Identification of Novel Human Dipeptidyl Peptidase-IV Inhibitors of Natural Origin (Part I): Virtual Screening and Activity Assays

Background

There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a) to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c) to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site.

Methodology/Principal Findings

We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity). Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives.

Conclusions/Significance

We have demonstrated that our virtual-screening protocol was successful in identifying novel lead compounds for developing more potent DPP-IV inhibitors.     

Evaluating Khaya senegalensis for Dipeptidyl Peptidase-IV Inhibition Using in Vitro Analysis and Molecular Dynamic Simulation of Identified Bioactive Compounds

The dipeptidyl peptidase-IV (DPP-IV) inhibitory activity of Khaya senegalensis extracts was evaluated. The DPP-IV from a rat kidney was purified to a purification fold of 2.3. Among extracts from K. senegalensis, the hexane extract had the best DPP-IV inhibitory activity, with IC₅₀ value of 1.56±0.61 μg/mL and was fractionated to eleven fractions (A–K). Fraction I had the best DPP-IV inhibition via uncompetitive pattern. GC-MS analysis of fraction I showed that the major bioactive compounds were 3-amino-3-hydroxyimino-N-phenylpropanamide ( 1 ) and 11-(2-cyclopenten-1-yl)undecanoic acid ( 2 ), with good binding affinities toward DPP-IV, based on molecular docking,. They were then subjected to molecular dynamic simulation using WEBGRO and utilizing a GROMACS system for 100 ns. The 3-amino-3-hydroxyimino-N-phenylpropanamide-DPP-IV complex was more stable and compact than the other complex. K. senegalensis contains compounds like 1 that might be used for the design of new DPP-IV inhibitors.     

Mutant HNF-1alpha and mutant HNF-1beta identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1alpha and mutant HNF-1beta in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1alpha and 13 mutant HNF-1alpha, as well as wild HNF-1beta and 2 mutant HNF-1beta, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1alpha and wild HNF-1beta significantly transactivated DPP-IV promoter, but mutant HNF-1alpha and mutant HNF-1beta exhibited low transactivation activity. Moreover, to study whether mutant HNF-1alpha and mutant HNF-1beta change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1alpha or wild HNF-1beta, or else respective dominant-negative mutant HNF-1alphaT539fsdelC or dominant-negative mutant HNF-1betaR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1alpha cells and wild HNF-1beta cells, whereas they decreased in HNF-1alphaT539fsdelC cells and HNF-1betaR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1alpha and wild HNF-1beta have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1alpha and mutant HNF-1beta attenuate the stimulatory effect.     

Identification of Novel Human Dipeptidyl Peptidase-IV Inhibitors of Natural Origin (Part II): In Silico Prediction in Antidiabetic Extracts

Background

Natural extracts play an important role in traditional medicines for the treatment of diabetes mellitus and are also an essential resource for new drug discovery. Dipeptidyl peptidase IV (DPP-IV) inhibitors are potential candidates for the treatment of type 2 diabetes mellitus, and the effectiveness of certain antidiabetic extracts of natural origin could be, at least partially, explained by the inhibition of DPP-IV.

Methodology/Principal Findings

Using an initial set of 29,779 natural products that are annotated with their natural source and an experimentally validated virtual screening procedure previously developed in our lab (Guasch et al.; 2012) [1], we have predicted 12 potential DPP-IV inhibitors from 12 different plant extracts that are known to have antidiabetic activity. Seven of these molecules are identical or similar to molecules with described antidiabetic activity (although their role as DPP-IV inhibitors has not been suggested as an explanation for their bioactivity). Therefore, it is plausible that these 12 molecules could be responsible, at least in part, for the antidiabetic activity of these extracts through their inhibitory effect on DPP-IV. In addition, we also identified as potential DPP-IV inhibitors 6 molecules from 6 different plants with no described antidiabetic activity but that share the same genus as plants with known antidiabetic properties. Moreover, none of the 18 molecules that we predicted as DPP-IV inhibitors exhibits chemical similarity with a group of 2,342 known DPP-IV inhibitors.

Conclusions/Significance

Our study identified 18 potential DPP-IV inhibitors in 18 different plant extracts (12 of these plants have known antidiabetic properties, whereas, for the remaining 6, antidiabetic activity has been reported for other plant species from the same genus). Moreover, none of the 18 molecules exhibits chemical similarity with a large group of known DPP-IV inhibitors.     

Identification of hydrophobic residues critical for DPP-IV dimerization

The prolyl dipeptidase DPP-IV plays diverse and important roles in cellular functions. It is a membrane-bound exoprotease involved in the proteolytic cleavage of several insulin-sensing hormones. The inhibition of its enzymatic activity has been proven effective in the treatment of type II diabetes. Homodimeric DPP-IV interacts extracellularly with adenosine deaminase, and this interaction is critical for adenosine signaling and T-cell proliferation. In this study, we investigated the contribution of hydrophobic interactions to the dimerization of DPP-IV. Hydrophobic residues F713, W734, and Y735 were found to be essential for DPP-IV dimerization. Moreover, the enzymatic activity of DPP-IV was correlated with its quaternary structure. Monomeric DPP-IV had only residual activity left, ranging from 1/30 to 1/1600 of the dimeric forms. Using a surface plasmon resonance technique, we demonstrated that the affinity of these DPP-IV monomers for adenosine deaminase was not significantly altered, compared to that of dimeric DPP-IV. The study not only identifies the hydrophobic interactions critical for DPP-IV dimer formation, but also reveals no global conformational change upon the formation of monomers as determined by the protein-protein interaction (Kd) of DPP-IV with adenosine deaminase.     

Dipeptidyl peptidase IV activity in commercial solutions of human serum albumin

Due to the heterogeneous nature of commercial human serum albumin (cHSA), other components, such as the protease dipeptidyl peptidase IV (DPP-IV), possibly contribute to the therapeutic effect of cHSA. Here, we provide evidence for the first time that DPP-IV activity contributes to the formation of aspartate–alanine diketopiperazine (DA-DKP), a known immunomodulatory molecule from the N terminus of human albumin. cHSA was assayed for DPP-IV activity using a specific DPP-IV substrate and inhibitor. DPP-IV activity was assayed at 37 and 60 °C because cHSA solutions are pasteurized at 60 °C. DPP-IV activity in cHSA was compared with other sources of albumin such as a recombinant albumin (rHSA). In addition, the production of DA-DKP was measured by negative electrospray ionization/liquid chromatography mass spectrometry (ESI⁻/LCMS). Significant levels of DPP-IV activity were present in cHSA. This activity was abolished using a specific DPP-IV inhibitor. Fully 70 to 80% DPP-IV activity remained at 60 °C compared with the 37 °C incubate. No DPP-IV activity was present in rHSA, suggesting that DPP-IV activity is present only in HSA produced using the Cohn fractionation process. The formation of DA-DKP at 60 °C was observed with the DPP-IV inhibitor significantly decreasing this formation. DPP-IV activity in cHSA results in the production of DA-DKP, which could account for some of the clinical effects of cHSA.     

Glutamic acid analogues as potent dipeptidyl peptidase IV and 8 inhibitors

To find potent and selective inhibitors of dipeptidyl peptidase IV (DPP-IV), we synthesized a series of 2-cyanopyrrolidine with P2-site 4-substituted glutamic acid derivatives and tested their activities against DPP-IV, DPP8, and DPP-II. Analogues that incorporated a bulky substituent at the first carbon position of benzylamine or isoquinoline showed over 30-fold selectivity for DPP-IV over both DPP8 and DPP-II. From structure-activity relationship studies, we speculate that the S2 site of DPP8 might be similar to that of DPP-IV, while DPP-IV inhibitor with N-substituted glycine in the P2 site and/or with a moiety involving in hydrophobic interaction with the side chain of Phe357 might provide a better selectivity for DPP-IV over DPP8.     

Reduced serum dipeptidyl peptidase-IV after metformin and pioglitazone treatments

Dipeptidyl peptidase-IV (DPP-IV) regulates metabolism by degrading incretins involved in nutritional regulation. Metformin and pioglitazone improve insulin sensitivity whereas glyburide promotes insulin secretion. Zucker diabetic rats were treated with these antidiabetic agents for 2 weeks and DPP-IV activity and expression were determined. Serum DPP-IV activity increased whereas tissue activity decreased as the rats aged. Treatment of rats with metformin, pioglitazone, and glyburide did not alter DPP-IV mRNA expression in liver or kidney. Metformin and pioglitazone significantly (P<0.05) reduced serum DPP-IV activity and glycosylated hemoglobin. Glyburide did not lower DPP-IV activity or glycosylated hemoglobin. Regression analysis showed serum DPP-IV activity correlated with glycosylated hemoglobin (r=0.92) and glucagon-like peptide-1 levels (r=-0.49). Metformin, pioglitazone, and glyburide had no effect on serum DPP-IV activity in vitro, indicating these are not competitive DPP-IV inhibitors. We propose the in vivo inhibitory effects observed with metformin and pioglitazone on serum DPP-IV activity results from reduced DPP-IV secretion.     

Synthesis and biological evaluation of homopiperazine derivatives with beta-aminoacyl group as dipeptidyl peptidase IV inhibitors

Compounds with homopiperazine skeleton are designed to find a potent DPP-IV inhibitor without inhibiting CYP. Thus a series of beta-aminoacyl-containing homopiperazine derivatives was synthesized and evaluated. Compounds with acid moiety were found to be potent inhibitors of DPP-IV without inhibiting CYP 3A4. More specifically, compound 7m showed nanomolar activity with no inhibition towards five subtypes of CYPs, was considered as a prototype for further derivatization. Based on its X-ray co-crystal structure with human DPP-IV, we identified compounds 7s and 7t which showed good in vitro activity, no CYP inhibition, and good selectivity.     

Synthesis and structure-activity relationships of potent 3- or 4-substituted-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP-IV) inhibitors have attracted attention as potential drugs for use in the treatment of type 2 diabetes because they prevent degradation of glucagon-like peptide-1 (GLP-1) and extend its duration of action. A series of 2-cyanopyrrolidines are among the most potent of DPP-IV inhibitors. We focused our attention on substitutions at the 3- or 4-position of 2-cyanopyrrolidines and synthesized and evaluated various derivatives. Among them, the 4-fluoro derivative was found to exhibit better DPP-IV inhibitory activity and higher plasma drug concentrations after oral administration to rats than the 4-unsubstituted derivative. We report here on the synthesis and biological data of the aforementioned derivatives.     

Synthesis and biological evaluation of all eight stereoisomers of DPP-IV inhibitor saxagliptin

All eight stereoisomers of saxagliptin have been synthesized and evaluated for their inhibitory activity against DPP-IV. It was unambiguously confirmed that the configuration of saxagliptin was critical to potent inhibition of DPP-IV. Docking study was performed to elucidate the configuration–activity relationship of saxagliptin stereoisomers. Tyr662 and Tyr470 have been suggested as the key residues of DPP-IV interacting with the inhibitors. This work provides valuable information for further inhibitor design against DPP-IV.     

Modulation of substance P signaling by dipeptidyl peptidase-IV enzymatic activity in human glioma cell lines

Dipeptidyl peptidase-IV (DPP-IV, CD26) is a serine protease almost ubiquitously expressed on cell surface and present in body fluids. DPP-IV has been suggested to proteolytically modify a number of biologically active peptides including substance P (SP) and the chemokine stromal cell derived factor-1alpha (SDF-1alpha, CXCL12). SP and SDF-1alpha have been implicated in the regulation of multiple biological processes and also induce responses that may be relevant for glioma progression. Both SP and SDF-1alpha are signaling through cell surface receptors and use intracellular calcium as a second messenger. The effect of DPP-IV on intracellular calcium mobilization mediated by SP and SDF-1alpha was monitored in suspension of wild type U373 and DPP-IV transfected U373DPPIV glioma cells using indicator FURA-2. Nanomolar concentrations of SP triggered a transient dose dependent increase in intracellular calcium rendering the cells refractory to repeated stimulation, while SDF-1 had no measurable effect. SP signaling in DPP-IV overexpressing U373DPPIV cells was not substantially different from that in wild type cells. However, preincubation of SP with the DPP-IV overexpressing cells lead to the loss of its signaling potential, which could be prevented with DPP-IV inhibitors. Taken together, DPP-IV may proteolytically inactivate local mediators involved in gliomagenesis.     

Discovery of potent and selective phenylalanine based dipeptidyl peptidase IV inhibitors

anti-Substituted beta-methylphenylalanine derived amides have been shown to be potent DPP-IV inhibitors exhibiting excellent selectivity over both DPP8 and DPP9. These are among the most potent compounds reported to date lacking an electrophilic trap. The most potent compound among these is 5-oxo-1,2,4-oxadiazole 44, which is a 3 nM DPP-IV inhibitor.     

(S)-gamma-(4-Aryl-1-piperazinyl)-l-prolyl]thiazolidines as a novel series of highly potent and long-lasting DPP-IV inhibitors

In the search for an inhibitor of dipeptidyl peptidase IV (DPP-IV) highly potent both in vitro and in vivo, we synthesized a series of L-prolylthiazolidine-based DPP-IV inhibitors having 4-arylpiperazine or 4-arylpiperidine at the gamma-position of the proline structure. Of these compounds, the 4-(5-nitro-2-pyridyl)piperazine analog 21e showed a sub-nanomolar (IC(50)=0.92 nmol/L) DPP-IV inhibitory activity and a long-lasting in vivo DPP-IV inhibition profile.     

Design and synthesis of potent amido- and benzyl-substituted cis-3-amino-4-(2-cyanopyrrolidide)pyrrolidinyl DPP-IV inhibitors

The cis-3-amino-4-(2-cyanopyrrolidide)-pyrrolidine template has been shown to afford low nanomolar inhibitors of human DPP-IV that exhibit a robust PK/PD profile. An X-ray co-crystal structure of 5 confirmed the proposed mode of binding. The potent single digit DPP-IV inhibitor 53 exhibited a preferred PK/PD profile in preclinical animal models and was selected for additional profiling.     

Dipeptidyl peptidase IV in the human lung and spinocellular lung cancer.

The isoelectric point and proportions of soluble and membrane bound dipeptidyl peptidase IV (DPP-IV) in human lung and spinocellular lung cancer tissue were tested. It was found that soluble DPP-IV is relatively less frequent in the cancer than in normal lung tissue. We demonstrated multiple molecular forms of DPP-IV in normal and cancer lung tissues, differing probably not only in the degree of sialylation. DPP-IV from lung cancer tissue consists of more basic molecular forms than that from normal lung tissue. These results suggest that the molecular properties of DPP-IV in normal and cancerous lung tissues may be different.     

(3R)-3-amino-4-(2,4,5-trifluorophenyl)-N-{4-[6-(2-methoxyethoxy)benzothiazol-2-yl]tetrahydropyran-4-yl}butanamide as a potent dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Novel series of 3-amino-N-(4-aryl-1,1-dioxothian-4-yl)butanamides and 3-amino-N-(4-aryltetrahydropyran-4-yl)butanamides were synthesized and evaluated as dipeptidyl peptidase IV (DPP-IV) inhibitors. Derivatives incorporating the 6-substituted benzothiazole group showed highly potent DPP-IV inhibitory activity. Oral administration of (3R)-3-amino-4-(2,4,5-trifluorophenyl)-N-{4-[6-(2-methoxyethoxy)benzothiazol-2-yl]tetrahydropyran-4-yl}butanamide (12u) reduced blood glucose excursion in an oral glucose tolerance test.     

Purification and properties of dipeptidyl peptidase IV from human urine

Dipeptidyl peptidase IV (DPP-IV) (EC 3.4.14.5) has been purified from normal human urine using immunoaffinity chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of urinary DPP-IV was estimated to be 280 kDa by gel filtration. The Km value for the hydrolysis of (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate was calculated to be 1.25 +/- 0.25 X 10(-4) M; the pH optimum and pI were 8.7 and 6.5, respectively. These properties were the same as those of renal DPP-IV. Both renal and this urinary DPP-IV were inhibited by diisopropylfluorophosphate and activated by phospholipid. From these results we suppose that urinary DPP-IV may be derived from the kidney rather than from the blood     

Crystal structures of DPP-IV (CD26) from rat kidney exhibit flexible accommodation of peptidase-selective inhibitors

Dipeptidyl peptidase IV (DPP-IV) belongs to a family of serine peptidases, and due to its indirect regulatory role in plasma glucose modulation, DPP-IV has become an attractive pharmaceutical target for diabetes therapy. DPP-IV inactivates the glucagon-like peptide (GLP-1) and several other naturally produced bioactive peptides that contain preferentially a proline or alanine residue in the second amino acid sequence position by cleaving the N-terminal dipeptide. To elucidate the details of the active site for structure-based drug design, we crystallized a natural source preparation of DPP-IV isolated from rat kidney and determined its three-dimensional structure using X-ray diffraction techniques. With a high degree of similarity to structures of human DPP-IV, the active site architecture provides important details for the design of inhibitory compounds, and structures of inhibitor-protein complexes offer detailed insight into three-dimensional structure-activity relationships that include a conformational change of Tyr548. Such accommodation is exemplified by the response to chemical substitution on 2-cyanopyrrolidine inhibitors at the 5 position, which conveys inhibitory selectivity for DPP-IV over closely related homologues. A similar conformational change is also observed in the complex with an unrelated synthetic inhibitor containing a xanthine core that is also selective for DPP-IV. These results suggest the conformational flexibility of Tyr548 is unique among protein family members and may be utilized in drug design to achieve peptidase selectivity.     

NVP-DPP728 (1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)- pyrrolidine), a slow-binding inhibitor of dipeptidyl peptidase IV.

Inhibition of dipeptidyl peptidase IV (DPP-IV) has been proposed recently as a therapeutic approach to the treatment of type 2 diabetes. N-Substituted-glycyl-2-cyanopyrrolidide compounds, typified by NVP-DPP728 (1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S )-p yrrolidine), inhibit degradation of glucagon-like peptide-1 (GLP-1) and thereby potentiate insulin release in response to glucose-containing meals. In the present study NVP-DPP728 was found to inhibit human DPP-IV amidolytic activity with a K(i) of 11 nM, a k(on) value of 1.3 x 10(5) M(-)(1) s(-)(1), and a k(off) of 1.3 x 10(-)(3) s(-)(1). Purified bovine kidney DPP-IV bound 1 mol/mol [(14)C]-NVP-DPP728 with high affinity (12 nM K(d)). The dissociation constant, k(off), was 1.0 x 10(-)(3) and 1.6 x 10(-)(3) s(-)(1) in the presence of 0 and 200 microM H-Gly-Pro-AMC, respectively (dissociation t(1/2) approximately 10 min). Through kinetic evaluation of DPP-IV inhibition by the D-antipode, des-cyano, and amide analogues of NVP-DPP728, it was determined that the nitrile functionality at the 2-pyrrolidine position is required, in the L-configuration, for maximal activity (K(i) of 11 nM vs K(i) values of 5.6 to >300 microM for the other analogues tested). Surprisingly, it was found that the D-antipode, despite being approximately 500-fold less potent than NVP-DPP728, displayed identical dissociation kinetics (k(off) of 1.5 x 10(-)(3) s(-)(1)). NVP-DPP728 inhibited DPP-IV in a manner consistent with a two-step inhibition mechanism. Taken together, these data suggest that NVP-DPP728 inhibits DPP-IV through formation of a novel, reversible, nitrile-dependent complex with transition state characteristics.