Dipeptidyl peptidase-4 inhibitors can inhibit angiotensin converting enzyme

European Journal of Pharmacology 862 (2019) 172638

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Dipeptidyl peptidase-4 inhibitors can inhibit angiotensin converting enzyme Mohamed Abouelkheir

a,b,∗

c,d

, Tarek H. El-Metwally

T

a

Department of Pharmacology and Therapeutics, College of Medicines, Jouf University, Sakaka, Saudi Arabia Pharmacology department, Faculty of Medicine, Mansoura University, Mansoura, Egypt Departments of Medical Biochemistry, Jouf University, Sakaka, Saudi Arabia d Faculty of Medicine, Assiut University, Assiut, Egypt b c

ARTICLE INFO

ABSTRACT

Keywords: Acute kidney injury Autodock Enalapril Hypotension Linagliptin Sitagliptin

Angiotensin-1 converting enzyme inhibitors (ACEIs) improve insulin sensitivity. Inhibitors of dipeptidyl peptidase-4 (DPP-4) are anti-diabetic drugs with several cardio-renal effects. Both ACE and DPP-4 share common features. Thus, we tested if they could be inhibited by one inhibitor. First, in silico screening was used to investigate the ability of different DPP-4 inhibitors or ACEIs to interact with DPP-4 and ACE. The results of screening were then extrapolated into animal study. Fifty Sprague Dawley rats were randomly assigned into 5 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 Angiotensin-1 converting enzyme inhibitors (ACEIs) are anti-hypertensive drugs with well-known ability to improve insulin sensitivity and reduce the development of new-onset type 2 diabetes (Favre et al., 2015; Gillespie et al., 2005; DREAM Trial Investigators et al., 2006; Paolisso et al., 1992). Various mechanisms have been proposed to explain the ability of renin-angiotensin system (RAS) blockade to improve insulin sensitivity (Leiter and Lewanczuk, 2005; Scheen, 2004; Tikellis et al., 2004; Ura et al., 1999). However, further studies did not support some of these mechanisms (Erbe et al., 2006; Muller-Fielitz et al., 2012). It seems that no mechanism stands alone to explain the positive effect of RAS blockade on insulin sensitivity. Dipeptidyl peptidase-4 (DPP-4) catabolizes the incretin, glucagonlike peptide-1 (GLP-1). Inhibitors of DPP-4 are used as anti-diabetic drugs (Meier et al., 2004). GLP-1, its analogues, and DPP-4 inhibitors have several cardiovascular benefits including reduction of blood pressure (Herzlinger and Horton, 2013; Papagianni and Tziomalos, 2015). These benefits are only partially explained by the resultant activation of GLP-1 receptors (Ban et al., 2008; Shah et al., 2011). The

∗

full-scale of mechanisms utilized for such blood pressure-lowering effects of DPP-4 inhibition encompasses improvement of endothelial function, increase nitric oxide, and change the balance of some of the biologically active peptides (Fadini and Avogaro, 2011; Lovshin and Zinman, 2014). However, none of these suggested mechanisms can explain the rapid, reversible hypotensive effects that have been reported with some DPP-4 inhibitors (Mistry et al., 2008). Moreover, these mechanisms did not explain why DPP-4 inhibitors are not equal regarding their blood pressure-lowering effects (Groop et al., 2013; Ogawa et al., 2011; Pacheco et al., 2011). Several observations suggest that RAS and GLP-1/DPP-4 could possibly share a common inhibitor. Beside the metabolic effects of ACEIs and the hypotensive effect of DPP-4 inhibitors, DPP-4 inhibitors aggravate ACEIs-induced angioedema (Brown et al., 2009). Similarly, linagliptin, a safe drug for patients with chronic renal impairment, can cause acute renal failure when added to treatment regimens of patients already treated with ACEIs (Kutoh, 2012; Nandikanti et al., 2016). At the molecular level, the activities of both DPP-4 and ACE are affected by the presence of the amino acid, proline, at the site of cleavage. For 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.

European Journal of Pharmacology 862 (2019) 172638

M. Abouelkheir and T.H. El-Metwally

some of DPP-4 inhibitors. Finally, both enzymes share many experimental inhibitors (Brenda Enzyme Database EC-Number 3.4.14.5; Brenda Enzyme Database EC-Number 3.4.15.1). The aim of the present study was to investigate whether DPP-4 inhibitors or ACEIs can simultaneously inhibit both DPP-4 and ACE. Providing evidence for such interaction can give a new explanation of the secondary actions produced by these drugs and discover crosstalk between medications used to treat hypertension and diabetes mellitus.

2.1.5. Docking into DPP-4 The same as with ACE, AutoGrid was used to create the 3D grid for DPP-4 and evaluate the binding energies between tested ligands and the enzyme. Regarding the used grid map, grid points were set as 126 × 126 × 126, grid point spacing as 0.400 Å, and central grid point of the map (47.633, 52.321, 27.307) was selected by default. Again, LGA was used (Morris et al., 1998) with the same parameters specified 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 Bioethics, Jouf University (approval number 2-2-4/40). Fifty Sprague Dawley female rats weighing 200–230 g (aging 8–9 weeks) were housed in an air-conditioned room on a light/dark cycle with free access to water and a standard pellet diet. After one week, animals were assigned into the following 5 groups (n = 10 in each): Group I: control group (received drug vehicle, saline, by gavage); Group II: received captopril (ACEI; Bristol Myers Squibb, USA), dissolved in drinking water; Group III: received enalapril (ACEI; Merck Sharp & Dohme, Italy); Group IV received linagliptin (DPP-4 inhibitor; Boehringer Ingelheim, USA) and Group V received sitagliptin (DPP-4 inhibitor; Merck Sharp & Dohme, Italy). Drugs in groups III-V were dissolved/suspended in saline and were given once daily by gavage. Treatment started with a low dose from each drug for one day (day 1). Following 6 days of drug-free interval and at day 8, a high dose was instituted daily for 7 days. Determination of “low” and “high” dose of each drug was after reviewing a large body of literatures. Low and high 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 enalapril, 3 and 10 mg/kg/day for linagliptin, 10 and 30 mg/kg/day for sitagliptin. Blood samples were obtained before treatment, and, at days 1, 8, 10 and 14 of therapy. Except for day 14, blood samples were collected aseptically, under isoflurane anesthesia, from the rat retro-orbital venous plexus at the specified time points 3 h after drug administration. By day 14 and under anesthesia, an indwelling catheter was inserted in the right jugular vein for blood sampling. An oral glucose tolerance test (OGTT) was performed after fasting for 8 h. The tested drugs were given 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 samples were collected just before administration of glucose and at 10, 30, 60, 120 min after oral glucose. The area under the glucose tolerance curve (AUC) was calculated using the trapezoidal rule. The blood samples collected before administration of glucose were used to assay DPP-4 enzymatic activity as well as basal glucose and GLP-1. Blood glucose was measured using blood glucose checker [GLUCOTREND®2 (Roche Group, UK)] and (AccuChek® Active) strips (Roche Group, UK). Blood samples intended for determination of the enzymatic activity were collected into dry tubes and centrifuged at 2500 g for 15 min at 4 °C. All samples were then stored at −80 °C until assy.

AutoDock 4.2.6 software (Morris et al., 1998) was used to dock the structures of different members of ACEIs and DPP-4 inhibitors into both ACE and DPP-4. Using AutoDock Tools (ADT), different ligands and enzymes were initially prepared. Discovery studio (2017 R2 client) was used to generate final figures. The tested DPP-4 inhibitors were linagliptin, sitagliptin, vildagliptin and anagliptin. The tested ACEIs were captopril, ramiprilat, enalaprilat, perindoprilat, benazeprilat, fosinoprilat, quinaprilat and zofenoprilat. Lisinopril was not tested because the number of rotatable bonds exceeds the capacity of the docking program. 2.1.1. Preparation of ligands All the ligands were obtained from Pubchem in SDF format, which was then converted into Mol2 format using Open Babel 2.3.1. All hydrogen atoms were added using ADT, which was also used to define torsion angles for each ligand. For better representation of ligand-enzyme interaction, ligands were considered flexible during docking. 2.1.2. Preparation of ACE We followed a previously described method (Endringer et al., 2014). The crystal structure of human testis ACE was downloaded from the Protein Data Bank (PDB code 2YDM) (Akif et al., 2011). With the exception of zinc and chloride ions, the ligand, water molecules, and other heteroatoms were completely removed using ADT. Formal charges and Van der Waals parameters for zinc and chloride ions were obtained from the AMBER database. The structure of the enzyme was set as rigid. Hydrogen atoms were added then all non-polar hydrogens were merged. 2.1.3. Preparation of DPP-4 The crystal structure of human DPP-4 was obtained from PDB (PDB code 3WQH) (Watanabe et al., 2015). The ligand, water molecules, and other heteroatoms were also removed using ADT. The structure of the enzyme was set as rigid. Hydrogen atoms were added then all non-polar hydrogens were merged. 2.1.4. Docking into ACE AutoGrid was used to create the 3D grid and evaluate the binding energies between different ligands and the enzyme. Regarding the used 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 the central grid point of the map. In order to model the binding between different ligands and ACE, Lamarckian genetic algorithm (LGA) was used (Morris et al., 1998) with the following specified parameters: 1) The initial population for LGA was 150 individuals, 2) the maximal number of energy evaluation was 2.5 × 106 and the maximal number of energy generation was 27000, 3) the rate of the gene mutations was 0.02 and the rate of the gene crossover was 0.8, 4) the selected number for docking rounds was 50, and, 5) free binding energy and the value of inhibition constant (Ki) for each ligand were used to compare the ability of these ligands to interact with ACE.

2.2.1. Assessment of ACE activity Serum ACE activity was measured according to the method described by Cushman and Cheung (1971). Hippuryl-His-Leu (HHL; Sigma) was used as a synthetic substrate. Rat serum (100 μl) was added to 150 μl of HHL (5 mM) in phosphate buffered saline (NaCl 300 mM) at pH 8.3. After 30 min of incubation with gentle horizontal shaking at 37 °C, the enzymatic reaction was terminated by adding 1N HCl (0.25 ml). The end product of ACE action on HHL, hippuric acid, was then extracted from acidified solution into 1.5 ml of ethyl acetate by vortex mixing. Following a brief centrifugation, 1 ml aliquots of each ethyl acetate layer was then transferred to a clean tube and heated for 2

European Journal of Pharmacology 862 (2019) 172638

M. Abouelkheir and T.H. El-Metwally

30 min at 120 °C. Hippuric acid was re-dissolved in distilled water (1 ml) and the amount formed was determined from its absorbance at 228 nm. ACE activity was expressed as a percentage in comparison to the mean of the baseline ACE activity in the control group.

that form S1 pocket (Ala354, Glu384, Tyr523) and S2 pocket (Glu281, His353, Lys511, His513) of ACE. Some DPP-4 inhibitors also interacted with S1‵ pocket (Glu162) and other amino acids that form the pharmacophore of ACE. Of note, linagliptin directly interacted with the zinc atom (Figs. 1–4). Regarding ACEIs, results of the docking calculations of different ACEIs into both DPP-4 and ACE were presented in Table 2. Unlike the docking results of DPP-4 inhibitors, the ability of ACEIs to occupy the active center of DPP-4 showed a wide variation. For captopril, fosinoprilat, perindoprilat ramiprilat and zofenoprilat, it seems that it is unlikely for these drugs to inhibit DPP-4 when used in therapeutic concentrations. Binding of these drugs to the active site of DPP-4 occur with a relatively low binding energy and at millimolar concentrations in comparison to micro- or nanomolar concentration for ACE inhibition. Captopril formed three hydrogen bonds with key amino acids in DPP-4 (Fig. 5A). The binding energy for interaction of enalaprilat with ACE was −7.12 kcal/mol, which was relatively far from that required for interaction with DPP-4 (−5.03 kcal/mol). Of note, enalaprilat could block the entrance of DPP-4 at a higher energy (−6.21 kcal/mol). Benazeprilat and, to lesser extent, quinaprilat were apparently the most promising among ACEIs that demonstrated DPP-4 inhibitory activity. They interacted with ACE at binding energies which were so close to that of DPP-4 binding. Enalaprilat formed 2 hydrogen bonds while each of quinaprilat and benazeprilat were able to form only one hydrogen bond with some of the key amino acids of DPP-4. In addition, they bind to amino acids from S1 pocket catalytic triad (His740, Asn710) and S2 pocket (Arg125, Glu206, Glu205) of DPP-4 using other bonds (Fig. 5B–D). The results of the in vivo study were somewhat different from the in silico screening. Testing different drugs against ACE revealed that small doses of either linagliptin or sitagliptin were able to produce mild reduction in serum ACE activity (P < 0.01; No. of animals in each group = 10) but without significant effects on plasma angiotensin II levels. Higher dose of either drugs were able to significantly inhibit ACE activity together with a reduction in angiotensin II levels (P < 0.01; No. of animals in each group = 10). In both dosage ranges, neither drug could attain the same ACE inhibition as captopril or enalapril. In comparison to linagliptin, higher doses of sitagliptin produced more reduction of ACE activity and angiotensin II levels (P < 0.01 for either parameter; Fig. 6). On the other side, the results of testing different drugs against DPP-4 in vivo were consistent with the in silico results. In comparison to linagliptin and sitagliptin, neither captopril nor enalapril was able to inhibit serum DPP-4 activity. The only exception is high dose of enalapril that slightly reduced DPP-4 activity at 10 and 14 days (P = 0.011 and < 0.01 respectively; No. of animals in each group = 10) without significantly affecting the level of active GLP-1 or OGTT results. For comparison, either doses of linagliptin and sitagliptin effectively inhibited DPP-4 activity. They prevented the degradation of plasma active GLP-1 mainly at 10 and 30 min after OGTT (P < 0.01). None of the tested drugs significantly affected blood glucose measures

2.2.2. Assessment of DPP-4 activity As previously described (Villhauer et al., 2003), the activity of DPP4 was determined by the cleavage rate of 7-amino-4-methylcoumarin (AMC) from the synthetic substrate H-glycyl-prolyl-AMC (Gly-Pro-AMC; Sigma). In brief, 5 μl of rat serum was mixed with 35 μl assay buffer [25 mmol/l HEPES, 140 mmol/l NaCl, 80 mmol/l MgCl2, 1% (w/v) BSA; pH 7.8]. At room temperature, the mixture was incubated for 5 min and the reaction was initiated by adding 40 μl of the assay buffer containing 0.1 mmol/l substrate Gly-Pro-AMC. Using a spectrofluorometer, fluorescence of the liberated AMC was continuously monitored at excitation 380 nm and emission 460 nm every 3 min for up to 18 min in a 96-well plate. DPP-4 activity was expressed as a percentage in comparison to the mean of the baseline DPP-4 activity in the control group. 2.2.3. Assessment of angiotensin II and active GLP-1 Blood samples intended for assay of the active GLP-1 and angiotensin II (Ang II) were collected in tubes containing EDTA and the DPP4 inhibitor valine pyrrolidide (Linco Research, USA). Plasma was then separated by centrifugation. Specific ELISA kits were used to determine serum levels of Ang II (Glory Science, China) and active GLP-1 (SinoGeneClon Biotech, China) according to the manufacturers’ instructions. 2.3. Statistical analysis SPSS version 21 was used for data analysis. Data was expressed as Mean ± S.D. Kolmogorov-Smirnov test was used to test the normal distribution of variables. Analysis of variance (ANOVA) followed by Bonferroni test were used to compare between groups. A P value of less than 0.05 was considered statistically significant. 3. Results The results of the docking calculations of different DPP-4 inhibitors into both DPP-4 and ACE are presented in Table 1. Interestingly, the predicted Ki value for interaction of vildagliptin with ACE (34.86 nM) was even lower than with DPP-4 (62.22 nM). The same applies for the binding energy, which was better when vildagliptin was tested against ACE than DPP-4. Although linagliptin scores for inhibition of DPP-4 and ACE were close, linagliptin could block the inlet of ACE at a better binding energy. However, the significance of such action in a real interaction is unknown. For sitagliptin, binding energy required for interaction with ACE (−8.22 kcal/mol) was close to that with DPP-4 (−8.9 kcal/mol). Except linagliptin, all tested DPP-4 inhibitors variably 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)

Linagliptin

DPP4 ACE DPP4 ACE DPP4 ACE DPP4 ACE ACE

−11.29 −11.2 −8.9 −8.22 −9.83 −10.17 −8.54 −8.16 −9.53

−0.32 −0.32 −0.32 −0.29 −0.45 −0.46 −0.31 −0.29 −0.34

5.31 nM 6.16 nM 300.51 nM 937.12 nM 62.22 nM 34.86 nM 547 nM 1.04 μM 102.84 nM

Sitagliptin Vildagliptin Anagliptin Ramiprilat

Number of conventionalH bonds with ACE

Remarks

0

Block inlet −11.73, 2.51 nM

5 4 6

3

Also −7.89, 1.65 μM Block inlet −9.11, 209.12 nm

European Journal of Pharmacology 862 (2019) 172638

M. Abouelkheir and T.H. El-Metwally

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). 4

European Journal of Pharmacology 862 (2019) 172638

M. Abouelkheir and T.H. El-Metwally

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 ACE DPP4 ACE DPP4 ACE DPP4 ACE DPP4 ACE DPP4 ACE DPP4 ACE DPP4 ACE

−6.95 −7.62 −3.93 −7.14 −5.03 −7.12 – −9.02 – −5.84 −6.24 −7.64 – −9.53 – −6.43

−0.24 −0.26 −0.28 −0.51 −0.2 −0.28 – −0.3 – −0.24 −0.21 −0.25 – −0.34 – −0.31

8.07 μM 2.59 μM 1.32 mM 5.82 μM 205.05 μM 6 μM – 246.18 nM – 52.71 μM 26.54 μM 2.5 μM – 102.84 nM – 19.44 μM

Molecule twisted on itself

Captopril Enalaprilat Fosinoprilat Perindoprilat Quinaprilat Ramiprilat Zofenoprilat Lisinopril

or AUC during OGTT (Fig. 7).

Blocks the main inlet −6.21, 28.14 μM Position 1 blocks the inlet −7.99, 1.39 μM Block the main inlet −6.68, 12.69 μM In mM Blocks the inlet −7.31, 4.4 μM In mM In mM Also −6.51, 16.8 μM The number of rotatable bonds exceeds the capacity of the program

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 inhibitors have potential inhibitory activity on ACE in concentrations close to those required for DPP-4 inhibition. The active site of ACE has three pockets: S1, S2, and S1‵. S1 pocket includes Ala354, Glu384 and Tyr523 residues, and S2 pocket includes Glu281, His353, Lys511, His513 and Tyr520 residues, while S1‵ is formed with Glu162 residue (Wu et al., 2015). Among different bonds and interactions, hydrogen bonds interaction forces appear to have the most important role in stabilizing the docking complex and affecting the enzyme catalytic reactions (Chaudhary et al., 2009; Girgih et al., 2014; Li et al., 2014). All tested DPP-4 inhibitors established up to six conventional hydrogen bonds with amino acids in the three pockets except linagliptin. Still, linagliptin actively bound to all amino acids from S1 pocket (Tyr523, Glu384, Ala354), one amino acid from S2 pocket (His353) and S1‵ pocket (Glu162) using seven non-conventional hydrogen bonds; all are less than 3.5Å. The binding energy and Ki of linagliptin-ACE interaction was so close to those of DPP-4 interaction. Another striking feature for linagliptin is that it is the only DPP-4 inhibitor that interacted with zinc (2.51Å) atom and the three amino acids attached to the zinc atom (Fig. 1). Zinc at the ACE active site coordinates with three ACE residues (His383, His387, Glu411) and is crucial for the enzyme activity (Jalkute et al., 2013). The ability of an inhibitor to directly interact with the zinc

4. Discussion The ability of the anti-hypertensive drugs, ACEIs, to improve insulin sensitivity and reduce the development of new-onset type 2 diabetes is well established (Favre et al., 2015). Similarly, the ability of some members of the anti-diabetic drugs, DPP-4 inhibitors, to produce cardiovascular and renal benefits has been investigated (Herzlinger and Horton, 2013; Papagianni and Tziomalos, 2015). Activation of GLP-1 receptors only provided partial explanation of the cardio-renal benefits of DPP-4 inhibitors. There was always a suggestion that, in comparison to GLP-1 receptor agonist, DPP-4 inhibitors have extra-mechanism(s) (Ban et al., 2008; Shah et al., 2011). Many of the suggested mechanisms did not explain the rapid, reversible hypotensive effects reported with some, but not all, DPP-4 inhibitors (Fadini and Avogaro, 2011; Groop et al., 2013; Lovshin and Zinman, 2014; Mistry et al., 2008; Ogawa et al., 2011; Pacheco et al., 2011). Moreover, not all DPP-4 inhibitors equally aggravated ACEIs-induced acute renal failure (Kutoh, 2012; Nandikanti et al., 2016); an incident awaiting further explanation. In a different path, similarities between ACE and DPP-4 at the molecular level stimulated the efforts to develop a dual inhibitor for both enzymes in order to provide a single useful drug for both of hypertension and diabetes (Sattigeri et al., 2017). Instead of developing a new drug, we 5

European Journal of Pharmacology 862 (2019) 172638

M. Abouelkheir and T.H. El-Metwally

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 DPP4 inhibitors were more effective than low doses (day 1) in producing mild reduction of serum ACE activity (P < 0.01). Only high doses of DPP-4 inhibitors 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 sitagliptin was more effective than linagliptin. 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.

6

European Journal of Pharmacology 862 (2019) 172638

M. Abouelkheir and T.H. El-Metwally

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.

regulation (Schoolwerth et al., 2001). However, this was not the situation in an earlier case report where a patient with mild renal impairment, suffered from transient AKI when linagliptin was added to ACEI (Kutoh, 2012). The patient was originally on sitagliptin and ACEI but AKI only developed when sitagliptin was substituted with linagliptin. In addition, the patient was not dehydrated so that the diuretic theory linagliptin is not applicable. In fact, sitagliptin have been also reported to have such diuretic effect (Girardi et al., 2008; Pacheco et al., 2011) suggesting that diuretic effect alone cannot explain the resultant AKI. These case reports might support our theory that linagliptin is a potential inhibitor of ACE. Of note, longer-acting ACEIs could cause higher incidences of renal function deterioration due to more complete or sustained ACE inhibition (Mason, 1990). In both case reports, the used ACEI was long acting, the half life of linagliptin is around 100 h, and that of sitagliptin is only 12.4 h (Linaglitpin prescribing information, 2019Sitagliptin prescribing information, 2019). Beside case reports, many striking features of linagliptin could support our assumption that linagliptin could affect ACE activity. First, linagliptin is probably the best among DPP-4 inhibitors in terms of renoprotective effects (Kanasaki, 2018). Second, whole body autoradiography to study tissue distribution of radio-labeled linagliptin revealed that its concentration in the kidney exceeds that of the plasma by several hundred times. Even in the absence of DPP-4, renal concentration of linagliptin was several times higher than the plasma (Fuchs et al., 2009). So, even if linagliptin produced mild inhibition of plasma ACE, it might significantly inhibit renal ACE. In addition, we found that linagliptin was the only DPP-4 inhibitor that directly interacts with zinc atom of ACE. It is interesting to know that binding of the ACEIs to zinc atom significantly enhance “tissue” ACE inhibitory effect (Lazar, 2005). Linagliptin should not be overlooked in searching for the optimal inhibitor of “renal” ACE. On the other side, it does not seem that most of ACEIs could interact with DPP-4. Only benazeprilat, and to lesser extent enalaprilat and

atom of ACE can augment the ACE inhibitory activity of such inhibitor, as reported with lisinopril (Jimsheena and Gowda, 2010; Pan et al., 2011). These results suggest that the DPP-4 inhibitors, including linagliptin, could effectively bind to the active site of ACE and interfere with its activity. In animal study, we decided to compare linagliptin, which interacts with zinc atom of ACE, to sitagliptin that binds to ACE with 5 hydrogen bonds. At the tested doses, sitagliptin was superior to linagliptin in inhibiting plasma ACE activity and reducing angiotensin II levels. Although we did not measure if sitagliptin reduced blood pressure, our results could explain the blood pressure lowering effect of sitagliptin that was reported in doses of 40 mg/kg/day (Pacheco et al., 2011). It can also explain why sitagliptin augmented the hypotensive effect of 5 mg dose of enalapril (Marney et al., 2010). It was found that treatment with sitagliptin could reduce blood pressure; a benefit which was lost when sitagliptin was replaced with linagliptin (Tojikubo and Tajiri, 2017). Still, clinical case reports (Kutoh, 2012; Nandikanti et al., 2016) and other observations could support the notion that linagliptin might additionally inhibit, at least, renal ACE. Due to minimal contribution of the kidney in eliminating linagliptin, the drug is considered safe in kidney impairment (Linaglitpin prescribing information, 2019). In one study, using linagliptin in patients with chronic kidney disease did not cause significant rise of acute kidney injury (AKI) incidence (McGill et al., 2013). However, the study did not include data about the use of ACEIs or angiotensin receptor blockers in the selected patients. In one case report (Nandikanti et al., 2016), it was reported that reduction of blood pressure, hyperkalemia and AKI rapidly developed after linagliptin was added on top of lisinopril in a patient with chronic kidney disease. Considering some weight loss, the authors attributed AKI to renal hypo-perfusion secondary to linagliptin-induced natriuresis (Girardi et al., 2008; Pacheco et al., 2011) and suggested that the role of lisinopril was augmentation of AKI by impairing the kidney auto7

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quinaprilat, could apparently interact with DPP-4 at binding energies relatively close to those of ACE interactions. The active site of DPP-4 is formed of key amino acids from S1 pocket catalytic triad (Ser630, Asn710 and His740) and those from S2 pocket (Glu205 and Glu206 dyad and Arg125). Other amino acids that surround the cavity of S2 are Val207, Ser209, Arg358 and Phe357 (Kuhn et al., 2007). Although different ACEIs were able to form hydrogen bonds with critical amino acids in either S1 or S2 pockets of DPP-4, the binding energy and Ki were relatively far from those required for ACE interaction. It seems that higher doses of ACEIs are required to achieve DPP-4 inhibition. Our animal study results demonstrated that only high doses of enalapril were able to slightly inhibit DPP-4 but without preventing the degradation of GLP-1 in rats; an effect that is unlikely to be achieved when therapeutic doses are to be used in the clinical settings. Unfortunately, we could not obtain benazepril or quinapril to test them in the animal study. Overall, it does not seem that the metabolic benefits of ACEIs could be attributed to inhibition of DPP-4. Our results explained why a lot of modification was required when an ACEI was selected to start with in developing dual inhibitor for both ACE and DPP-4 (Sattigeri et al., 2017). Of note, the present study was conducted on normal non-diabetic non-hypertensive rats as it focused on screening dual enzyme inhibition of different ACEIs and DPP-4 inhibitors. Owing to the discrepancies between in silico and in vivo results, other members of either drug groups should be screened in animals. However, using normal rats could not evaluate the actual contribution of the suggested mechanism in the cardiovascular/metabolic benefits of these drugs in disease models. Depending on the successful drugs, further investigations require the use of properly selected hypertensive or diabetic models. Taking sitagliptin as example, we need to test it at least in two models of hypertension as the contribution of RAS in the pathophysiology of hypertension models in rats show significant variation (Pinto et al., 1998).

enzyme in complex with a selenium analogue of captopril. FEBS J. 278, 3644–3650. 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. Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through both glucagon-like peptide 1 receptor-dependent and -independent pathways. Circulation 117, 2340–2350. http://doi.org/10.1161/CIRCULATIONAHA. 107.739938. Peptidyl-dipeptidase A (EC-Number 3.4.15.1). Dipeptidyl-peptidase IV (EC-Number 3.4.14.5). Brown, N.J., Byiers, S., Carr, D., Maldonado, M., Warner, B.A., 2009. Dipeptidyl peptidase-IV inhibitor use associated with increased risk of ACE inhibitor-associated angioedema. Hypertension 54, 516–523. http://doi.org/10.1161/ HYPERTENSIONAHA.109.134197. 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 angiotensinconverting enzyme inhibitors. Bioorg. Med. Chem. Lett 19, 4364–4366. http://doi. org/10.1016/j.bmcl.2009.05.079. Cushman, D.W., Cheung, H.S., 1971. Spectrophotometric assay and properties of the 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., Dagenais, G., Diaz, R., Avezum, A., Lanas, F., Probstfield, J., Fodor, G., Holman, R.R., 2006. Effect of ramipril on the incidence of diabetes. N. Engl. J. Med. 355, 1551–1562. http://doi.org/10.1056/NEJMoa065061. Endringer, D., Oliveira, O., Braga, F., 2014. In Vitro and in Silico Inhibition of Angiotensin-Converting Enzyme by Carbohydrates and Cyclitols, Chemical. Papers. pp. 37–45. https://doi.org/10.2478/s11696-013-0407-8. 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 1receptor antagonists. Vasc. Pharmacol. 45, 154–162. http://doi.org/10.1016/j.vph. 2006.05.002. Fadini, G.P., Avogaro, A., 2011. Cardiovascular effects of DPP-4 inhibition: beyond GLP1. Vasc. Pharmacol. 55, 10–16. http://doi.org/10.1016/j.vph.2011.05.001. Favre, G.A., Esnault, V.L., Van Obberghen, E., 2015. Modulation of glucose metabolism by the renin-angiotensin-aldosterone system. Am. J. Physiol. Endocrinol. Metab. 308, 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 BI 1356 is dominated by saturable binding to its target in rats. Biopharm Drug Dispos. 30, 229–240. http://doi.org/10.1002/bdd.662. Gillespie, E.L., White, C.M., Kardas, M., Lindberg, M., Coleman, C.I., 2005. The impact of ACE inhibitors or angiotensin II type 1 receptor blockers on the development of newonset type 2 diabetes. Diabetes Care 28, 2261–2266. Girardi, A.C., Fukuda, L.E., Rossoni, L.V., Malnic, G., Reboucas, N.A., 2008. Dipeptidyl peptidase IV inhibition downregulates Na+ - H+ exchanger NHE3 in rat renal proximal tubule. Am. J. Physiol. Renal. Physiol. 294, F414–F422. http://doi.org/10. 1152/ajprenal.00174.2007. 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 (Cannabis sativa L.) peptides. J. Agric. Food Chem. 62, 4135–4144. http://doi.org/ 10.1021/jf5002606. Groop, P.H., Cooper, M.E., Perkovic, V., Emser, A., Woerle, H.J., von Eynatten, M., 2013. Linagliptin lowers albuminuria on top of recommended standard treatment in patients with type 2 diabetes and renal dysfunction. Diabetes Care 36, 3460–3468. http://doi.org/10.2337/dc13-0323. Herzlinger, S., Horton, E.S., 2013. Extraglycemic effects of glp-1-based therapeutics: addressing metabolic and cardiovascular risks associated with type 2 diabetes. 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 simulation and molecular docking studies of Angiotensin converting enzyme with inhibitor lisinopril and amyloid Beta Peptide. Protein J. 32, 356–364. http://doi.org/ 10.1007/s10930-013-9492-3. Jimsheena, V.K., Gowda, L.R., 2010. Arachin derived peptides as selective angiotensin Iconverting enzyme (ACE) inhibitors: structure-activity relationship. Peptides 31, 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 effects 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 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 artery bypass patient. Ann. Thorac. Surg. 79, 1081–1089. http://doi.org/10.1016/j. athoracsur.2004.05.046. 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. http://doi.org/10.1016/j.amjhyper.2004.07.001. 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/ 10.1016/j.procbio.2014.02.007. Tradjenta® (Linaglitpin) prescribing information. Lovshin, J.A., Zinman, B., 2014. Blood pressure-lowering effects of incretin-based diabetes therapies. Can. J. Diabetes 38, 364–371. http://doi.org/10.1016/j.jcjd.2014. 05.001. Marney, A., Kunchakarra, S., Byrne, L., Brown, N.J., 2010. Interactive hemodynamic effects of dipeptidyl peptidase-IV inhibition and angiotensin-converting enzyme inhibition in humans. Hypertension 56, 728–733. http://doi.org/10.1161/

5. Conclusion While some DPP-4 inhibitors could inhibit ACE, it is unlikely that the reverse could happen. Final proof requires a well-designed clinical study to find out whether the results of the present study could be extrapolated in clinical setting. Providing evidence of such interaction could explain the cardio-renal benefits, as well as adverse effect, of some DPP-4 inhibitors. It could change guidelines for safe and effective use of individual DPP-4 inhibitors. Considering the interaction of DPP-4 inhibitors and ACE will allow proper selection of DPP-4 inhibitors for diabetic, hypertensive and renal patient and save billions on the ongoing attempts to develop a dual inhibitor of DPP-4 and ACE. Declaration of interest The authors declare no conflict of interest. Acknowledgement This project was funded by the Deanship for Scientific Research, Jouf University, Sakaka, Saudi Arabia (grant# 269/39). The authors greatly appreciate the limit-less help during the accomplishment of this work from Medical Experimental Research Center (MERC) and Dr. Samah Fouad, MERC, Faculty of Medicine, Mansoura University, Egypt. References Morris, G.M., Goodsell, D.S., Halliday, R.S., Huey, R., Hart, W.E., Belew, R.K., Olson, A.J., 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19, 1639–1662. https://doi.org/10. 1002/(SICI)1096-987X(19981115)19:14. Akif, M., Masuyer, G., Schwager, S.L., Bhuyan, B.J., Mugesh, G., Isaac, R.E., Sturrock, E.D., Acharya, K.R., 2011. Structural characterization of angiotensin I-converting

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M. Abouelkheir and T.H. El-Metwally HYPERTENSIONAHA.110.156554. Mason, N.A., 1990. Angiotensin-converting enzyme inhibitors and renal function. DICP 24, 496–505. McGill, J.B., Sloan, L., Newman, J., Patel, S., Sauce, C., von Eynatten, M., Woerle, H.J., 2013. Long-term efficacy and safety of linagliptin in patients with type 2 diabetes and severe renal impairment: a 1-year, randomized, double-blind, placebo-controlled study. Diabetes Care 36, 237–244. http://doi.org/10.2337/dc12-0706. Meier, J.J., Nauck, M.A., Kranz, D., Holst, J.J., Deacon, C.F., Gaeckler, D., Schmidt, W.E., Gallwitz, B., 2004. Secretion, degradation, and elimination of glucagon-like peptide 1 and gastric inhibitory polypeptide in patients with chronic renal insufficiency and healthy control subjects. Diabetes 53, 654–662. Mistry, G.C., Maes, A.L., Lasseter, K.C., Davies, M.J., Gottesdiener, K.M., Wagner, J.A., Herman, G.A., 2008. Effect of sitagliptin, a dipeptidyl peptidase-4 inhibitor, on blood pressure in nondiabetic patients with mild to moderate hypertension. J. Clin. Pharmacol. 48, 592–598. http://doi.org/10.1177/0091270008316885. Muller-Fielitz, H., Landolt, J., Heidbreder, M., Werth, S., Vogt, F.M., Johren, O., Raasch, W., 2012. Improved insulin sensitivity after long-term treatment with AT1 blockers is not associated with PPARgamma target gene regulation. Endocrinology 153, 1103–1115. http://doi.org/10.1210/en.2011-0183. Nandikanti, D.K., Gosmanova, E.O., Gosmanov, A.R., 2016. Acute kidney injury associated with linagliptin. Case. Rep. Endocrinol. 5695641. 2016. http://doi.org/10. 1155/2016/5695641. Ogawa, S., Ishiki, M., Nako, K., Okamura, M., Senda, M., Mori, T., Ito, S., 2011. Sitagliptin, a dipeptidyl peptidase-4 inhibitor, decreases systolic blood pressure in Japanese hypertensive patients with type 2 diabetes. Tohoku J. Exp. Med. 223, 133–135. Pacheco, B.P., Crajoinas, R.O., Couto, G.K., Davel, A.P., Lessa, L.M., Rossoni, L.V., Girardi, A.C., 2011. Dipeptidyl peptidase IV inhibition attenuates blood pressure rising in young spontaneously hypertensive rats. J. Hypertens. 29, 520–528. http://doi.org/ 10.1097/HJH.0b013e328341939d. Pan, D., Guo, H., Zhao, B., Cao, J., 2011. The molecular mechanisms of interactions between bioactive peptides and angiotensin-converting enzyme. Bioorg. Med. Chem. Lett 21, 3898–3904. http://doi.org/10.1016/j.bmcl.2011.05.033. Paolisso, G., Gambardella, A., Verza, M., D'Amore, A., Sgambato, S., Varricchio, M., 1992. ACE inhibition improves insulin-sensitivity in aged insulin-resistant hypertensive patients. J. Hum. Hypertens. 6, 175–179. Papagianni, M., Tziomalos, K., 2015. Cardiovascular effects of dipeptidyl peptidase-4 inhibitors. Hippokratia 19, 195–199. Pinto, Y.M., Paul, M., Ganten, D., 1998. Lessons from rat models of hypertension: from Goldblatt to genetic engineering. Cardiovasc. Res. 39, 77–88. https://doi.org/10. 1016/S0008-6363(98)00077-7. Sattigeri, J.A., Sethi, S., Davis, J.A., Ahmed, S., Rayasam, G.V., Jadhav, B.G., Chilla, S.M.,

Datta, D., Gadhave, A., Tulasi, V.K., Jain, T., Voleti, S., Benjamin, B., Udupa, S., Jain, G., Singh, Y., Srinivas, K., Bansal, V.S., Ray, A., Bhatnagar, P.K., Cliffe, I.A., 2017. Approaches towards the development of chimeric DPP4/ACE inhibitors for treating metabolic syndrome. Bioorg. Med. Chem. Lett 27, 2313–2318. http://doi.org/10. 1016/j.bmcl.2017.04.036. Scheen, A.J., 2004. Renin-angiotensin system inhibition prevents type 2 diabetes mellitus. Part 2. Overview of physiological and biochemical mechanisms. Diabetes Metab. 30, 498–505. Schoolwerth, A.C., Sica, D.A., Ballermann, B.J., Wilcox, C.S., Council on the Kidney in Cardiovascular, D., the Council for High Blood Pressure Research of the American Heart, A., 2001. Renal considerations in angiotensin converting enzyme inhibitor therapy: a statement for healthcare professionals from the council on the kidney in cardiovascular disease and the council for high blood pressure Research of the American heart association. Circulation 104, 1985–1991. Shah, Z., Pineda, C., Kampfrath, T., Maiseyeu, A., Ying, Z., Racoma, I., Deiuliis, J., Xu, X., Sun, Q., Moffatt-Bruce, S., Villamena, F., Rajagopalan, S., 2011. Acute DPP-4 inhibition modulates vascular tone through GLP-1 independent pathways. Vasc. Pharmacol. 55, 2–9. http://doi.org/10.1016/j.vph.2011.03.001. Januvia® (sitagliptin), prescribing information. Tikellis, C., Wookey, P.J., Candido, R., Andrikopoulos, S., Thomas, M.C., Cooper, M.E., 2004. Improved islet morphology after blockade of the renin- angiotensin system in the ZDF rat. Diabetes 53, 989–997. Tojikubo, M., Tajiri, Y., 2017. Different effects of linagliptin and sitagliptin on blood pressure and renal function in Japanese patients with type 2 diabetes mellitus. Diabetol. Int. 8, 397–401. http://doi.org/10.1007/s13340-017-0320-4. Ura, N., Higashiura, K., Shimamoto, K., 1999. The mechanisms of insulin sensitivity improving effects of angiotensin converting enzyme inhibitor. Immunopharmacology 44, 153–159. Villhauer, E.B., Brinkman, J.A., Naderi, G.B., Burkey, B.F., Dunning, B.E., Prasad, K., Mangold, B.L., Russell, M.E., Hughes, T.E., 2003. 1-[[(3-hydroxy-1-adamantyl) amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J. Med. Chem. 46, 2774–2789. http://doi.org/10.1021/jm030091l. Watanabe, Y.S., Yasuda, Y., Kojima, Y., Okada, S., Motoyama, T., Takahashi, R., Oka, M., 2015. Anagliptin, a potent dipeptidyl peptidase IV inhibitor: its single-crystal structure and enzyme interactions. J. Enzym. Inhib. Med. Chem. 30, 981–988. http://doi. org/10.3109/14756366.2014.1002402. Wu, Q., Jia, J., Yan, H., Du, J., Gui, Z., 2015. A novel angiotensin-capital I, Ukrainian converting enzyme (ACE) inhibitory peptide from gastrointestinal protease hydrolysate of silkworm pupa (Bombyx mori) protein: biochemical characterization and molecular docking study. Peptides 68, 17–24. http://doi.org/10.1016/j.peptides. 2014.07.026.

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