Gene_locus Report for: human-DPP4

Starting from the lead isodaphnetin, a natural product inhibitor of DPP-4 discovered through a target fishing docking based approach, a series of novel 2-phenyl-3,4-dihydro-2H-benzo[f]chromen-3-amine derivatives as potent DPP-4 inhibitors are rationally designed utilizing highly efficient 3D molecular similarity based scaffold hopping as well as electrostatic complementary methods. Those ingenious drug design strategies bring us approximate 7400-fold boost in potency. Compounds 22a and 24a are the most potent ones (IC50 approximately 2.0 nM) with good pharmacokinetic profiles. Compound 22a demonstrated stable pharmacological effect. A 3 mg/kg oral dose provided >80% inhibition of DPP-4 activity within 24 h, which is comparable to the performance of the long-acting control omarigliptin. Moreover, the efficacy of 22a in improving the glucose tolerance is also comparable with omarigliptin. In this study, not only promising DPP-4 inhibitors as long acting antidiabetic that are clinically on demand are identified, but the target fish docking and medicinal chemistry strategies were successfully implemented.

The worldwide prevalence of diabetes has spurred numerous studies on the development of new antidiabetic medicines. As a result, dipeptidyl peptidase IV (DPP4) has been recognized as a validated target. In our efforts to discover new DPP4 inhibitors, we analyzed the complexed structures of DPP4 available in Protein Data Bank and designed a series of triazole compounds. After enzyme activity assays and crystallographic verification of the binding interaction patterns, we found that the triazole compounds can inhibit DPP4 with micromolar IC50 values. Liver microsome stability and cytochrome P450 metabolic tests were performed on this series, revealing undesirable pharmacokinetic profiles for the triazole compounds. To overcome this liability, we substituted the triazole ring with an amide or urea group to produce a new series of DPP4 inhibitors. Based on its enzyme activity, metabolic stability, and selectivity over DPP8 and DPP9, we selected compound 21 r for further study of its in vivo effects in mice using an oral glucose tolerance test (OGTT). The results show that 21 r has efficacy similar to that of sitagliptin at a dose of 3 mg kg(-1) . The crystal structure of 21 r bound to DPP4 also reveals that the trifluoromethyl group is directed toward a subpocket different from the subsite bound by sitagliptin, providing clues for the design of new DPP4 inhibitors.

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the gamma-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-yl carbonyl]thiazolidine (8 g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8 g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S(2) extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8 g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8 g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.

Poor medication adherence is one of the leading causes of suboptimal glycaemic control in approximately half of the patients with type 2 diabetes mellitus (T2DM). Long-acting antidiabetic drugs are clinically needed for improving patients' compliance. Dipeptidyl peptidase-4 (DPP-4) inhibitors play an increasingly important role in the treatment of T2DM because of their favorable properties of weight neutrality and hypoglycemia avoidance. Herein, we report the successful discovery and scale-up synthesis of compound 5, a structurally novel, potent, and long-acting DPP-4 inhibitor for the once-weekly treatment of T2DM. Inhibitor 5 has fast-associating and slow-dissociating binding kinetics profiles as well as slow clearance rate and long terminal half-life pharmacokinetic properties. A single-dose oral administration of 5 (3 mg/kg) inhibited >80% of DPP-4 activity for more than 7 days in diabetic mice. The long-term antidiabetic efficacies of 5 (10 mg/kg, qw) were better than those of the once-weekly trelagliptin and omarigliptin, especially in decreasing the hemoglobin A1c level.

Aims: Since 2006, DPP-4 inhibitors have become established therapy for the treatment of type 2 diabetes. Despite sharing a common mechanism of action, considerable chemical diversity exists amongst members of the DPP-4 inhibitor class, raising the question as to whether structural differences may result in differentiated enzyme inhibition and antihyperglycaemic activity. Methods: We have compared the binding properties of the most commonly used inhibitors and have investigated the relationship between their inhibitory potency at the level of the enzyme and their acute glucose-lowering efficacy. Results: Firstly, using a combination of published crystal structures and in-house data, we demonstrated that the binding site utilized by all of the DPP-4 inhibitors assessed was the same as that used by neuropeptide Y, supporting the hypothesis that DPP-4 inhibitors are able to competitively inhibit endogenous substrates for the enzyme. Secondly, we ascertained that the enzymatic cleft of DPP-4 is a relatively large cavity which displays conformational flexibility to accommodate structurally diverse inhibitor molecules. Finally, we found that for all inhibitors, irrespective of their chemical structure, the inhibition of plasma DPP-4 enzyme activity correlates directly with acute plasma glucose lowering in mice. Conclusion: The common binding site utilized by different DPP-4 inhibitors enables similar competitive inhibition of the cleavage of the endogenous DPP-4 substrates. Furthermore, despite chemical diversity and a range of binding potencies observed amongst the DPP-4 inhibitors, a direct relationship between enzyme inhibition in the plasma and glucose lowering is evident in mice for each member of the classes studied.

Evogliptin ((R)-4-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(tert-butoxymethyl) piperazine-2-one)) is a highly potent selective inhibitor of dipeptidyl peptidase IV (DPP4) that was approved for the treatment of type 2 diabetes in South Korea. In this study, we report the crystal structures of Evogliptin, DA-12166, and DA-12228 (S,R diastereomer of Evogliptin) complexed to human DPP4. Analysis of both the structures and inhibitory activities suggests that the binding of the trifluorophenyl moiety in the S1 pocket and the piperazine-2-one moiety have hydrophobic interactions with Phe357 in the S2 extensive subsite, and that the multiple hydrogen bonds made by the (R)-beta-amine group in the S2 pocket and the contacts made by the (R)-tert-butyl group with Arg125 contribute to the high potency observed for Evogliptin.

Novel potent and selective 5,6,5- and 5,5,6-tricyclic pyrrolidine dipeptidyl peptidase IV (DPP-4) inhibitors were identified. Structure-activity relationship (SAR) efforts focused on improving the intrinsic DPP-4 inhibition potency, increasing protease selectivity, and demonstrating clean ion channel and cytochrome P450 profiles while trying to achieve a pharmacokinetic profile suitable for once weekly dosing in humans.

Trelagliptin (SYR-472), a novel dipeptidyl peptidase-4 inhibitor, shows sustained efficacy by once-weekly dosing in type 2 diabetes patients. In this study, we characterized in vitro properties of trelagliptin, which exhibited approximately 4- and 12-fold more potent inhibition against human dipeptidyl peptidase-4 than alogliptin and sitagliptin, respectively, and >10,000-fold selectivity over related proteases including dipeptidyl peptidase-8 and dipeptidyl peptidase-9. Kinetic analysis revealed reversible, competitive and slow-binding inhibition of dipeptidyl peptidase-4 by trelagliptin (t1/2 for dissociation approximately 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. Taken together, potent dipeptidyl peptidase inhibition may partially contribute to sustained efficacy of trelagliptin.

Starting from the lead isodaphnetin, a natural product inhibitor of DPP-4 discovered through a target fishing docking based approach, a series of novel 2-phenyl-3,4-dihydro-2H-benzo[f]chromen-3-amine derivatives as potent DPP-4 inhibitors are rationally designed utilizing highly efficient 3D molecular similarity based scaffold hopping as well as electrostatic complementary methods. Those ingenious drug design strategies bring us approximate 7400-fold boost in potency. Compounds 22a and 24a are the most potent ones (IC50 approximately 2.0 nM) with good pharmacokinetic profiles. Compound 22a demonstrated stable pharmacological effect. A 3 mg/kg oral dose provided >80% inhibition of DPP-4 activity within 24 h, which is comparable to the performance of the long-acting control omarigliptin. Moreover, the efficacy of 22a in improving the glucose tolerance is also comparable with omarigliptin. In this study, not only promising DPP-4 inhibitors as long acting antidiabetic that are clinically on demand are identified, but the target fish docking and medicinal chemistry strategies were successfully implemented.

Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

In our efforts to develop second generation DPP-4 inhibitors, we endeavored to identify distinct structures with long-acting (once weekly) potential. Taking advantage of X-ray cocrystal structures of sitagliptin and other DPP-4 inhibitors, such as alogliptin and linagliptin bound to DPP-4, and aided by molecular modeling, we designed several series of heterocyclic compounds as initial targets. During their synthesis, an unexpected chemical transformation provided a novel tricyclic scaffold that was beyond our original design. Capitalizing on this serendipitous discovery, we have elaborated this scaffold into a very potent and selective DPP-4 inhibitor lead series, as highlighted by compound 17c.

UNLABELLED: The novel emerging coronavirus Middle East respiratory syndrome coronavirus (MERS-CoV) binds to its receptor, dipeptidyl peptidase 4 (DPP4), via 14 interacting amino acids. We previously showed that if the five interacting amino acids which differ between hamster and human DPP4 are changed to the residues found in human DPP4, hamster DPP4 does act as a receptor. Here, we show that the functionality of hamster DPP4 as a receptor is severely decreased if less than 4 out of 5 amino acids are changed. IMPORTANCE: The novel emerging coronavirus MERS-CoV has infected >1,600 people worldwide, and the case fatality rate is approximately 36%. In this study, we show that by changing 4 amino acids in hamster DPP4, this protein functions as a receptor for MERS-CoV. This work is vital in the development of new small-animal models, which will broaden our understanding of MERS-CoV and be instrumental in the development of countermeasures.

The single-crystal structure of anagliptin, N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methyl pyrazolo[1,5-a]pyrimidine-6-carboxamide, was determined. Two independent molecules were held together by intermolecular hydrogen bonds, and the absolute configuration of the 2-cyanopyrrolidine ring delivered from l-prolinamide was confirmed to be S. The interactions of anagliptin with DPP-4 were clarified by the co-crystal structure solved at 2.85 A resolution. Based on the structure determined by X-ray crystallography, the potency and selectivity of anagliptin were discussed, and an SAR study using anagliptin derivatives was performed.

In our effort to discover DPP-4 inhibitors with added benefits over currently commercially available DPP-4 inhibitors, MK-3102 (omarigliptin), was identified as a potent and selective dipeptidyl peptidase 4 (DPP-4) inhibitor with an excellent pharmacokinetic profile amenable for once-weekly human dosing and selected as a clinical development candidate. This manuscript summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for omarigliptin, which is currently in phase 3 clinical development.

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.

Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through binding the receptor binding domain (RBD) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hDPP4). Here, we report identification of critical residues on hDPP4 for RBD binding and virus entry through analysis of a panel of hDPP4 mutants. Based on the RBD-hDPP4 crystal structure we reported, the mutated residues were located at the interface between RBD and hDPP4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hDPP4, thereby interfering or disrupting their interaction with RBD. Using surface plasmon resonance (SPR) binding analysis and pseudovirus infection assay, we showed that several residues in hDPP4-RBD binding interface were important on hDPP4-RBD binding and viral entry. These results provide atomic insights into the features of interactions between hDPP4 and MERS-CoV RBD, and also provide potential explanation for cellular and species tropism of MERS-CoV infection.

The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) is phylogenetically closely related to the bat coronaviruses (BatCoVs) HKU4 and HKU5. However, the evolutionary pathway of MERS-CoV is still unclear. A receptor binding domain (RBD) in the MERS-CoV envelope-embedded spike protein specifically engages human CD26 (hCD26) to initiate viral entry. The high sequence identity in the viral spike protein prompted us to investigate if HKU4 and HKU5 can recognize hCD26 for cell entry. We found that HKU4-RBD, but not HKU5-RBD, binds to hCD26, and pseudotyped viruses embedding HKU4 spike can infect cells via hCD26 recognition. The structure of the HKU4-RBD/hCD26 complex revealed a hCD26-binding mode similar overall to that observed for MERS-RBD. HKU4-RBD, however, is less adapted to hCD26 than MERS-RBD, explaining its lower affinity for receptor binding. Our findings support a bat origin for MERS-CoV and indicate the need for surveillance of HKU4-related viruses in bats.

Dipeptidyl peptidase IV (DPP4) inhibitors are proven in the treatment of type 2 diabetes. We designed and synthesized a series of novel indole compounds that selectively inhibit the activity of DPP4 over dipeptidyl peptidase 9 (DPP9) (>200 fold). We further co-crystallized DPP4 with indole sulfonamide (compound 1) to confirm a proposed binding mode. Good metabolic stability of the indole compounds represents another positive attribute for further development.

Optimization of a 5-oxopyrrolopyridine series based upon structure-activity relationships (SARs) developed from our previous efforts on a number of related bicyclic series yielded compound 2s (BMS-767778) with an overall activity, selectivity, efficacy, PK, and developability profile suitable for progression into the clinic. SAR in the series and characterization of 2s are described.

The newly emergent Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans, representing the second example of a highly pathogenic coronavirus, the first being SARS-CoV. CD26 (also known as dipeptidyl peptidase 4, DPP4) was recently identified as the cellular receptor for MERS-CoV. The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus-cell fusion, thereby initiating infection. Here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Furthermore, binding between the RBD and CD26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nM. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 beta-propeller. The atomic details at the interface between the two binding entities reveal a surprising protein-protein contact mediated mainly by hydrophilic residues. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition.

In recent years, various dipeptidyl peptidase IV (DPP-4) inhibitors have been released as therapeutic drugs for type 2 diabetes in many countries. In spite of their diverse chemical structures, no comparative studies of their binding modes in the active site of DPP-4 have been disclosed. We determined the co-crystal structure of vildagliptin with DPP-4 by X-ray crystallography and compared the binding modes of six launched inhibitors in DPP-4. The inhibitors were categorized into three classes on the basis of their binding subsites: (i) vildagliptin and saxagliptin (Class 1) form interactions with the core S1 and S2 subsites and a covalent bond with Ser630 in the catalytic triad; (ii) alogliptin and linagliptin (Class 2) form interactions with the S1' and/or S2' subsites in addition to the S1 and S2 subsites; and (iii) sitagliptin and teneligliptin (Class 3) form interactions with the S1, S2 and S2 extensive subsites. The present study revealed that the additional interactions with the S1', S2' or S2 extensive subsite may increase DPP-4 inhibition beyond the level afforded by the fundamental interactions with the S1 and S2 subsites and are more effective than forming a covalent bond with Ser630.

The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor, dipeptidyl peptidase 4 (DPP4). Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike, which mediates this interaction. We report the 3.0 A-resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4. Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain. The receptor-binding subdomain interacts with DPP4 beta-propeller but not its intrinsic hydrolase domain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor-binding subdomain. Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell. The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction, which can guide development of therapeutics and vaccines against MERS-CoV infection.

The worldwide prevalence of diabetes has spurred numerous studies on the development of new antidiabetic medicines. As a result, dipeptidyl peptidase IV (DPP4) has been recognized as a validated target. In our efforts to discover new DPP4 inhibitors, we analyzed the complexed structures of DPP4 available in Protein Data Bank and designed a series of triazole compounds. After enzyme activity assays and crystallographic verification of the binding interaction patterns, we found that the triazole compounds can inhibit DPP4 with micromolar IC50 values. Liver microsome stability and cytochrome P450 metabolic tests were performed on this series, revealing undesirable pharmacokinetic profiles for the triazole compounds. To overcome this liability, we substituted the triazole ring with an amide or urea group to produce a new series of DPP4 inhibitors. Based on its enzyme activity, metabolic stability, and selectivity over DPP8 and DPP9, we selected compound 21 r for further study of its in vivo effects in mice using an oral glucose tolerance test (OGTT). The results show that 21 r has efficacy similar to that of sitagliptin at a dose of 3 mg kg(-1) . The crystal structure of 21 r bound to DPP4 also reveals that the trifluoromethyl group is directed toward a subpocket different from the subsite bound by sitagliptin, providing clues for the design of new DPP4 inhibitors.

Dipeptidyl peptidase IV (DPP-4) inhibitors have been shown to enhance GLP-1 levels and thereby improve hyperglycemia in type II diabetes. From a small fragment hit, using structure-based design, we have discovered a new class of non-covalent, potent and selective DPP-4 inhibitors.

Novel deazaxanthine-based DPP-4 inhibitors have been identified that are potent (IC(50) <10nM) and highly selective versus other dipeptidyl peptidases. Their synthesis and SAR are reported, along with initial efforts to improve the PK profile through decoration of the deazaxanthine core. Optimisation of compound 3a resulted in the identification of compound (S)-4i, which displayed an improved in vitro and ADME profile. Further enhancements to the PK profile were possible by changing from the deazahypoxanthine to the deazaxanthine template, culminating in compound 12g, which displayed good ex vivo DPP-4 inhibition and a superior PK profile in rat, suggestive of once daily dosing in man.

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the gamma-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-yl carbonyl]thiazolidine (8 g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8 g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S(2) extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8 g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8 g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.

Hypoglycemic agents with a mechanism of depeptidyl peptidase IV (DPP-4) inhibition are suitable for once daily oral dosing. It is difficult to strike a balance between inhibitory activity and duration of action in plasma for inhibitors bearing an electrophilic nitrile group. We explored fused bicyclic heteroarylpiperazine substituted at the gamma-position of the proline structure in the investigation of L-prolylthiazolidines lacking the electrophilic nitrile. Among them, 2-trifluoroquinolyl compound 8g is the most potent, long-lasting DPP-4 inhibitor (IC(50) = 0.37 nmol/L) with high selectivity against other related peptidases. X-ray crystal structure determination of 8g indicates that CH-pi interactions generated between the quinolyl ring and the guanidinyl group of Arg358 enhances the DPP-4 inhibitory activity and selectivity.

The design, synthesis, and structure-activity relationships of a new class of potent and orally active non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones, are described. We hypothesized that the 4-phenyl group of the isoquinolone occupies the S1 pocket of the enzyme, the 3-aminomethyl group forms an electrostatic interaction with the S2 pocket, and the introduction of a hydrogen bond donor onto the 6- or 7-substituent provides interaction with the hydrophilic region of the enzyme. Based on this hypothesis, intensive research focused on developing new non-peptide DPP-4 inhibitors has been carried out. Among the compounds designed in this study, we identified 2-[(3-aminomethyl-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-6-isoquinolinyl)o xy]acetamide (35a) as a potent, selective, and orally bioavailable DPP-4 inhibitor, which exhibited in vivo efficacy in diabetic model rats. Finally, X-ray crystallography of 35a in a complex with the enzyme validated our hypothesized binding mode and identified Lys554 as a new target-binding site available for DPP-4 inhibitors.

Dipeptidyl peptidase IV (DPP-4) inhibition is a validated therapeutic option for type 2 diabetes, exhibiting multiple antidiabetic effects with little or no risk of hypoglycemia. In our studies involving non-covalent DPP-4 inhibitors, a novel series of quinoline-based inhibitors were designed based on the co-crystal structure of isoquinolone 2 in complex with DPP-4 to target the side chain of Lys554. Synthesis and evaluation of designed compounds revealed 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]piperazine- 2,5-dione (1) as a potent, selective, and orally active DPP-4 inhibitor (IC(5)(0)=1.3 nM) with long-lasting ex vivo activity in dogs and excellent antihyperglycemic effects in rats. A docking study of compound 1 revealed a hydrogen-bonding interaction with the side chain of Lys554, suggesting this residue as a potential target site useful for enhancing DPP-4 inhibition.

Inhibition of dipeptidyl peptidase IV (DPP-4) is an exciting new approach for the treatment of diabetes. To date there has been no DPP-4 chemotype possessing a carboxy group that has progressed into clinical trials. Originating from the discovery of the structurally novel quinoline derivative 1, we designed novel pyridine derivatives containing a carboxy group. In our design, the carboxy group interacted with the targeted amino acid residues around the catalytic region and thereby increased the inhibitory activity. After further optimization, we identified a hydrate of [5-(aminomethyl)-6-(2,2-dimethylpropyl)-2-ethyl-4-(4-methylphenyl)pyridin-3-yl]ac etic acid (30c) as a potent and selective DPP-4 inhibitor. The desired interactions with the critical active-site residues, such as a salt-bridge interaction with Arg125, were confirmed by X-ray cocrystal structure analysis. In addition, compound 30c showed a desired preclinical safety profile, and it was encoded as TAK-100.

A series of beta-aminoacyl containing thiazolidine derivatives was synthesized and evaluated for their ability to inhibit DPP-IV. Several thiazolidine derivatives with an acid moiety were found to be potent DPP-IV inhibitors. Among them, compound 2da is the most active in this series with an IC(50) value of 1 nM, and it showed excellent selectivity over DPP-IV related enzymes including DPP-2, DPP-8, and DPP-9. Compound 2da is chemically and metabolically stable, and showed no CYP inhibition, hERG binding or cytotoxicity. Compound 2db, an ester prodrug of 2da, showed good in vivo DPP-IV inhibition after oral administration in rat and dog models.

The dipeptidyl peptidase (DPP) family members, including DPP-IV, DPP8, DPP9 and others, cleave the peptide bond after the penultimate proline residue and are drug target rich. The dimerization of DPP-IV is required for its activity. A propeller loop located at the dimer interface is highly conserved within the family. Here we carried out site-directed mutagenesis on the loop of DPPIV and identified several residues important for dimer formation and enzymatic activity. Interestingly, the corresponding residues on DPP9 have a different impact whereby the mutations decrease activity without changing dimerization. Thus the propeller loop seems to play a varying role in different DPPs.

Design, synthesis, and SAR of 7-oxopyrrolopyridine-derived DPP4 inhibitors are described. The preferred stereochemistry of these atropisomeric biaryl analogs has been identified as Sa. Compound (+)-3t, with a K(i) against DPP4, DPP8, and DPP9 of 0.37 nM, 2.2, and 5.7 muM, respectively, showed a significant improvement in insulin response after single doses of 3 and 10 mumol/kg in ob/ob mice.

The discovery of two classes of heterocyclic dipeptidyl peptidase IV (DPP-4) inhibitors, pyrimidinones and pyrimidinediones, is described. After a single oral dose, these potent, selective, and noncovalent inhibitors provide sustained reduction of plasma DPP-4 activity and lowering of blood glucose in animal models of diabetes. Compounds 13a, 27b, and 27j were selected for development.

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.

Design, synthesis, and SAR are described for a class of DPP-IV inhibitors based on aminobenzo[a]quinolizines with non-aromatic substituents in the S1 specificity pocket. One representative thereof, carmegliptin (8p), was chosen for clinical development. Its X-ray structure in complex with the enzyme and early efficacy data in animal models of type 2 diabetes are also presented.

Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.

A series of 4-substituted proline amides was synthesized and evaluated as inhibitors of dipeptidyl pepdidase IV for the treatment of type 2 diabetes. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrr olidin-2-yl]-methanone (5) emerged as a potent (IC(50) = 13 nM) and selective compound, with high oral bioavailability in preclinical species and low plasma protein binding. Compound 5, PF-00734200, was selected for development as a potential new treatment for type 2 diabetes.

A new series of DPP-4 inhibitors derived from piperidine-fused benzimidazoles and imidazopyridines is described. Optimization of this class of DPP-4 inhibitors led to the discovery of imidazopyridine 34. The potency, selectivity, cross-species DMPK profiles, and in vivo efficacy of 34 is reported.

Modifications of DPP-4 inhibitor 5, that was discovered by structure based design, are described and structure-activity relationships discussed. With analogue 7k one of the most potent non-covalent inhibitors of DPP-4 reported to date (IC(50)=0.38nM) was discovered. X-ray structure of inhibitor 7k bound to DPP-4 revealed a hydrogen bonding interaction with Q553. First successful efforts in balancing overall properties, as demonstrated by improved metabolic stability, highlight the potential of this series.

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.

The synthesis, selectivity, rat pharmacokinetic profile, and drug metabolism profiles of a series of potent fluoroolefin-derived DPP-4 inhibitors (4) are reported. A radiolabeled fluoroolefin 33 was shown to possess a high propensity to form reactive metabolites, thus revealing a potential liability for this class of DPP-4 inhibitors.

A series of pyrazoline derivatives with beta-amino acyl group were synthesized and evaluated for their ability to inhibit dipeptidyl peptidase IV. Several pyrazoline derivatives exhibited submicromolar inhibitory activities against DPP-IV. X-ray co-crystal structure of initial hit compound 1h was determined. Among this series, carboxylic acid substituted pyrazoline derivative 2u was the most active and greatly decreased the inhibitory activity toward CYP3A4 enzyme.

Probing with tool molecules, and by modeling and X-ray crystallography the binding modes of two structurally distinct series of DPP-4 inhibitors led to the discovery of a rare aromatic fluorine H-bond and the spatial requirement for better biaryl binding in the DPP-4 enzyme active site. These newly found binding elements were successfully incorporated into novel DPP-4 inhibitors.

The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 A). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an approximately 1000-fold reduction in affinity relative to DPP-IV WT, while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at approximately 14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.

A novel series of non-covalent, benzimidazole-based inhibitors of DPP-4 has been developed from a small fragment hit using structure-based drug design. A highly versatile synthetic route was created for the development of SAR, which led to the discovery of potent and selective inhibitors with excellent pharmaceutical properties.

Inhibitors of dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes. A series of beta-aminoacyl-containing cyclic hydrazine derivatives were synthesized and evaluated as DPP-IV inhibitors. One member of this series, (R)-3-amino-1-(2-benzoyl-1,2-diazepan-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one (10f), showed potent in vitro activity, good selectivity and in vivo efficacy in mouse models. Also, the binding mode of compound 10f was determined by X-ray crystallography.

A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable parallel chemistry, and protein co-crystal structural data guided the optimization of N-substituents. Employing this strategy, a >400x improvement in potency over the initial hit was realized in rapid fashion. Optimized compounds are potent and selective inhibitors with excellent pharmacokinetic profiles. Compound 30 was efficacious in vivo, lowering blood glucose in ZDF rats that were allowed to feed freely on a mixed meal.

Replacement of the triazolopiperazine ring of sitagliptin (DPP-4 IC(50)=18nM) with 3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one gave dipeptidyl peptidase IV (DPP-4) inhibitor 1 which is potent (DPP-4 IC(50)=2.6nM), selective, and efficacious in an oral glucose tolerance test in mice. It was selected for extensive preclinical development as a potential back-up candidate to sitagliptin.

Molecular modeling was used to design a rigid analog of sitagliptin 1. The X-ray crystal structure of sitagliptin bound to DPP-4 suggested that the central beta-amino butyl amide moiety could be replaced with a cyclohexylamine group. This was confirmed by structural analysis and the resulting analog 2a was synthesized and found to be a potent DPP-4 inhibitor (IC(50)=21 nM) with excellent in vivo activity and pharmacokinetic profile.

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.

A novel series of 4-aminophenylalanine and 4-aminocyclohexylalanine derivatives were designed and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4). The phenylalanine series afforded compounds such as 10 that were potent and selective (DPP-4, IC(50)=28nM), but exhibited limited oral bioavailability. The corresponding cyclohexylalanine derivatives such as 25 afforded improved PK exposure and efficacy in a murine OGTT experiment. The X-ray crystal structure of 25 bound to the DPP-4 active site is presented.

A new chemical class of potent DPP-4 inhibitors structurally derived from the xanthine scaffold for the treatment of type 2 diabetes has been discovered and evaluated. Systematic structural variations have led to 1 (BI 1356), a highly potent, selective, long-acting, and orally active DPP-4 inhibitor that shows considerable blood glucose lowering in different animal species. 1 is currently undergoing clinical phase IIb trials and holds the potential for once-daily treatment of type 2 diabetics.

Alogliptin is a potent, selective inhibitor of the serine protease dipeptidyl peptidase IV (DPP-4). Herein, we describe the structure-based design and optimization of alogliptin and related quinazolinone-based DPP-4 inhibitors. Following an oral dose, these noncovalent inhibitors provide sustained reduction of plasma DPP-4 activity and a lowering of blood glucose in animal models of diabetes. Alogliptin is currently undergoing phase III trials in patients with type 2 diabetes.

Dipeptidyl peptidase IV (DPPIV), which belongs to the prolyl oligopeptidase family of serine proteases, is known to have a variety of regulatory biological functions and has been shown to be implicated in type 2 diabetes. It is therefore important to develop selective human DPPIV (hDPPIV) inhibitors. In this study, we determined the crystal structure of apo hDPPIV at 1.9 A resolution. Our high-resolution crystal structure of apo hDPPIV revealed the presence of sodium ion and glycerol molecules at the active site. In order to elucidate the hDPPIV binding mode and substrate specificity, we determined the crystal structure of hDPPIV-diprotin B (Val-Pro-Leu) complex at 2.1 A resolution, and clarified the difference in binding mode between diprotin B and diprotin A (Ile-Pro-Ile) into the active site of hDPPIV. Comparison between our crystal structures and the reported apo hDPPIV structures revealed that positively charged functional groups and conserved water molecules contributed to the interaction of ligands with hDPPIV. These results are useful for the design of potent hDPPIV inhibitors.

A novel series of 4-arylcyclohexylalanine DPP-4 inhibitors was synthesized and tested for inhibitory activity as well as selectivity over the related proline-specific enzymes DPP-8 and DPP-9. Optimization of this series led to 28 (DPP-4 IC(50)=4.8 nM), which showed an excellent pharmacokinetic profile across several preclinical species. Evaluation of 28 in an oral glucose tolerance test demonstrated that this compound effectively reduced glucose excursion in lean mice.

Synthesis of a novel series of DPPIV inhibitors with 1,2,4- and 1,3,4-oxadiazolyl ketone derivatives and its structure-activity relationships are discussed. Compound 18h showed good inhibitory activity against DPPIV and favorable pharmacokinetic properties. In vivo pharmacodynamic efficacy and co-crystal structure of compound 18h with DPPIV is also described.

Various beta-amino amides containing triazolopiperazine heterocycles have been prepared and evaluated as potent, selective, orally active dipeptidyl peptidase IV (DPP-4) inhibitors. These compounds display excellent oral bioavailability and good overall pharmacokinetic profiles in preclinical species. Moreover, in vivo efficacy in an oral glucose tolerance test in lean mice is demonstrated.

In a search for novel DPP-IV inhibitors, 2-aminobenzo[a]quinolizines were identified as submicromolar HTS hits. Due to the difficult synthetic access to this compound class, 1,3-disubstituted 4-aminopiperidines were used as model compounds for optimization. The developed synthetic methodology and the SAR could be transferred to the 2-aminobenzo[a]quinolizine series, leading to highly active DPP-IV inhibitors.

Dipeptidyl peptidase IV (DPP4) inhibitors are emerging as a new class of therapeutic agents for the treatment of type 2 diabetes. They exert their beneficial effects by increasing the levels of active glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are two important incretins for glucose homeostasis. Starting from a high-throughput screening hit, we were able to identify a series of piperidinone- and piperidine-constrained phenethylamines as novel DPP4 inhibitors. Optimized compounds are potent, selective, and have good pharmacokinetic profiles.

A series of non-covalent inhibitors of the serine protease dipeptidyl peptidase IV (DPP-IV) were found to adopt a U-shaped binding conformation in X-ray co-crystallization studies. Remarkably, Tyr547 undergoes a 70 degrees side-chain rotation to accommodate the inhibitor and allows access to a previously unexposed area of the protein backbone for hydrogen bonding.

A series of pyrrolidine based inhibitors of dipeptidyl peptidase IV were developed from a high throughput screening hit for the treatment of type 2 diabetes. Potency, selectivity, and pharmacokinetic properties were optimized resulting in the identification of a pre-clinical candidate for further profiling.

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.

A series of beta-substituted biarylphenylalanine amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the metabolic profile of early analogues led to the discovery of (2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]tr iazolo[1,5-a]pyridin-6-ylphenyl)butanamide (6), a potent, orally active DPP-4 inhibitor (IC(50) = 6.3 nM) with excellent selectivity, oral bioavailability in preclinical species, and in vivo efficacy in animal models. Compound 6 was selected for further characterization as a potential new treatment for type 2 diabetes.

Dipeptidyl peptidase-IV (DPP-IV) inhibitors are poised to be the next major drug class for the treatment of type 2 diabetes. Structure-activity studies of substitutions at the C5 position of the 2-cyanopyrrolidide warhead led to the discovery of potent inhibitors of DPP-IV that lack activity against DPP8 and DPP9. Further modification led to an extremely potent (Ki(DPP)(-)(IV) = 1.0 nM) and selective (Ki(DPP8) > 30 microM; Ki(DPP9) > 30 microM) clinical candidate, ABT-279, that is orally available, efficacious, and remarkably safe in preclinical safety studies.

Dipeptidyl peptidase IV (DPP4) deactivates glucose-regulating hormones such as GLP-1 and GIP, thus, DPP4 inhibition has become a useful therapy for type 2 diabetes. Optimization of the high-throughput screening lead 6 led to the discovery of 25 (ABT-341), a highly potent, selective, and orally bioavailable DPP4 inhibitor. When dosed orally, 25 dose-dependently reduced glucose excursion in ZDF rats. Amide 25 is safe in a battery of in vitro and in vivo tests and may represent a new therapeutic agent for the treatment of type 2 diabetes.

A series of (5-substituted pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidine (C5-Pro-Pro) analogues was discovered as dipeptidyl peptidase IV (DPPIV) inhibitors as a potential treatment of diabetes and obesity. X-ray crystallography data show that these inhibitors bind to the catalytic site of DPPIV with the cyano group forming a covalent bond with the serine residue of DPPIV. The C5-substituents make various interactions with the enzyme and affect potency, chemical stability, selectivity, and PK properties of the inhibitors. Optimized analogues are extremely potent with subnanomolar K(i)'s, are chemically stable, show very little potency decrease in the presence of plasma, and exhibit more than 1,000-fold selectivity against related peptidases. The best compounds also possess good PK and are efficacious in lowering blood glucose in an oral glucose tolerance test in ZDF rats.

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.

Dipeptidyl peptidase-IV (DPP-IV) inhibitors, or glucagon-like peptide-1 (GLP-1) enhancers, are looked to as a potential new class of antidiabetic agents. In particular, potent and long-acting inhibitors might offer advantages in exploiting DPP-IV inhibition. The series of [(S)-gamma-(arylamino)prolyl]-(S)-2-cyanopyrrolidine compounds on which we reported previously has a highly potent inhibitory activity but seemed to be unstable in neutral aqueous solution. Here, we describe [(S)-gamma-(arylamino)prolyl]thiazolidine compounds as a novel series of potent and stable DPP-IV inhibitors. They are the thiazolidine analogs of [(S)-gamma-(arylamino)prolyl]-(S)-2-cyanopyrrolidine but with the electrophilic nitrile removed to improve chemical stability in aqueous solution. Of the compounds investigated in the present study, the [((S)-gamma-3,4-dicyanophenylamino)prolyl]thiazolidine 12 m was the most potent. The structure-activity relationship (SAR) of the gamma-substituent in the proline moiety of the thiazolidide was similar to that obtained with the (S)-2-cyanopyrrolidide. The gamma-substituent in the proline moiety of both the (S)-2-cyanopyrrolidide and the thiazolidide may engage with the S(2) binding pocket of DPP-IV and thereby achieve hydrophobic interaction in the same manner. Based on pharmacokinetic experiments in rats, the representative compound 11, which displayed high oral bioavailability (BA=83.9%) and long half-life in plasma (t(1/2)=5.27 h), was found to have an excellent pharmacokinetic profile.

A novel series of oxadiazole based amides have been shown to be potent DPP-4 inhibitors. The optimized compound 43 exhibited excellent selectivity over a variety of DPP-4 homologs.

A novel series of beta-amino amides incorporating fused heterocycles, i.e., triazolopiperazines, were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)- yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (1) is a potent, orally active DPP-IV inhibitor (IC(50) = 18 nM) with excellent selectivity over other proline-selective peptidases, oral bioavailability in preclinical species, and in vivo efficacy in animal models. MK-0431, the phosphate salt of compound 1, was selected for development as a potential new treatment for type 2 diabetes.

CD26 or dipeptidyl-peptidase IV (DPPIV) is engaged in immune functions by co-stimulatory effects on activation and proliferation of T lymphocytes, binding to adenosine deaminase, and regulation of various chemokines and cytokines. DPPIV peptidase activity is inhibited by both Tat protein from human immunodeficiency virus (HIV)-1 and its N-terminal nonapeptide Tat-(1-9) with amino acid sequence MDPVDPNIE, suggesting that DPPIV mediates immunosuppressive effects of Tat protein. The 2.0- and 3.15-A resolution crystal structures of the binary complex between human DPPIV and nonapeptide Tat-(1-9) and the ternary complex between the variant MWPVDPNIE, called Trp(2)-Tat-(1-9), and DPPIV bound to adenosine deaminase show that Tat-(1-9) and Trp(2)-Tat-(1-9) are located in the active site of DPPIV. The interaction pattern of DPPIV with Trp(2)-Tat-(1-9) is tighter than that with Tat-(1-9), in agreement with inhibition constants (K(i)) of 2 x 10(-6) and 250 x 10(-6) m, respectively. Both peptides cannot be cleaved by DPPIV because the binding pockets of the N-terminal 2 residues are interchanged compared with natural substrates: the N-terminal methionine occupies the hydrophobic S1 pocket of DPPIV that normally accounts for substrate specificity by binding the penultimate residue. Because the N-terminal sequence of the thromboxane A2 receptor resembles the Trp(2)-Tat-(1-9) peptide, a possible interaction with DPPIV is postulated.

Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates, including many chemokines, neuropeptides, and peptide hormones. Specific inhibition of DPPIV is being investigated in human trials for the treatment of type II diabetes. To understand better the molecular determinants that underlie enzyme catalysis and substrate specificity, we report the crystal structures of DPPIV in the free form and in complex with the first 10 residues of the physiological substrate, Neuropeptide Y (residues 1-10; tNPY). The crystal structure of the free form of the enzyme reveals two potential channels through which substrates could access the active site-a so-called propeller opening, and side opening. The crystal structure of the DPPIV/tNPY complex suggests that bioactive peptides utilize the side opening unique to DPPIV to access the active site. Other structural features in the active site such as the presence of a Glu motif, a well-defined hydrophobic S1 subsite, and minimal long-range interactions explain the substrate recognition and binding properties of DPPIV. Moreover, in the DPPIV/tNPY complex structure, the peptide is not cleaved but trapped in a tetrahedral intermediate that occurs during catalysis. Conformational changes of S630 and H740 between DPPIV in its free form and in complex with tNPY were observed and contribute to the stabilization of the tetrahedral intermediate. Our results facilitate the design of potent, selective small molecule inhibitors of DPPIV that may yield compounds for the development of novel drugs to treat type II diabetes.

Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr(547) and a water molecule positioned in close proximity to Tyr(547). To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases.

Dipeptidyl peptidase IV (DPPIV) is a serine protease, a member of the prolyl oligopeptidase (POP) family, and has been implicated in several diseases. Therefore, it seems important to develop selective inhibitors for human DPPIV (hDPPIV) that are able to control the biological function of hDPPIV. In order to elucidate the binding mode and substrate specificity, we determined the crystal structure complex of hDPPIV and diprotin A (IIe-Pro-IIe), a slowly hydrolyzed substrate of hDPPIV, at 2.2 A resolution. In this paper, we discuss the molecular interaction mechanism of diprotin A with hDPPIV based on the X-ray crystal structure.

The influence of aromatic substitution on a newly discovered class of inhibitors of dipeptidyl peptidase IV was investigated. A 10(5)-fold increase in potency was achieved by the optimization of aromatic substituents in a parallel chemistry program. The observed SAR could be explained by an X-ray structure of the protein-ligand complex.

Dipeptidyl-peptidase IV (DPPIV or CD26) is a homodimeric type II membrane glycoprotein in which the two monomers are subdivided into a beta-propeller domain and an alpha/beta-hydrolase domain. As dipeptidase, DPPIV modulates the activity of various biologically important peptides and, in addition, DPPIV acts as a receptor for adenosine deaminase (ADA), thereby mediating co-stimulatory signals in T-lymphocytes. The 3.0-A resolution crystal structure of the complex formed between human DPPIV and bovine ADA presented here shows that each beta-propeller domain of the DPPIV dimer binds one ADA. At the binding interface, two hydrophobic loops protruding from the beta-propeller domain of DPPIV interact with two hydrophilic and heavily charged alpha-helices of ADA, giving rise to the highest percentage of charged residues involved in a protein-protein contact reported thus far. Additionally, four glycosides linked to Asn229 of DPPIV bind to ADA. In the crystal structure of porcine DPPIV, the observed tetramer formation was suggested to mediate epithelial and lymphocyte cell-cell adhesion. ADA binding to DPPIV could regulate this adhesion, as it would abolish tetramerization.

Dipeptidyl peptidase IV (DPPIV) is a serine protease, a member of the prolyl oligopeptidase (POP) family, and has been implicated in several diseases. Therefore, the development of DPPIV selective inhibitors, which are able to control the biological function of DPPIV, is important. We determined the crystal structure of human DPPIV at 2.6A resolution. The molecule consists of a unique eight-bladed beta-propeller domain in the N-terminal region and a serine protease domain in the C-terminal region. Also, the large "cave" structure, which is thought to control the access of the substrate, is found on the side of the beta-propeller fold. Comparison of the overall amino acid sequence between human DPPIV and POP shows low homology (12.9%). In this paper, we report the structure of human DPPIV, especially focusing on a unique eight-bladed beta-propeller domain. We also discuss the way for the access of the substrate to this domain.

Dipeptidyl peptidase IV is a multifunctional type II transmembrane serine protease glycoprotein. The high-resolution crystal structure of the homodimeric human apo dipeptidyl peptidase IV has been determined at 1.9 A resolution. In addition, the structure of the binary complex with 1-[([2-[(5-iodopyridin-2-yl)amino]-ethyl]amino)-acetyl]-2-cyano-(S)-pyrrolidine has been solved, revealing the nature of the covalent interaction with the active-site serine.

Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 A structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.

Inhibition of dipeptidyl peptidase IV (DPP-IV), the main glucagon-like peptide 1 (GLP1)-degrading enzyme, has been proposed for the treatment of type II diabetes. We expressed and purified the ectodomain of human DPP-IV in Pichia pastoris and determined the X-ray structure at 2.1 A resolution. The enzyme consists of two domains, the catalytic domain, with an alpha/beta hydrolase fold, and a beta propeller domain with an 8-fold repeat of a four-strand beta sheet motif. The beta propeller domain contributes two important functions to the molecule that have not been reported for such structures, an extra beta sheet motif that forms part of the dimerization interface and an additional short helix with a double Glu sequence motif. The Glu motif provides recognition and a binding site for the N terminus of the substrates, as revealed by the complex structure with diprotin A, a substrate with low turnover that is trapped in the tetrahedral intermediate of the reaction in the crystal.

The dipeptidyl peptidase IV gene encodes a plasma-membrane exopeptidase that is highly expressed in small intestine, lung and kidney. In order to better understand the mechanisms responsible for this tissue-specific expression we cloned, sequenced and functionally characterized the 5'-flanking region of the human dipeptidyl peptidase IV gene. The first 500 bases of the 5'-flanking sequence constituted an unmethylated CpG island, contained several Sp1-binding sites and lacked a consensus TATA box, all characteristics of gene promoters lacking tissue-specific expression. RNase-protection analysis using both small intestinal and Caco2 cell RNA indicated that the dipeptidyl peptidase IV transcript was initiated from no fewer than six major and 12 minor start sites. The 5'-flanking sequence also exhibited functional promoter activity in transient transfection experiments. Here, various lengths of the sequence were cloned upstream of a luciferase gene and introduced into cultured cells using lipofectin. A region located between bases -150 and -109 relative to the start of translation was found to be important for high-level promoter activity in both Caco2 and HepG2 cells. Moreover, Caco2 cells and HepG2 cells, which express high levels of dipeptidyl peptidase IV activity, exhibited much higher normalized luciferase activity after transfection than did 3T3, Jurkat or COS-7 cells, which have low enzyme levels. Sodium butyrate was found to increase both enzyme activity and normalized luciferase in HepG2 cells. Thus the dipeptidyl peptidase IV promoter possesses the ability to initiate transcription in a tissue-specific fashion in spite of having the sequence characteristics of a housekeeping gene promoter.

CD26 is a lymphocyte cell surface antigen which is increased during T-cell activation and is also expressed in other tissues. It is an atypical serine protease belonging to the prolyl oligopeptidase family. CD26 has been implicated in a variety of biological functions including T-cell activation, cell-to-cell adhesion, and recently in HIV infection. This paper describes, through the isolation and partial sequencing of eight human CD26 genomic clones, the first information on the genomic organization of the prolyl oligopeptidase family. We have established that the human CD26 gene spans approximately 70 kilobases (kb) and contains 26 exons, ranging in size from 45 base pairs (bp) to 1.4 kb. The nucleotides that encode the serine recognition site (G-W-S-Y-G) are split between two exons. This clearly distinguishes the genomic organization of the prolyl oligopeptidase family from that of the classical serine protease family. The 5' flanking domain of the CD26 gene contains neither a TATA box nor a CAAT box, but a 300 bp region extremely rich in C and G (72%) contains potential binding sites for several transcriptional factors. The human CD26 gene encodes two messages sized at about 4.2 and 2.8 kb. These are both expressed at high levels in the placenta and kidney and at moderate levels in the lung and liver. Only the 4.2 kb mRNA was expressed at low levels in skeletal muscle, heart, brain, and pancreas. Fluorescence in situ hybridization on metaphase chromosome spreads located the human CD26 gene to the long arm of chromosome 2(2q24.3).

A cDNA (DPCR1) specific for human intestinal dipeptidyl peptidase IV (DPP IV) has been isolated. This 1.7-kilobase cDNA, together with a previously published partial sequence, covers the entire open reading frame of human DPP IV plus 67 base pairs of the 3'-untranslated end. Human DPP IV is a 766-amino acid polypeptide with a high degree of homology with the rat liver protein. The characterization of this molecular probe allowed us to definitively confirm the identity of DPP IV with CD 26, a mouse thymocyte activation antigen, a conclusion strengthened by the fact that we observed identical patterns on Southern blot of human genomic DNA hybridized either with human DPP IV or mouse CD 26 cDNA probe. Using this new tool, we have investigated the expression of DPP IV during the onset of enterocytic differentiation of two cultured human colon cancer cell lines, HT-29 and Caco-2. Whatever the cell line and the culture conditions, DPP IV expression strictly correlates with the presence of a differentiated phenotype, as shown by enzyme activity and the steady state amount of the protein measured by indirect immunofluorescence and Western blot. Accordingly, DPP IV biosynthesis exclusively increases in cells that display an enterocytic differentiation. Neither the glycosylation nor the stability of the protein appear to be dependent on the state of enterocytic differentiation. The DPP IV mRNA level remains very low in undifferentiated cell populations and specifically increases in cells that undergo an enterocytic differentiation. These results strongly suggest that DPP IV gene expression is controlled at the transcriptional or posttranscriptional level during intestinal differentiation.

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell activation.

We report the nucleotide sequence and derived amino-acid sequence of a cDNA clone encoding the 3' end of human intestinal dipeptidylpeptidase IV (DPP-IV). This cDNA probe identifies a 4 kb mRNA in the human colon cancer cell line Caco-2. We demonstrate here an extensive homology between this human DPP-IV cDNA and the recently published rat liver DPP-IV cDNA. Using the human DPP-IV cDNA to probe genomic DNA from a panel of somatic cell hybrids we have assigned the gene encoding human DPP-IV to chromosome 2.