European Journal of Pharmacology 862 (2019) 172638

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European Journal of Pharmacology

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Full length article

Dipeptidyl peptidase-4 inhibitors can inhibit angiotensin converting enzyme T

a,b,∗ c,d
Mohamed Abouelkheir , Tarek H. El-Metwally
a
Department of Pharmacology and Therapeutics, College of Medicines, Jouf University, Sakaka, Saudi Arabia
b
Pharmacology department, Faculty of Medicine, Mansoura University, Mansoura, Egypt
c
Departments of Medical Biochemistry, Jouf University, Sakaka, Saudi Arabia
d
Faculty of Medicine, Assiut University, Assiut, Egypt

ARTICLE INFO ABSTRACT

Keywords: Angiotensin-1 converting enzyme inhibitors (ACEIs) improve insulin sensitivity. Inhibitors of dipeptidyl pepti-
Acute kidney injury dase-4 (DPP-4) are anti-diabetic drugs with several cardio-renal effects. Both ACE and DPP-4 share common
Autodock features. Thus, we tested if they could be inhibited by one inhibitor. First, in silico screening was used to
Enalapril investigate the ability of different DPP-4 inhibitors or ACEIs to interact with DPP-4 and ACE. The results of
Hypotension
screening were then extrapolated into animal study. Fifty Sprague Dawley rats were randomly assigned into 5
Linagliptin
Sitagliptin
groups treated with vehicle, captopril, enalapril, linagliptin or sitagliptin. Both low and high doses of each drug
were tested. Baseline blood samples and samples at days 1, 8, 10, 14 were used to measure plasma DPP-4 and
ACE activities and angiotensin II levels. Active glucagon-like peptide-1 (GLP-1) levels were measured after oral
glucose challenge. All tested DPP-4 inhibitors could interact with ACE at a relatively reasonable binding energy
while most of the ACEIs only interacted with DPP-4 at a predicted high inhibition constant. In rats, high dose of
sitagliptin was able to inhibit ACE activity and reduce angiotensin II levels while linagliptin had only a mild
effect. ACEIs did not significantly affect DPP-4 activity or prevent GLP-1 degradation. It seems that some DPP-4
inhibitors could inhibit ACE and this could partially explain the cardio-renal effects of these drugs. Further
studies are required to determine if such inhibition could take place in clinical settings.

1. Introduction full-scale of mechanisms utilized for such blood pressure-lowering ef-

fects of DPP-4 inhibition encompasses improvement of endothelial
Angiotensin-1 converting enzyme inhibitors (ACEIs) are anti-hy- function, increase nitric oxide, and change the balance of some of the
pertensive drugs with well-known ability to improve insulin sensitivity biologically active peptides (Fadini and Avogaro, 2011; Lovshin and
and reduce the development of new-onset type 2 diabetes (Favre et al., Zinman, 2014). However, none of these suggested mechanisms can
2015; Gillespie et al., 2005; DREAM Trial Investigators et al., 2006; explain the rapid, reversible hypotensive effects that have been re-
Paolisso et al., 1992). Various mechanisms have been proposed to ex- ported with some DPP-4 inhibitors (Mistry et al., 2008). Moreover,
plain the ability of renin-angiotensin system (RAS) blockade to improve these mechanisms did not explain why DPP-4 inhibitors are not equal
insulin sensitivity (Leiter and Lewanczuk, 2005; Scheen, 2004; Tikellis regarding their blood pressure-lowering effects (Groop et al., 2013;
et al., 2004; Ura et al., 1999). However, further studies did not support Ogawa et al., 2011; Pacheco et al., 2011).
some of these mechanisms (Erbe et al., 2006; Muller-Fielitz et al., Several observations suggest that RAS and GLP-1/DPP-4 could
2012). It seems that no mechanism stands alone to explain the positive possibly share a common inhibitor. Beside the metabolic effects of
effect of RAS blockade on insulin sensitivity. ACEIs and the hypotensive effect of DPP-4 inhibitors, DPP-4 inhibitors
Dipeptidyl peptidase-4 (DPP-4) catabolizes the incretin, glucagon- aggravate ACEIs-induced angioedema (Brown et al., 2009). Similarly,
like peptide-1 (GLP-1). Inhibitors of DPP-4 are used as anti-diabetic linagliptin, a safe drug for patients with chronic renal impairment, can
drugs (Meier et al., 2004). GLP-1, its analogues, and DPP-4 inhibitors cause acute renal failure when added to treatment regimens of patients
have several cardiovascular benefits including reduction of blood already treated with ACEIs (Kutoh, 2012; Nandikanti et al., 2016). At
pressure (Herzlinger and Horton, 2013; Papagianni and Tziomalos, the molecular level, the activities of both DPP-4 and ACE are affected
2015). These benefits are only partially explained by the resultant ac- by the presence of the amino acid, proline, at the site of cleavage. For
tivation of GLP-1 receptors (Ban et al., 2008; Shah et al., 2011). The this reason, proline was used to build up most of the early ACEIs and

∗
Corresponding author. College of Medicine, Jouf University, King Khalid Road, Sakaka, 72345, Saudi Arabia.
E-mail addresses: [email protected], [email protected] (M. Abouelkheir).

https://doi.org/10.1016/j.ejphar.2019.172638
Received 28 May 2019; Received in revised form 30 August 2019; Accepted 2 September 2019
Available online 03 September 2019
0014-2999/ © 2019 Elsevier B.V. All rights reserved.
M. Abouelkheir and T.H. El-Metwally European Journal of Pharmacology 862 (2019) 172638

some of DPP-4 inhibitors. Finally, both enzymes share many experi- 2.1.5. Docking into DPP-4
mental inhibitors (Brenda Enzyme Database EC-Number 3.4.14.5; The same as with ACE, AutoGrid was used to create the 3D grid for
Brenda Enzyme Database EC-Number 3.4.15.1). DPP-4 and evaluate the binding energies between tested ligands and the
The aim of the present study was to investigate whether DPP-4 in- enzyme. Regarding the used grid map, grid points were set as
hibitors or ACEIs can simultaneously inhibit both DPP-4 and ACE. 126 × 126 × 126, grid point spacing as 0.400 Å, and central grid point
Providing evidence for such interaction can give a new explanation of of the map (47.633, 52.321, 27.307) was selected by default. Again,
the secondary actions produced by these drugs and discover crosstalk LGA was used (Morris et al., 1998) with the same parameters specified
between medications used to treat hypertension and diabetes mellitus. for ACE and the selected number for docking rounds was 50. As with
ACE, free binding energy and the value of Ki were used to compare the
ability of these ligands to inhibit DPP-4.
2. Materials and methods

2.2. Animal study

2.1. In silico study

The experimental protocol was approved by the Local Committee of

AutoDock 4.2.6 software (Morris et al., 1998) was used to dock the
Bioethics, Jouf University (approval number 2-2-4/40). Fifty Sprague
structures of different members of ACEIs and DPP-4 inhibitors into both
Dawley female rats weighing 200–230 g (aging 8–9 weeks) were housed
ACE and DPP-4. Using AutoDock Tools (ADT), different ligands and
in an air-conditioned room on a light/dark cycle with free access to
enzymes were initially prepared. Discovery studio (2017 R2 client) was
water and a standard pellet diet. After one week, animals were assigned
used to generate final figures. The tested DPP-4 inhibitors were li-
into the following 5 groups (n = 10 in each): Group I: control group
nagliptin, sitagliptin, vildagliptin and anagliptin. The tested ACEIs were
(received drug vehicle, saline, by gavage); Group II: received captopril
captopril, ramiprilat, enalaprilat, perindoprilat, benazeprilat, fosino-
(ACEI; Bristol Myers Squibb, USA), dissolved in drinking water; Group
prilat, quinaprilat and zofenoprilat. Lisinopril was not tested because
III: received enalapril (ACEI; Merck Sharp & Dohme, Italy); Group IV
the number of rotatable bonds exceeds the capacity of the docking
received linagliptin (DPP-4 inhibitor; Boehringer Ingelheim, USA) and
program.
Group V received sitagliptin (DPP-4 inhibitor; Merck Sharp & Dohme,
Italy). Drugs in groups III-V were dissolved/suspended in saline and
2.1.1. Preparation of ligands were given once daily by gavage.
All the ligands were obtained from Pubchem in SDF format, which Treatment started with a low dose from each drug for one day (day
was then converted into Mol2 format using Open Babel 2.3.1. All hy- 1). Following 6 days of drug-free interval and at day 8, a high dose was
drogen atoms were added using ADT, which was also used to define instituted daily for 7 days. Determination of “low” and “high” dose of
torsion angles for each ligand. For better representation of ligand-en- each drug was after reviewing a large body of literatures. Low and high
zyme interaction, ligands were considered flexible during docking. doses for captopril, given in drinking water, were 100 and 380 mg/l/
day, respectively. The low and high doses were 3 and 10 mg/kg/day for
2.1.2. Preparation of ACE enalapril, 3 and 10 mg/kg/day for linagliptin, 10 and 30 mg/kg/day for
We followed a previously described method (Endringer et al., 2014). sitagliptin.
The crystal structure of human testis ACE was downloaded from the Blood samples were obtained before treatment, and, at days 1, 8, 10
Protein Data Bank (PDB code 2YDM) (Akif et al., 2011). With the ex- and 14 of therapy. Except for day 14, blood samples were collected
ception of zinc and chloride ions, the ligand, water molecules, and other aseptically, under isoflurane anesthesia, from the rat retro-orbital ve-
heteroatoms were completely removed using ADT. Formal charges and nous plexus at the specified time points 3 h after drug administration.
Van der Waals parameters for zinc and chloride ions were obtained By day 14 and under anesthesia, an indwelling catheter was inserted in
from the AMBER database. The structure of the enzyme was set as rigid. the right jugular vein for blood sampling. An oral glucose tolerance test
Hydrogen atoms were added then all non-polar hydrogens were (OGTT) was performed after fasting for 8 h. The tested drugs were given
merged. dissolved/suspended in saline 60 min before OGTT. The only exception
was captopril which was already added to drinking water. At 0 time
point, animals were administered with 2 g/kg glucose by gavage. Blood
2.1.3. Preparation of DPP-4 samples were collected just before administration of glucose and at 10,
The crystal structure of human DPP-4 was obtained from PDB (PDB 30, 60, 120 min after oral glucose. The area under the glucose tolerance
code 3WQH) (Watanabe et al., 2015). The ligand, water molecules, and curve (AUC) was calculated using the trapezoidal rule. The blood
other heteroatoms were also removed using ADT. The structure of the samples collected before administration of glucose were used to assay
enzyme was set as rigid. Hydrogen atoms were added then all non-polar DPP-4 enzymatic activity as well as basal glucose and GLP-1. Blood
hydrogens were merged. glucose was measured using blood glucose checker [GLUCOTREND®2
(Roche Group, UK)] and (AccuChek® Active) strips (Roche Group, UK).
2.1.4. Docking into ACE Blood samples intended for determination of the enzymatic activity
AutoGrid was used to create the 3D grid and evaluate the binding were collected into dry tubes and centrifuged at 2500 g for 15 min at
energies between different ligands and the enzyme. Regarding the used 4 °C. All samples were then stored at −80 °C until assy.
grid map, grid points were set as 126 × 126 × 126, grid point spacing
as 0.300 Å, and Zinc ion (15.447, −4.643, −19.597) was selected as 2.2.1. Assessment of ACE activity
the central grid point of the map. In order to model the binding between Serum ACE activity was measured according to the method de-
different ligands and ACE, Lamarckian genetic algorithm (LGA) was scribed by Cushman and Cheung (1971). Hippuryl-His-Leu (HHL;
used (Morris et al., 1998) with the following specified parameters: 1) Sigma) was used as a synthetic substrate. Rat serum (100 μl) was added
The initial population for LGA was 150 individuals, 2) the maximal to 150 μl of HHL (5 mM) in phosphate buffered saline (NaCl 300 mM) at
number of energy evaluation was 2.5 × 106 and the maximal number of pH 8.3. After 30 min of incubation with gentle horizontal shaking at
energy generation was 27000, 3) the rate of the gene mutations was 37 °C, the enzymatic reaction was terminated by adding 1N HCl
0.02 and the rate of the gene crossover was 0.8, 4) the selected number (0.25 ml). The end product of ACE action on HHL, hippuric acid, was
for docking rounds was 50, and, 5) free binding energy and the value of then extracted from acidified solution into 1.5 ml of ethyl acetate by
inhibition constant (Ki) for each ligand were used to compare the ability vortex mixing. Following a brief centrifugation, 1 ml aliquots of each
of these ligands to interact with ACE. ethyl acetate layer was then transferred to a clean tube and heated for

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M. Abouelkheir and T.H. El-Metwally European Journal of Pharmacology 862 (2019) 172638

30 min at 120 °C. Hippuric acid was re-dissolved in distilled water that form S1 pocket (Ala354, Glu384, Tyr523) and S2 pocket (Glu281,
(1 ml) and the amount formed was determined from its absorbance at His353, Lys511, His513) of ACE. Some DPP-4 inhibitors also interacted
228 nm. ACE activity was expressed as a percentage in comparison to with S1‵ pocket (Glu162) and other amino acids that form the phar-
the mean of the baseline ACE activity in the control group. macophore of ACE. Of note, linagliptin directly interacted with the zinc
atom (Figs. 1–4).
2.2.2. Assessment of DPP-4 activity Regarding ACEIs, results of the docking calculations of different
As previously described (Villhauer et al., 2003), the activity of DPP- ACEIs into both DPP-4 and ACE were presented in Table 2. Unlike the
4 was determined by the cleavage rate of 7-amino-4-methylcoumarin docking results of DPP-4 inhibitors, the ability of ACEIs to occupy the
(AMC) from the synthetic substrate H-glycyl-prolyl-AMC (Gly-Pro-AMC; active center of DPP-4 showed a wide variation. For captopril, fosino-
Sigma). In brief, 5 μl of rat serum was mixed with 35 μl assay buffer prilat, perindoprilat ramiprilat and zofenoprilat, it seems that it is un-
[25 mmol/l HEPES, 140 mmol/l NaCl, 80 mmol/l MgCl2, 1% (w/v) likely for these drugs to inhibit DPP-4 when used in therapeutic con-
BSA; pH 7.8]. At room temperature, the mixture was incubated for centrations. Binding of these drugs to the active site of DPP-4 occur
5 min and the reaction was initiated by adding 40 μl of the assay buffer with a relatively low binding energy and at millimolar concentrations
containing 0.1 mmol/l substrate Gly-Pro-AMC. Using a spectro- in comparison to micro- or nanomolar concentration for ACE inhibition.
fluorometer, fluorescence of the liberated AMC was continuously Captopril formed three hydrogen bonds with key amino acids in DPP-4
monitored at excitation 380 nm and emission 460 nm every 3 min for (Fig. 5A). The binding energy for interaction of enalaprilat with ACE
up to 18 min in a 96-well plate. DPP-4 activity was expressed as a was −7.12 kcal/mol, which was relatively far from that required for
percentage in comparison to the mean of the baseline DPP-4 activity in interaction with DPP-4 (−5.03 kcal/mol). Of note, enalaprilat could
the control group. block the entrance of DPP-4 at a higher energy (−6.21 kcal/mol). Be-
nazeprilat and, to lesser extent, quinaprilat were apparently the most
promising among ACEIs that demonstrated DPP-4 inhibitory activity.
2.2.3. Assessment of angiotensin II and active GLP-1
They interacted with ACE at binding energies which were so close to
Blood samples intended for assay of the active GLP-1 and angio-
that of DPP-4 binding. Enalaprilat formed 2 hydrogen bonds while each
tensin II (Ang II) were collected in tubes containing EDTA and the DPP-
of quinaprilat and benazeprilat were able to form only one hydrogen
4 inhibitor valine pyrrolidide (Linco Research, USA). Plasma was then
bond with some of the key amino acids of DPP-4. In addition, they bind
separated by centrifugation. Specific ELISA kits were used to determine
to amino acids from S1 pocket catalytic triad (His740, Asn710) and S2
serum levels of Ang II (Glory Science, China) and active GLP-1
pocket (Arg125, Glu206, Glu205) of DPP-4 using other bonds
(SinoGeneClon Biotech, China) according to the manufacturers’ in-
(Fig. 5B–D).
structions.
The results of the in vivo study were somewhat different from the in
silico screening. Testing different drugs against ACE revealed that small
2.3. Statistical analysis doses of either linagliptin or sitagliptin were able to produce mild re-
duction in serum ACE activity (P < 0.01; No. of animals in each
SPSS version 21 was used for data analysis. Data was expressed as group = 10) but without significant effects on plasma angiotensin II
Mean ± S.D. Kolmogorov-Smirnov test was used to test the normal levels. Higher dose of either drugs were able to significantly inhibit ACE
distribution of variables. Analysis of variance (ANOVA) followed by activity together with a reduction in angiotensin II levels (P < 0.01;
Bonferroni test were used to compare between groups. A P value of less No. of animals in each group = 10). In both dosage ranges, neither drug
than 0.05 was considered statistically significant. could attain the same ACE inhibition as captopril or enalapril. In
comparison to linagliptin, higher doses of sitagliptin produced more
3. Results reduction of ACE activity and angiotensin II levels (P < 0.01 for either
parameter; Fig. 6).
The results of the docking calculations of different DPP-4 inhibitors On the other side, the results of testing different drugs against DPP-4
into both DPP-4 and ACE are presented in Table 1. Interestingly, the in vivo were consistent with the in silico results. In comparison to li-
predicted Ki value for interaction of vildagliptin with ACE (34.86 nM) nagliptin and sitagliptin, neither captopril nor enalapril was able to
was even lower than with DPP-4 (62.22 nM). The same applies for the inhibit serum DPP-4 activity. The only exception is high dose of en-
binding energy, which was better when vildagliptin was tested against alapril that slightly reduced DPP-4 activity at 10 and 14 days
ACE than DPP-4. Although linagliptin scores for inhibition of DPP-4 and (P = 0.011 and < 0.01 respectively; No. of animals in each
ACE were close, linagliptin could block the inlet of ACE at a better group = 10) without significantly affecting the level of active GLP-1 or
binding energy. However, the significance of such action in a real in- OGTT results. For comparison, either doses of linagliptin and sitagliptin
teraction is unknown. For sitagliptin, binding energy required for in- effectively inhibited DPP-4 activity. They prevented the degradation of
teraction with ACE (−8.22 kcal/mol) was close to that with DPP-4 plasma active GLP-1 mainly at 10 and 30 min after OGTT (P < 0.01).
(−8.9 kcal/mol). Except linagliptin, all tested DPP-4 inhibitors variably None of the tested drugs significantly affected blood glucose measures
interacted, via hydrogen bonds, with some of the important amino acids

Table 1
Estimated binding energy, inhibition constant (Ki) and ligand efficiency obtained by docking of different DPP-4 inhibitors into DPP-4 and ACE. Results of docking
ramiprilat into ACE were provided for comparison.
Drug Enzyme Binding energy (kcal/mol) Ligand efficiency Inhibition constant (Ki) Number of conventionalH bonds with ACE Remarks

Linagliptin DPP4 −11.29 −0.32 5.31 nM

ACE −11.2 −0.32 6.16 nM 0 Block inlet −11.73, 2.51 nM
Sitagliptin DPP4 −8.9 −0.32 300.51 nM
ACE −8.22 −0.29 937.12 nM 5
Vildagliptin DPP4 −9.83 −0.45 62.22 nM
ACE −10.17 −0.46 34.86 nM 4
Anagliptin DPP4 −8.54 −0.31 547 nM Also −7.89, 1.65 μM
ACE −8.16 −0.29 1.04 μM 6 Block inlet −9.11, 209.12 nm
Ramiprilat ACE −9.53 −0.34 102.84 nM

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M. Abouelkheir and T.H. El-Metwally European Journal of Pharmacology 862 (2019) 172638

Fig. 1. Docking of linagliptin (yellow) into ACE showing linagliptin occupying the active site of the enzyme. The Zn atom of the active site is represented in light
green label. At binding energy of −11.2 kcal/mol, linagliptin was able to form variable bonds with some key amino acids from S1 pocket (Tyr523, Glu384, Ala354),
S2 pocket (His353), and S1‵ pocket (Glu162) in addition to other amino acids. Linagliptin was the only tested DPP-4 inhibitor that actively bound Zn atom of ACE.

Fig. 2. Docking sitagliptin (yellow) into ACE showing sitagliptin occupying the active site of the enzyme. At a relatively high binding energy, sitagliptin was able to
form hydrogen bond with some amino acids from S2 pocket (Lys511, Glu281) in addition to other amino acids. Using other types of bonds, sitagliptin was able to
bind to other key amino acids in S2 and S1‵ pockets. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of
this article.)

Fig. 3. Docking of vildagliptin (yellow) into ACE showing vildagliptin occupying the active site of the enzyme. At binding energy of −10.17 kcal/mol, vildagliptin
was able to bind, using hydrogen bond, to some amino acids from S1 pocket (Ala354), S2 pocket (His353, His513) in addition to other amino acids. Sitagliptin forms
other types of bonds with other pharmacophore amino acids including those binding Zn atom (His383, Glu411).

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M. Abouelkheir and T.H. El-Metwally European Journal of Pharmacology 862 (2019) 172638

Fig. 4. Docking of anagliptin (yellow) into ACE showing the drug occupying the active site of the enzyme. At binding energy of −8.16 kcal/mol, anagliptin actively
bound to some amino acids from S1 pocket (Tyr523, Glu384, Ala354), S2 pocket (His353, Glu281) in addition to other amino acids including those binding Zn atom
(His383, His387, Glu411).

Table 2
Estimated binding energy, inhibition constant (Ki) and ligand efficiency obtained by docking of different ACE inhibtors into DPP4 and ACE.
Drug Enzyme Binding energy (kcal/mol) Ligand efficiency Inhibition constant (Ki) Remarks

Benazeprilat DPP4 −6.95 −0.24 8.07 μM Molecule twisted on itself

ACE −7.62 −0.26 2.59 μM
Captopril DPP4 −3.93 −0.28 1.32 mM
ACE −7.14 −0.51 5.82 μM
Enalaprilat DPP4 −5.03 −0.2 205.05 μM Blocks the main inlet −6.21, 28.14 μM
ACE −7.12 −0.28 6 μM Position 1 blocks the inlet −7.99, 1.39 μM
Fosinoprilat DPP4 – – – Block the main inlet −6.68, 12.69 μM
ACE −9.02 −0.3 246.18 nM
Perindoprilat DPP4 – – – In mM
ACE −5.84 −0.24 52.71 μM Blocks the inlet −7.31, 4.4 μM
Quinaprilat DPP4 −6.24 −0.21 26.54 μM
ACE −7.64 −0.25 2.5 μM
Ramiprilat DPP4 – – – In mM
ACE −9.53 −0.34 102.84 nM
Zofenoprilat DPP4 – – – In mM
ACE −6.43 −0.31 19.44 μM Also −6.51, 16.8 μM
Lisinopril The number of rotatable bonds exceeds the capacity of the program

or AUC during OGTT (Fig. 7). hypothesized that existing ACEIs or DPP-4 inhibitors could act as a dual
inhibitor of both enzymes.
In the present study, we have demonstrated that many DPP-4 in-
4. Discussion
hibitors have potential inhibitory activity on ACE in concentrations
close to those required for DPP-4 inhibition. The active site of ACE has
The ability of the anti-hypertensive drugs, ACEIs, to improve insulin
three pockets: S1, S2, and S1‵. S1 pocket includes Ala354, Glu384 and
sensitivity and reduce the development of new-onset type 2 diabetes is
Tyr523 residues, and S2 pocket includes Glu281, His353, Lys511,
well established (Favre et al., 2015). Similarly, the ability of some
His513 and Tyr520 residues, while S1‵ is formed with Glu162 residue
members of the anti-diabetic drugs, DPP-4 inhibitors, to produce car-
(Wu et al., 2015). Among different bonds and interactions, hydrogen
diovascular and renal benefits has been investigated (Herzlinger and
bonds interaction forces appear to have the most important role in
Horton, 2013; Papagianni and Tziomalos, 2015). Activation of GLP-1
stabilizing the docking complex and affecting the enzyme catalytic re-
receptors only provided partial explanation of the cardio-renal benefits
actions (Chaudhary et al., 2009; Girgih et al., 2014; Li et al., 2014). All
of DPP-4 inhibitors. There was always a suggestion that, in comparison
tested DPP-4 inhibitors established up to six conventional hydrogen
to GLP-1 receptor agonist, DPP-4 inhibitors have extra-mechanism(s)
bonds with amino acids in the three pockets except linagliptin. Still,
(Ban et al., 2008; Shah et al., 2011). Many of the suggested mechanisms
linagliptin actively bound to all amino acids from S1 pocket (Tyr523,
did not explain the rapid, reversible hypotensive effects reported with
Glu384, Ala354), one amino acid from S2 pocket (His353) and S1‵
some, but not all, DPP-4 inhibitors (Fadini and Avogaro, 2011; Groop
pocket (Glu162) using seven non-conventional hydrogen bonds; all are
et al., 2013; Lovshin and Zinman, 2014; Mistry et al., 2008; Ogawa
less than 3.5Å. The binding energy and Ki of linagliptin-ACE interaction
et al., 2011; Pacheco et al., 2011). Moreover, not all DPP-4 inhibitors
was so close to those of DPP-4 interaction. Another striking feature for
equally aggravated ACEIs-induced acute renal failure (Kutoh, 2012;
linagliptin is that it is the only DPP-4 inhibitor that interacted with zinc
Nandikanti et al., 2016); an incident awaiting further explanation. In a
(2.51Å) atom and the three amino acids attached to the zinc atom
different path, similarities between ACE and DPP-4 at the molecular
(Fig. 1). Zinc at the ACE active site coordinates with three ACE residues
level stimulated the efforts to develop a dual inhibitor for both enzymes
(His383, His387, Glu411) and is crucial for the enzyme activity (Jalkute
in order to provide a single useful drug for both of hypertension and
et al., 2013). The ability of an inhibitor to directly interact with the zinc
diabetes (Sattigeri et al., 2017). Instead of developing a new drug, we

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M. Abouelkheir and T.H. El-Metwally European Journal of Pharmacology 862 (2019) 172638

Fig. 5. Docking of ACE inhibitors captopril (A),

enalaprilat (B), quinaprilat (C) and benazeprilat (D)
into DPP-4. Except for benazeprliat, most of ACE
inhibitors require binding energy score to occupy
the active site on DPP-4 which were relatively far
from those of ACE. Enalaprilat was able to form two
hydrogen bonds with amino acids forming the S2
pocket of DPP-4. Quinaprilat was able to bind to
some key amino acids from S1 pocket (His740) and
S2 pocket (Arg125, Glu206); only one is hydrogen
bond. At a higher binding energy, benazeprilat
bound to some key amino acids from S1 pocket
(Asn710) and S2 pocket (Arg125, Glu205, Glu206)
with only one hydrogen bond with Asn710.

Fig. 6. Comparing the effect of different drugs on

serum ACE activity (A) and plasma angiotesin II
levels (B). High doses (days 8 and 10) of tested DPP-
4 inhibitors were more effective than low doses (day
1) in producing mild reduction of serum ACE ac-
tivity (P < 0.01). Only high doses of DPP-4 in-
hibitors could interfere with ACE activity to levels
enough to reduce plasma angiotesin II levels
(P < 0.01). Neither drug could attain the same
ACE inhibition as captopril or enalapril but si-
tagliptin was more effective than linagliptin. Data is
expressed as Mean ± S.D.; * significant from con-
trol, # significant from captopril, ◊ significant from
enalapril, ○ significant from linagliptin. No. of ani-
mals in each group = 10.

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M. Abouelkheir and T.H. El-Metwally European Journal of Pharmacology 862 (2019) 172638

Fig. 7. Comparing the effect of different drugs on serum DPP-4 activity (A), plasma active GLP-1 levels (B), blood glucose after OGTT (C) and area under the curve
(AUC) of glucose tolerance (D). Linagliptin and sitagliptin effectively inhibited DPP-4 activity (P < 0.01) and higher doses (days 10 and 14) of either drugs
prevented the degradation of active GLP-1 (P < 0.01). While high dose of enalapril significantly inhibited DPP-4 activity at 10 (P = 0.011) and 14 (P < 0.01) days,
neither captopril nor enalapril were able to produce significant inhibition DPP-4 to a level that could prevent the degradation of active GLP-1. None of the tested
drugs significantly affected blood glucose measures or AUC during OGTT although linagliptin and sitagliptin had apparently lower AUC after glucose challenge. Data
is expressed as Mean ± S.D.; * significant from control, # significant from captopril, ◊ significant from enalapril, ○ significant from linagliptin. No. of animals in each
group = 10.

atom of ACE can augment the ACE inhibitory activity of such inhibitor, regulation (Schoolwerth et al., 2001). However, this was not the si-
as reported with lisinopril (Jimsheena and Gowda, 2010; Pan et al., tuation in an earlier case report where a patient with mild renal im-
2011). These results suggest that the DPP-4 inhibitors, including li- pairment, suffered from transient AKI when linagliptin was added to
nagliptin, could effectively bind to the active site of ACE and interfere ACEI (Kutoh, 2012). The patient was originally on sitagliptin and ACEI
with its activity. but AKI only developed when sitagliptin was substituted with li-
In animal study, we decided to compare linagliptin, which interacts nagliptin. In addition, the patient was not dehydrated so that the
with zinc atom of ACE, to sitagliptin that binds to ACE with 5 hydrogen diuretic theory linagliptin is not applicable. In fact, sitagliptin have
bonds. At the tested doses, sitagliptin was superior to linagliptin in been also reported to have such diuretic effect (Girardi et al., 2008;
inhibiting plasma ACE activity and reducing angiotensin II levels. Pacheco et al., 2011) suggesting that diuretic effect alone cannot ex-
Although we did not measure if sitagliptin reduced blood pressure, our plain the resultant AKI. These case reports might support our theory
results could explain the blood pressure lowering effect of sitagliptin that linagliptin is a potential inhibitor of ACE. Of note, longer-acting
that was reported in doses of 40 mg/kg/day (Pacheco et al., 2011). It ACEIs could cause higher incidences of renal function deterioration due
can also explain why sitagliptin augmented the hypotensive effect of to more complete or sustained ACE inhibition (Mason, 1990). In both
5 mg dose of enalapril (Marney et al., 2010). It was found that treat- case reports, the used ACEI was long acting, the half life of linagliptin is
ment with sitagliptin could reduce blood pressure; a benefit which was around 100 h, and that of sitagliptin is only 12.4 h (Linaglitpin pre-
lost when sitagliptin was replaced with linagliptin (Tojikubo and Tajiri, scribing information, 2019Sitagliptin prescribing information, 2019).
2017). Beside case reports, many striking features of linagliptin could
Still, clinical case reports (Kutoh, 2012; Nandikanti et al., 2016) and support our assumption that linagliptin could affect ACE activity. First,
other observations could support the notion that linagliptin might ad- linagliptin is probably the best among DPP-4 inhibitors in terms of re-
ditionally inhibit, at least, renal ACE. Due to minimal contribution of noprotective effects (Kanasaki, 2018). Second, whole body auto-
the kidney in eliminating linagliptin, the drug is considered safe in radiography to study tissue distribution of radio-labeled linagliptin re-
kidney impairment (Linaglitpin prescribing information, 2019). In one vealed that its concentration in the kidney exceeds that of the plasma by
study, using linagliptin in patients with chronic kidney disease did not several hundred times. Even in the absence of DPP-4, renal con-
cause significant rise of acute kidney injury (AKI) incidence (McGill centration of linagliptin was several times higher than the plasma
et al., 2013). However, the study did not include data about the use of (Fuchs et al., 2009). So, even if linagliptin produced mild inhibition of
ACEIs or angiotensin receptor blockers in the selected patients. In one plasma ACE, it might significantly inhibit renal ACE. In addition, we
case report (Nandikanti et al., 2016), it was reported that reduction of found that linagliptin was the only DPP-4 inhibitor that directly inter-
blood pressure, hyperkalemia and AKI rapidly developed after li- acts with zinc atom of ACE. It is interesting to know that binding of the
nagliptin was added on top of lisinopril in a patient with chronic kidney ACEIs to zinc atom significantly enhance “tissue” ACE inhibitory effect
disease. Considering some weight loss, the authors attributed AKI to (Lazar, 2005). Linagliptin should not be overlooked in searching for the
renal hypo-perfusion secondary to linagliptin-induced natriuresis optimal inhibitor of “renal” ACE.
(Girardi et al., 2008; Pacheco et al., 2011) and suggested that the role of On the other side, it does not seem that most of ACEIs could interact
lisinopril was augmentation of AKI by impairing the kidney auto- with DPP-4. Only benazeprilat, and to lesser extent enalaprilat and

7
M. Abouelkheir and T.H. El-Metwally European Journal of Pharmacology 862 (2019) 172638

quinaprilat, could apparently interact with DPP-4 at binding energies enzyme in complex with a selenium analogue of captopril. FEBS J. 278, 3644–3650.
relatively close to those of ACE interactions. The active site of DPP-4 is http://doi.org/10.1111/j.1742-4658.2011.08276.x.
Ban, K., Noyan-Ashraf, M.H., Hoefer, J., Bolz, S.S., Drucker, D.J., Husain, M., 2008.
formed of key amino acids from S1 pocket catalytic triad (Ser630, Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are
Asn710 and His740) and those from S2 pocket (Glu205 and Glu206 mediated through both glucagon-like peptide 1 receptor-dependent and -independent
dyad and Arg125). Other amino acids that surround the cavity of S2 are pathways. Circulation 117, 2340–2350. http://doi.org/10.1161/CIRCULATIONAHA.
107.739938.
Val207, Ser209, Arg358 and Phe357 (Kuhn et al., 2007). Although Peptidyl-dipeptidase A (EC-Number 3.4.15.1).
different ACEIs were able to form hydrogen bonds with critical amino Dipeptidyl-peptidase IV (EC-Number 3.4.14.5).
acids in either S1 or S2 pockets of DPP-4, the binding energy and Ki Brown, N.J., Byiers, S., Carr, D., Maldonado, M., Warner, B.A., 2009. Dipeptidyl pepti-
dase-IV inhibitor use associated with increased risk of ACE inhibitor-associated an-
were relatively far from those required for ACE interaction. It seems gioedema. Hypertension 54, 516–523. http://doi.org/10.1161/
that higher doses of ACEIs are required to achieve DPP-4 inhibition. HYPERTENSIONAHA.109.134197.
Our animal study results demonstrated that only high doses of enalapril Chaudhary, S., Vats, I.D., Chopra, M., Biswas, P., Pasha, S., 2009. Effect of varying chain
length between P(1) and P(1') position of tripeptidomimics on activity of angiotensin-
were able to slightly inhibit DPP-4 but without preventing the de-
converting enzyme inhibitors. Bioorg. Med. Chem. Lett 19, 4364–4366. http://doi.
gradation of GLP-1 in rats; an effect that is unlikely to be achieved when org/10.1016/j.bmcl.2009.05.079.
therapeutic doses are to be used in the clinical settings. Unfortunately, Cushman, D.W., Cheung, H.S., 1971. Spectrophotometric assay and properties of the
we could not obtain benazepril or quinapril to test them in the animal angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol. 20, 1637–1648.
DREAM Trial Investigators, Bosch, J., Yusuf, S., Gerstein, H.C., Pogue, J., Sheridan, P.,
study. Overall, it does not seem that the metabolic benefits of ACEIs Dagenais, G., Diaz, R., Avezum, A., Lanas, F., Probstfield, J., Fodor, G., Holman, R.R.,
could be attributed to inhibition of DPP-4. Our results explained why a 2006. Effect of ramipril on the incidence of diabetes. N. Engl. J. Med. 355,
lot of modification was required when an ACEI was selected to start 1551–1562. http://doi.org/10.1056/NEJMoa065061.
Endringer, D., Oliveira, O., Braga, F., 2014. In Vitro and in Silico Inhibition of
with in developing dual inhibitor for both ACE and DPP-4 (Sattigeri Angiotensin-Converting Enzyme by Carbohydrates and Cyclitols, Chemical. Papers.
et al., 2017). pp. 37–45. https://doi.org/10.2478/s11696-013-0407-8.
Of note, the present study was conducted on normal non-diabetic Erbe, D.V., Gartrell, K., Zhang, Y.L., Suri, V., Kirincich, S.J., Will, S., Perreault, M., Wang,
S., Tobin, J.F., 2006. Molecular activation of PPARgamma by angiotensin II type 1-
non-hypertensive rats as it focused on screening dual enzyme inhibition receptor antagonists. Vasc. Pharmacol. 45, 154–162. http://doi.org/10.1016/j.vph.
of different ACEIs and DPP-4 inhibitors. Owing to the discrepancies 2006.05.002.
between in silico and in vivo results, other members of either drug Fadini, G.P., Avogaro, A., 2011. Cardiovascular effects of DPP-4 inhibition: beyond GLP-
1. Vasc. Pharmacol. 55, 10–16. http://doi.org/10.1016/j.vph.2011.05.001.
groups should be screened in animals. However, using normal rats Favre, G.A., Esnault, V.L., Van Obberghen, E., 2015. Modulation of glucose metabolism
could not evaluate the actual contribution of the suggested mechanism by the renin-angiotensin-aldosterone system. Am. J. Physiol. Endocrinol. Metab. 308,
in the cardiovascular/metabolic benefits of these drugs in disease E435–E449. http://doi.org/10.1152/ajpendo.00391.2014.
Fuchs, H., Binder, R., Greischel, A., 2009. Tissue distribution of the novel DPP-4 inhibitor
models. Depending on the successful drugs, further investigations re-
BI 1356 is dominated by saturable binding to its target in rats. Biopharm Drug Dispos.
quire the use of properly selected hypertensive or diabetic models. 30, 229–240. http://doi.org/10.1002/bdd.662.
Taking sitagliptin as example, we need to test it at least in two models Gillespie, E.L., White, C.M., Kardas, M., Lindberg, M., Coleman, C.I., 2005. The impact of
of hypertension as the contribution of RAS in the pathophysiology of ACE inhibitors or angiotensin II type 1 receptor blockers on the development of new-
onset type 2 diabetes. Diabetes Care 28, 2261–2266.
hypertension models in rats show significant variation (Pinto et al., Girardi, A.C., Fukuda, L.E., Rossoni, L.V., Malnic, G., Reboucas, N.A., 2008. Dipeptidyl
1998). peptidase IV inhibition downregulates Na+ - H+ exchanger NHE3 in rat renal
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1152/ajprenal.00174.2007.
5. Conclusion Girgih, A.T., He, R., Aluko, R.E., 2014. Kinetics and molecular docking studies of the
inhibitions of angiotensin converting enzyme and renin activities by hemp seed
While some DPP-4 inhibitors could inhibit ACE, it is unlikely that (Cannabis sativa L.) peptides. J. Agric. Food Chem. 62, 4135–4144. http://doi.org/
10.1021/jf5002606.
the reverse could happen. Final proof requires a well-designed clinical Groop, P.H., Cooper, M.E., Perkovic, V., Emser, A., Woerle, H.J., von Eynatten, M., 2013.
study to find out whether the results of the present study could be ex- Linagliptin lowers albuminuria on top of recommended standard treatment in pa-
trapolated in clinical setting. Providing evidence of such interaction tients with type 2 diabetes and renal dysfunction. Diabetes Care 36, 3460–3468.
http://doi.org/10.2337/dc13-0323.
could explain the cardio-renal benefits, as well as adverse effect, of
Herzlinger, S., Horton, E.S., 2013. Extraglycemic effects of glp-1-based therapeutics:
some DPP-4 inhibitors. It could change guidelines for safe and effective addressing metabolic and cardiovascular risks associated with type 2 diabetes.
use of individual DPP-4 inhibitors. Considering the interaction of DPP-4 Diabetes Res. Clin. Pract. 100, 1–10. http://doi.org/10.1016/j.diabres.2012.11.009.
Jalkute, C.B., Barage, S.H., Dhanavade, M.J., Sonawane, K.D., 2013. Molecular dynamics
inhibitors and ACE will allow proper selection of DPP-4 inhibitors for
simulation and molecular docking studies of Angiotensin converting enzyme with
diabetic, hypertensive and renal patient and save billions on the on- inhibitor lisinopril and amyloid Beta Peptide. Protein J. 32, 356–364. http://doi.org/
going attempts to develop a dual inhibitor of DPP-4 and ACE. 10.1007/s10930-013-9492-3.
Jimsheena, V.K., Gowda, L.R., 2010. Arachin derived peptides as selective angiotensin I-
converting enzyme (ACE) inhibitors: structure-activity relationship. Peptides 31,
Declaration of interest 1165–1176. http://doi.org/10.1016/j.peptides.2010.02.022.
Kanasaki, K., 2018. The role of renal dipeptidyl peptidase-4 in kidney disease: renal ef-
The authors declare no conflict of interest. fects of dipeptidyl peptidase-4 inhibitors with a focus on linagliptin. Clin. Sci. (Lond.)
132, 489–507. http://doi.org/10.1042/CS20180031.
Kuhn, B., Hennig, M., Mattei, P., 2007. Molecular recognition of ligands in dipeptidyl
Acknowledgement peptidase IV. Curr. Top. Med. Chem. 7, 609–619.
Kutoh, E., 2012. Potential linagliptin-induced renal impairment. JMC 3, 361–364.
Lazar, H.L., 2005. Role of angiotensin-converting enzyme inhibitors in the coronary ar-
This project was funded by the Deanship for Scientific Research, tery bypass patient. Ann. Thorac. Surg. 79, 1081–1089. http://doi.org/10.1016/j.
Jouf University, Sakaka, Saudi Arabia (grant# 269/39). The authors athoracsur.2004.05.046.
greatly appreciate the limit-less help during the accomplishment of this Leiter, L.A., Lewanczuk, R.Z., 2005. Of the renin-angiotensin system and reactive oxygen
species Type 2 diabetes and angiotensin II inhibition. Am. J. Hypertens. 18, 121–128.
work from Medical Experimental Research Center (MERC) and Dr.
http://doi.org/10.1016/j.amjhyper.2004.07.001.
Samah Fouad, MERC, Faculty of Medicine, Mansoura University, Egypt. Li, P., Jia, J., Fang, M., Zhang, L., Guo, M., Xie, J., Xia, Y., Zhou, L., Wei, D., 2014. In vitro
and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of
identified peptide binding with ACE. Process Biochem. 49, 898–904. https://doi.org/
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