classical MM

1. chemical active part treated by QM methods

2. Interface region 3. large environment that is modeled by a classical force field

interface

QM

Basic modeling Strategies

Receptor Structure
Unknown Generate 3D structures, Similarity/dissimilarity Homology modelling HTS, Comb. Chemistry
(Build the lock, then find the key)

Known Active Site Search Receptor Based DD de NOVO design, 3D searching

(Build or find the key that fits the lock)

Unknown

Ligand Structure
Known

Indirect DD Ligand-Based DD Analogs Design

2D/3D QSAR & Pharmacophore

Rational Drug Design (Structure-based DD) Molecular Docking (Drug-Receptor interaction)

Computer Aided Drug Design Techniques

- Physicochemical Properties Calculations

- Partition Coefficient (LogP), Dissociation Constant (pKa) etc. - Drug Design - Ligand Based Drug Design - QSARs - Pharmacophore Perception - Structure Based Drug Design - Docking & Scoring - de-novo drug design - Pharmacokinetic Modeling (QSPRs) - Absorption, Metabolism, Distribution and Toxicity etc. - Cheminformatics - Database Management - Similarity / Diversity Searches -All techniques joins together to form VIRTUAL SCREENING protocols

Quantitative Structure Activity Relationships (QSAR)

QSARs are the mathematical relationships linking chemical structures with biological activity using physicochemical or any other derived property as an interface.

Biological Activity = f (Physico-chemical properties)

Mathematical Methods used in QSAR includes various regression and pattern recognition techniques.
Physicochemical or any other property used for generating QSARs is termed as Descriptors and treated as independent variable. Biological property is treated as dependent variable.

QSAR and Drug Design

Compounds + biological activity

QSAR

New compounds with improved biological activity

WHY QSAR.?

Chemical Space Issue

Virtual Hit Series, Lead Series Identification, clinical candidate selection

Figure: Stage-by-stage quality assessment to reduce costly late-stage attrition.

(Ref: Nature Review-Drug Discovery vol. 2, May 2003, 369)

Types of QSARs
Two Dimensional QSAR
- Classical Hansh Analysis - Two dimensional molecular properties

Three Dimensional QSAR

- Three dimensional molecular properties - Molecular Field Analysis

- Molecular Shape Analysis

- Distance Geometry - Receptor Surface Analysis

QSAR ASSUMPTIONS
The Effect is produced by model compound and not its metabolites. The proposed conformation is the bioactive one. The binding site is same for all modeled compounds. The Bioactivity explain the direct interaction of molecule and target.

Pharmacokinetics aspects, solvent effects, diffusion, transport are not under consideration.

QSAR Generation Process

1. Selection of training set
2. Enter biological activity data

3. Generate conformations
4. Calculate descriptors 5. Selection of statistical method 6. Generate a QSAR equation 7. Validation of QSAR equation 8. Predict for Unknown

Descriptors
1. 2. 3. 4. 5. 6. 7. 8. Structural descriptors Electronic descriptors Quantum Mech. descriptors Thermodynamic descriptors Shape descriptors Spatial descriptors Conformational descriptors Receptor descriptors

Selection of Descriptors 1. 2. 3. 4. What is particularly relevant to the therapeutic target? What variation is relevant to the compound series? What property data can be readily measured? What can be readily calculated?

QSAR EQUATION

Molecular Field Analysis

Activity = 0.947055 - 0.258821(Ele/401) + 0.085612(vdW/392) + 0.122799(Ele/391) - 0.7848(vdW/350)

Comparative Molecular Field Analysis

Electrostatic Field

Steric Field

Blue (electropositive) Red (electronegative)

Green (favourable) Yellow (repulsive) regions

COMFA studies on oxazolone derivatives

q2 = 0.688 and r2 = 0.969

alignment

Steric

Electrostatic

COMSIA studies on imidazole derivatives

alignment Electrostatic

steric Hydrophobic/ hydrophilic

q2 =0.761 and r2 = 0.945

3D-QSAR - RECEPTOR SURFACE MODEL

Hypothetical receptor surface model constructed from training set molecules 3D shape and activity data. The best model can be derived by optimizing various parameters like atomic partial charges and surface fit. Descriptors like van der Waals energy, electrostatic energy, and total non-bonded energy can be used to derived series of QSAR equations using G/PLS statistical method

PHARMACOPHORE APPROCH
Pharmacophore:
The Spatial orientation of various functional groups or features in 3D necessary to show biological activity.

Types of Pharmacophore Models

Distance Geometry based Qualitative Common Feature Hypothesis.

Quantitative Predictive Pharmacophores from a training set with known biological activities.

Pharmacophore-based Drug Design

Examine features of inactive small molecules (ligands) and the features of active small molecules. Generate a hypothesis about what chemical groups on the ligand are necessary for biological function; what chemical groups suppress biological function. Generate new ligands which have the same necessary chemical groups in the same 3D locations. (Mimic the active groups)

Advantage: Dont need to know the biological target structure

Pharmacophore Generation Process

Five Steps Training set selection. Features selection Conformation Generation

Common feature Alignments

Validation

Considerations/Assumptions
Training Set Molecules should be - Diverse in structure - Contain maximum structural information. - Most potent within series.

Features should be selected on the basis of SAR studies of training set

Each training set molecule should be represented by a set of low energy conformations. Conformations generation technique ensures broad coverage of conformational space. Align the active conformations of the training set molecules to find the best overlay of the corresponding features. Judge by statistical profile & visual inspection of model.

Pharmacophore Features
HB Acceptor & HB Donor Hydrophobic Hydrophobic aliphatic Hydrophobic aromatic Positive charge/Pos. Ionizable Negative charge/Neg. Ionizable Ring Aromatic
Each feature consists of four parts: 1. Chemical function 2. Location and orientation in 3D space 3. Tolerance in location 4. Weight

Pharmacophore Generation
Input Structures SAR Data

Conformational Modelling

Generate model

Evaluate Hypothesis

Training set
N
N N O O N H O N H O N N O N N N N HO O N N N N N

N N N N

BMS - 23
O OH N O

IRBESARTAN Sanofi
O OH

TELMISARTAN Boehringer
N N NH2 O Br

Forasartan
N N O Br O N N N N Cl OH

N N N N
N

O F F F H N S O O

OH

VALSARTAN
Novartis

EPROSARTAN
GSK

SAPRISARTAN
GSK

Zolasartan

OH

N N Cl OH N N N N
N O N N N N O N
N

O N N N

COOH N N N N

N N N N

Losartan-Merck

Candesartan-Astra

Tisartan

Tosartan

Pharmacophore Hypothesis Mapped on Active Molecule

Receptor-based Drug Design

Examine the 3D structure of the biological target (an X-ray/ NMR structure. Hopefully one where the target is complexed with a small molecule ligand (Co-crystallized) Look for specific chemical groups that could be part of an attractive interaction between the target protein and the ligand. Design a new ligands that will have sites of complementary interactions with the biological target. Advantage: Visualization allows direct design of molecules

Docking Process
Put a compound in the approximate area where binding occurs Docking algorithm encodes orientation of compound and conformations. Optimize binding to protein
Minimize energy Hydrogen bonding Hydrophobic interactions

Scoring

Docking compounds into proteins computationally

De Novo Drug Design

Build compounds that are complementary to a target binding site on a protein via random combination of small molecular fragments to make complete molecule with better binding profile.

 Can pursue both receptor and pharmacophore-based approaches independently If the binding mode of the ligand and target is known, information from each approach can be used to help the other Ideally, identify a structural model that explains the biological activities of the known small molecules on the basis of their interactions with the 3D structure of the target protein.

Typical projects are not purely receptor or pharmacophore-based; they use combination of information, hopefully synergistically

Cheminformatics - Data Management

Need to be able to store chemical structure and biological data for millions of data points Computational representation of 2D structure Need to be able to organize thousands of active compounds into meaningful groups Group similar structures together and relate to activity Need to learn as much information as possible from the data (data mining) Apply statistical methods to the structures and related information

Chemical Library Issues

Which R-groups to choose Which libraries to make Fill out existing compound collection? Targeted to a particular protein? As many compounds as possible? Computational profiling of libraries can help Virtual libraries can be assessed on computer

VIRTUAL SCREENING PROTOCOL

Objective - To search chemical compounds similar to active structure. Essential components of protocol are as follows Substructure Hypothesis Pharmacophore Hypothesis Shape Similarity Hypothesis Electronic Similarity Hypothesis - VIRTUAL SCREENING Library of ~ 2 lac compounds was screened Initially 800 compounds were short listed applying above filters. Further 30 compounds were selected by applying diversity & similarity analysis. Compounds have been in vitro screened and found various new scaffolds

Virtual Screening
Build a computational model of activity for a particular target Use model to score compounds from virtual or real libraries Use scores to decide which to make and pass through a real screen We may want to virtual screen - All of a companys in-house compounds, to see which to screen first - A compound collection that could be purchased - A potential chemistry library, to see if it is worth making, and if so which to make

Virtual Screening

1970s: no biological target structures known, so all pharmacophore-based approaches. 1990s: recombinant DNA, cloning, etc. helped the generation of 3D structural data of biological targets. Present: plenty of structural data of biological targets, but also improved technology to increase pharmacophorebased projects.

Drug Discovery overview (LI & LO)

Lead discovery. Identification of a compound that triggers specific biological actions.

Lead optimization. Properties of the lead are tested with biological assays; new molecules are designed and synthesized to obtain the desired properties

Pharmacokinetic Modeling (QSPRs)

In-Silico ADMET Models

Computational methods can predict compound properties important to ADMET Solubility Permeability Absorption Cytochrome p450 metabolism Toxicity

Estimates can be made for millions of compounds, helping reduce attrition the failure rate of compounds in late stage

Drug Design Successes (Fruits of QSAR)

Name of the drug discovered 1. Erythromycin analogs 2. New Sulfonamide dervs. 3. Rifampicin dervs. 4. Napthoquinones 5. Mitomycins 6. Pyridine 2-methanols 7. Cyclopropalamines 8. -Carbolines 9. Phenyl oxazolidines 10.Hydantoin dervs. 11.Quinolones Biol. Activity Antibacterial Antibacterial Anti-T.B. Antimalerials Antileukemia Spasmolytics MAO inhibitors MAO Inhibitors Radioprotectives Anti CNS-tumors Antibacterial

Drug Design Successes

While we are still waiting for a drug totally designed from scratch, many drugs have been developed with major contributions from computational methods
O F N HN N Et CO 2H MeO MeO N S O2 S O NH SO2NH 2

norfloxacin (1983) antibiotic first of the 6-fluoroquinolones QSAR studies

HO Cl N N Bu

donepezil (1996) Alzheimer's treatment acetylcholinesterase inhibitor shape analysis and docking studies

dorzolamide [Trusopt] (1994) glaucoma treatment carbonic anhydrase inhibitor SBLD and ab initio calcs

N NH N N

O NH O H

H N NMe 2

losartan [Cozaar] (1995) angiotensin II antagonist anti-hypertensive Modeling Angiotensin II octapeptide

zolmatriptan [Zomig] 1995 5-HT1D agonist migraine treatment Molecular modeling

Drug Design Successes-2

HIV-1 protease inhibitors
N N N H N O OH Ph H N O OH N S O N N Me H Ph H N O Ph OH N H O O S N

indinavir [Crixivan] (Merck, 1996) X-ray data from enzyme and molecular mechanics
SPh O N H H N

ritonavir [Norvir] (Abbott, 1995) peptidomimetic strategy

Ph O H N

Me O HO

OH H

N O

H N

N H OH CONH 2

H H

nelfinivir [Viracept] (Agouron, 1996)

saquinavir [Invirase, Fortovase] (Roche, 1990) transition state mimic of enzyme substrate

SUMMARY
Drug Discovery is a multidisciplinary, complex, costly and intellect intensive process. Modern drug design techniques can make drug discovery process more fruitful & rational. Knowledge management and technique specific expertise can save time & cost, which is a paramount need of the hour.

CADD Facility at TORRENT

HARDWARE: SGI Indigo2, O2 OCTANE SOFTWARE: Cerius2 (Version 4.10) Catalyst (Version 4.10) Daylight (ClogP ver. 4.1) ACD/Lab (pKa & logD suite) TOPKAT (6.2) DATABASES: ACD, NCI, MayBridge, MiniBiobyte, CAPScreening
UNIX, LINUX & (Oracle support)

Acknowledgment
Dr. C. Dutt Dr. Sunil Nadkarni Dr. Vijay Chauthaiwale Dr .Deepa Joshi Dr. R.C. Gupta Mr. Davinder Tuli Dr. Pankaj Sharma

THANK YOU