Discovery, SAR, and X-ray structure of novel biaryl-based dipeptidyl peptidase IV inhibitors

The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.     

Inhibition of dipeptidyl peptidase IV (DPP IV) is one of the mechanisms explaining the hypoglycemic effect of berberine

Berberine was investigated as an inhibitor of human dipeptidyl peptidase IV (DPP IV) in an attempt to explain its anti-hyperglycemic activities. The investigation included simulated docking experiments to fit berberine within the binding pocket of DPP IV. Berberine was found to readily fit within the binding pocket of DPP IV in a low energy orientation characterized with optimal electrostatic attractive interactions bridging the isoquinolinium positively charged nitrogen atom (berberine) and the negatively charged acidic residue of glutamic acid-205 (GLU205) of DPP IV. Experimentally, berberine was found to inhibit human recombinant DPP IV in vitro with IC₅₀ = 13.3 μM. Our findings suggest that DPP IV inhibition is, at least, one of the mechanisms that explain the anti-hyperglycemic activity of berberine. The fact that berberine was recently reported to potently inhibit the pro-diabetic target human protein tyrosine phosphatase 1B (h-PTP 1B) discloses a novel dual natural h-PTP 1B/DPP IV inhibitor.     

Discovery of C-(1-aryl-cyclohexyl)-methylamines as selective,orally available inhibitors of dipeptidyl peptidase IV

The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPP IV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral pharmacokinetic profile in the rat.     

Acylated Gly-(2-cyano)pyrrolidines as inhibitors of fibroblast activation protein (FAP) and the issue of FAP/prolyl oligopeptidase (PREP)-selectivity

A series of N-acylated glycyl-(2-cyano)pyrrolidines were synthesized with the aim of generating structure-activity relationship (SAR) data for this class of compounds as inhibitors of fibroblast activation protein (FAP). Specifically, the influence of (1) the choice of the N-acyl group and (2) structural modification of the 2-cyanopyrrolidine residue were investigated. The inhibitors displayed inhibitory potency in the micromolar to nanomolar range and showed good to excellent selectivity with respect to the proline selective dipeptidyl peptidases (DPPs) DPP IV, DPP9 and DPP II. Additionally, selectivity for FAP with respect to prolyl oligopeptidase (PREP) is reported. Not unexpectedly, the latter data suggest significant overlap in the pharmacophoric features that define FAP or PREP-inhibitory activity and underscore the importance of systematically evaluating the FAP/PREP-selectivity index for inhibitors of either of these two enzymes. Finally, this study forwards several compounds that can serve as leads or prototypic structures for future FAP-selective-inhibitor discovery.     

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.     

Evidence for a role of dipeptidyl peptidase IV in fibronectin-mediated interactions of hepatocytes with extracellular matrix.

Dipeptidyl peptidase IV (DPP IV) is a cell surface glycoprotein which has been implicated in hepatocyte-extracellular matrix interactions [Hixson, DeLourdes, Ponce, Allison & Walborg (1984) Exp. Cell Res. 152, 402-414; Walborg, Tsuchida, Weeden, Thomas, Barrick, McEntire, Allison & Hixson (1985) Exp. Cell Res. 158, 509-518; Hanski, Huhle & Reutter (1985) Biol. Chem. Hoppe-Seyler 366, 1169-1176]. However, its proteolytic substrate(s) and/or binding protein(s) which mediate this influence have not been conclusively identified. Nitrocellulose binding assays using 125I-labelled DPP IV that was purified to homogeneity from rat hepatocytes revealed a direct interaction of DPP IV with fibronectin. Although fibronectin could mediate an indirect binding of DPP IV to collagen, no evidence was found for a direct binding of DPP IV to native or denatured Type I collagen. Fibronectin appeared to bind DPP IV at a site distinct from its exopeptidase substrate recognition site since protease inhibitors such as competitive peptide substrates and phenylmethanesulphonyl fluoride enhanced binding, possibly as a result of an altered conformation of DPP IV. To determine if fibronectin binding to DPP IV is involved in the interaction of fibronectin with the hepatocyte surface, the effect of various DPP IV inhibitors on 125I-fibronectin binding to isolated hepatocytes in suspension was examined. Kinetic studies revealed that inhibitors of DPP IV which enhanced fibronectin binding in vitro accelerated the initial binding of fibronectin to the cell surface where it was subsequently cross-linked (presumably by tissue transglutaminase) to as yet undefined components. Immunolocalization of fibronectin and DPP IV in normal rat liver sections showed that both proteins were present along the hepatocyte sinusoidal membrane. These observations, coupled with previous results showing that DPP IV is tightly bound to biomatrix isolated from rat liver (Hixson et al., 1984; Walborg et al., 1985), suggest that DPP IV binding to fibronectin may play a role in interactions of hepatocytes with extracellular matrix in vivo and possibly in matrix assembly.     

Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone-releasing factor and analogs.

The kinetics and selectivity of proteolysis of synthetic human growth hormone-releasing factor and analogs by purified human placental dipeptidyl peptidase IV (DPP IV) were studied by HPLC. The initial rates of Ala2-Asp3 cleavage (pH 7.8, 37 degrees C, So = 0.15 mM) were all approx. 5 mumol min-1 mg-1 for the parent hormone, GRF(1-44)-NH2, and the fragments, GRF(1-29)-NH2 and GRF(1-20)-NH2. Lower activities observed for GRF(1-11)-OH, GRF(1-3)-OH, and cyclic lactam analogs indicate S1'-Sn' binding. Assays of [Trp6]-GRF(1-29)-NH2 versus [D-Trp6]-GFR(1-29)-NH2 indicate an S4' binding cavity. Peptides with D-configuration at P2, P1 or P1' and desNH2Tyr1 and N-MeTyr1 analogs of GRF were not cleaved. Catalytic parameters for the P1-substituted analogs [X2,Ala15]-GRF(1-29)-NH2 were found to vary with X as follows, Km: Abu less than Ala less than Pro less than Val less than Ser less than Gly much less than Leu; kcat: Pro greater than Ala greater than Abu greater than Ser greater than Gly much greater than Leu greater than Val; kcat/Km: Abu greater than Pro greater than Ala much greater than Ser greater than Gly = Val much greater than Leu. Km is at a minimum and kcat/Km at a maximum, for a hydrophobic P1 side-chain of about 0.25 nm in length, i.e., the ethyl side-chain of alpha-aminobutyric acid (Abu) is very close to optimal. These results further define the S1 selectivity of DPP IV and may be useful in the design of DPP IV resistant GRF analogs that can be produced by recombinant DNA methods and the design of DPP IV inhibitors.     

Aryl- and heteroaryl-substituted aminobenzo[a]quinolizines as dipeptidyl peptidase IV inhibitors

Synthesis and SAR are described for a structurally distinct class of DPP–IV inhibitors based on aminobenzo[a]quinolizines bearing (hetero-)aromatic substituents in the S1 specificity pocket. The m-(fluoromethyl)-phenyl derivative (S,S,S)-2g possesses the best fit in the S1 pocket. However, (S,S,S)-2i, bearing a more hydrophilic 5-methyl-pyridin-2-yl residue as substituent for the S1 pocket, displays excellent in vivo activity and superior drug-like properties.     

Direct evidence for the binding of rat liver DPP IV to collagen in vitro

Previous studies have shown that the tripeptide Gly-Pro-Ala, a substrate for dipeptidyl peptidase IV (DPP IV, EC 3.3.14.5), interferes with initial spreading of hepatocytes on a matrix consisting of fibronectin and denatured collagen. In the present investigation we report that the tripeptide as well as the anti-DPP IV antibody inhibits the initial spreading of hepatocytes also on native collagen. This effect appears to be due to the interaction of DPP IV from hepatocyte plasma membrane with native collagen. It is shown in vitro by immunohistochemistry, catalytic histochemistry, and by affinity chromatography of solubilized plasma membrane on collagen-Sepharose that DPP IV has a binding affinity to collagen. This binding does not affect the activity of DPP IV.     

Rational design and synthesis of potent and long-lasting glutamic acid-based dipeptidyl peptidase IV inhibitors

A series of (2S)-cyanopyrrolidines with glutamic acid derivatives at the P2 site have been prepared and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV). The structure–activity relationships (SAR) led to the discovery of potent 3-substituted glutamic acid analogues, providing enhanced chemical stability and excellent selectivity over the closely related enzymes, DPP8, DPP-II and FAP. Compound 13f exhibited the ability to both significantly decrease the glucose excursion and inhibit plasma DPP-IV activity.     

Computational screening of dipeptidyl peptidase IV inhibitors from micoroalgal metabolites by pharmacophore modeling and molecular docking

Dipeptidyl peptidase IV (DPP‐IV) catalyzes conversion of GLP‐1 (glucagon like peptide 1) to inert structure, which results in insufficient secretion of insulin and increase in postprandial blood glucose level. The present study attempts to identify novel inhibitors from bioactive metabolites present in microalgae against DPP‐IV through virtual screening, molecular docking, and pharmacophore modeling for the active target. Possible binding modes of all 60 ligands against DPP‐IV receptor were constructed using MTiOpenScreen virtual screening server. Pharmacophore model was built based on identified 38 DPP‐IV test ligands by using the web‐based PharmaGist program which encompasses hydrogen‐bond acceptors, hydrophobic groups, spatial features, and aromatic rings. The pharmacophore model having highest scores was selected to screen active DPP‐IV ligands. Highest scoring model was used as a query in ZincPharmer screening. All identified ligands were filtered, based on the Lipinski's rule‐of‐five and were subjected to docking studies. In the process of docking analyses, we considered different bonding modes of one ligand with multiple active cavities of DPP‐IV with the help of AutoDock 4.0. The docking analyses indicate that the bioactive constituents, namely, β‐stigmasterol, barbamide, docosahexaenoic acid, arachidonic acid, and harman showed the best binding energies on DPP‐IV receptor and hydrogen bonding with ASP545, GLY741, TYR754, TYR666, ARG125, TYR547, SER630, and LYS554 residues. This study concludes that docosahexaenoic acid, arachidonic acid, β‐stigmasterol, barbamide, harman, ZINC58564986, ZINC56907325, ZINC69432950, ZINC69431828, ZINC73533041, ZINC84287073, ZINC69849395, and ZINC10508406 act as possible DPP‐IV inhibitors.     

Novel N-substituted 4-hydrazino piperidine derivative as a dipeptidyl peptidase IV inhibitor

A novel class of N-substituted 4-hydrazino piperidine derivatives were designed, synthesized and evaluated for DPP IV inhibition. The SAR studies on the N-substituted piperidine led to the discovery of compound 22e as a potent DPP IV inhibitor (IC₅₀ 88 nM), which is highly selective over other peptidases. In vivo efficacy indicates that compound 22e stimulates insulin release in response to glucose load and improves glucose tolerance in n5-STZ and Zucker Diabetic Fatty (ZDF) rats.     

Diprolyl nitriles as potent dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP4) is a multifunctional type II transmembrane serine peptidase which regulates various physiological processes, most notably plasma glucose homeostasis by cleaving peptide hormones glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. Inhibition of DPP4 is a potentially valuable therapy for type 2 diabetes. Synthesis and structure-activity relationships of a series of substituted diprolyl nitriles are described, leading to the identification of compound 1 with a measured DPP4 K(i) of 3.6 nM.     

Dipeptidyl-peptidase IV (CD26)--role in the inactivation of regulatory peptides

Dipeptidyl-peptidase IV (DPP IV/CD26) has a dual function as a regulatory protease and as a binding protein. Its role in the inactivation of bioactive peptides was recognized 20 years ago due to its unique ability to liberate Xaa-Pro or Xaa-Ala dipeptides from the N-terminus of regulatory peptides, but further examples are now emerging from in vitro and vivo experiments. Despite the minimal N-terminal truncation by DPP IV, many mammalian regulatory peptides are inactivated--either totally or only differentially--for certain receptor subtypes. Important DPP IV substrates include neuropeptides like neuropeptide Y or endomorphin, circulating peptide hormones like peptide YY, growth hormone-releasing hormone, glucagon-like peptides(GLP)-1 and -2, gastric inhibitory polypeptide as well as paracrine chemokines like RANTES (regulated on activation normal T cell expressed and secreted), stromal cell-derived factor, eotaxin and macrophage-derived chemokine. Based on these findings the potential clinical uses of selective DPP IV inhibitors or DPP IV-resistant analogues, especially for the insulinotropic hormone GLP-1, have been tested to enhance insulin secretion and to improve glucose tolerance in diabetic animals. Thus, DPP IV appears to be a major physiological regulator for some regulatory peptides, neuropeptides, circulating hormones and chemokines.     

Ultrastructural localization of dipeptidyl peptidase IV in rat salivary glands by immunocytochemistry

Summary The ultrastructural localization of dipeptidyl peptidase IV (DPP IV) (EC 3.4.14.5) in rat submandibular and parotid glands was studied immunocytochemically by the peroxidase-antiperoxidase (PAP) method, using a monospecific antiserum against rat kidney DPP IV. There were no differences in the immunocytochemical localization of DPP IV between submandibular and parotid glands. In these glands, DPP IV was primarily found to be associated with the luminal and intercellular canalicular plasma membranes of acinar cells and with the luminal plasma membranes of intercalated and striated duct cells. Occasionally, immunoreaction of DPP IV was detected in cytoplasmic vesicles (vacuoles), lysosomes, and multivesicular bodies in some acinar cells as well as in ductal epithelial cells. Furthermore, the reaction product was also found within the lumina of peri-acinar and peri-ductal capillaries and in the cytoplasm of some fibroblasts in the interstitial connective tissue. These data suggest that DPP IV in the submandibular and parotid glands may play some role in the secretion or reabsorption processes of secretory proteins and peptides in these glands.     

Insulin-dependent phosphorylation of DPP IV in liver. Evidence for a role of compartmentalized c-Src

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model aimed at elucidating protein sorting signals. We identify here, by MS, several tyrosine-phosphorylated proteins in a rat liver Golgi/endosome (G/E) fraction including DPP IV. We show that a pool of DPP IV is tyrosine-phosphorylated. Maximal phosphorylation was observed after 2 min following intravenous insulin injection. DPP IV coimmunoprecipitated with the cellular tyrosine kinase Src (c-Src) with maximal association also observed after 2 min following insulin injection. DPP IV was found phosphorylated after incubation of nonsolubilized G/E membranes with [gamma-32P]ATP. The c-Src inhibitor PP2 inhibited DPP IV phosphorylation. Oriented proteolysis experiments indicate that a large pool of c-Src is protected in G/E fractions. Following injection of the protein-tyrosine phosphatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40% both in plasma membrane and G/E fractions. In the fraction designated Lh, DPP IV levels decreased by 50% 15 min following insulin injection. Therefore, a pool of DPP IV is tyrosine-phosphorylated in an insulin-dependent manner. The results suggest the presence of a yet to be characterized signalling mechanism whereby DPP IV has access to c-Src-containing signalling platforms.     

Evidence that thymocyte-activating molecule is mouse CD26 (dipeptidyl peptidase IV).

We previously described a developmentally regulated, Mr 115,000 (reduced) and 110,000/128,000 (nonreduced) mouse T cell-activating molecule (THAM) also expressed on a variety of epithelial cell surfaces, and associated with neutral exoaminopeptidase activity. In the present study, we show that THAM is the mouse counterpart of the human T cell-activating ectoenzyme CD26 (dipeptidyl peptidase IV, DPP IV) and that highly purified THAM lacks neutral exoaminopeptidase activity. This conclusion is based on the following: 1) the N-terminal segments of the THAM Mr 110,000 and 128,000 components shared the same amino acid sequence with the rat DPP IV. These N-termini comprised a short intracytoplasmic tail of six residues followed by a downstream hydrophobic transmembrane segment. 2) THAM-specific mAb H194-112-Affi-Gel immunoadsorbent was capable of removing DPP IV enzymatic activity from mouse thymoma cell detergent extracts. 3) H194-112 reactivity pattern on developing thymocytes was found to parallel that previously reported for membrane-bound DPP IV enzymatic activity. The extent of THAM N-glycosylation, as measured by N-glycanase treatment of H194-112 immunoprecipitates, was found to be similar to that of human and rat DPP IV (i.e., approximately 20 kDa). Cross-linking experiments indicated that THAM was expressed at the cell surface as a dimer of approximately 220 kDa. Its two subunits were found to be structurally related but not identical as shown by their different Mr under nonreducing conditions and by their slightly distinct peptide profiles after proteolytic cleavage. We conclude from these data that DPP IV, in addition to its extracellular matrix receptor and ectoenzymatic functions, is a T cell-activating structure in both human and mouse species.     

CD26/dipeptidyl-peptidase IV down-regulates the eosinophil chemotactic potency, but not the anti-HIV activity of human eotaxin by affecting its interaction with CC chemokine receptor 3

Chemokines attract and activate distinct sets of leukocytes. The CC chemokine eotaxin has been characterized as an important mediator in allergic reactions because it selectively attracts eosinophils, Th2 lymphocytes, and basophils. Human eotaxin has a penultimate proline, indicating that it might be a substrate for dipeptidyl-peptidase IV (CD26/DPP IV). In this study we demonstrate that eotaxin is efficiently cleaved by CD26/DPP IV and that the NH2-terminal truncation affects its biological activity. CD26/DPP IV-truncated eotaxin(3-74) showed reduced chemotactic activity for eosinophils and impaired binding and signaling properties through the CC chemokine receptor 3. Moreover, eotaxin(3-74) desensitized calcium signaling and inhibited chemotaxis toward intact eotaxin. In addition, HIV-2 infection of CC chemokine receptor 3-transfected cells was inhibited to a similar extent by eotaxin and eotaxin(3-74). Thus, CD26/DPP IV differently regulates the chemotactic and antiviral potencies of eotaxin by the removal of two NH2-terminal residues. This physiological processing may be an important down-regulatory mechanism, limiting eotaxin-mediated inflammatory responses.     

Identification of diverse dipeptidyl peptidase IV inhibitors via structure-based virtual screening

Dipeptidyl peptidase IV (DPP4) is an important target for the treatment of type II diabetes mellitus. Inhibition of DPP4 will improve glycemic control in such patients by preventing the rapid breakdown and thereby prolonging the physiological actions of incretin hormones. Known DPP4 inhibitors (including marketed drugs and those drug candidates) appear to share similar structural features: the cyanopyrrolidine moieties, the xanthenes/pyrimidine parts and amino-like linkages. In this study, a multi-step virtual screening strategy including both rigid and flexible docking was employed to search for novel structures with DPP4 inhibition. From SPECS database, consisting of over 190,000 commercially available compounds, 99 virtual hits were picked up and 15 of them were eventually identified to have DPP4 inhibitory activities at 5?~?50?μM. Diverse structures of our compounds were out of usual structural categories. Hence a pharmacophore model was built to further explore their common binding features on the enzyme. The results provided a new pathway for the discovery of DPP4 inhibitors and would be helpful for further optimization of DPP4 inhibitors.     

Novel tetrahydropyran analogs as dipeptidyl peptidase IV inhibitors: Profile of clinical candidate (2R,3S,5R)-2-(2,5-difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine (23)

A series of novel tri-2,3,5-substituted tetrahydropyran analogs were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the series provided inhibitors with good DPP-4 potency and selectivity over other peptidases (QPP, DPP8, and FAP). Compound 23, which is very potent, selective, efficacious in the diabetes PD model, and has an excellent pharmacokinetic profile, is selected as a clinical candidate.