Potent and selective proline derived dipeptidyl peptidase IV inhibitors

In-house screening of the Merck sample collection identified proline derived homophenylalanine 3 as a DPP-IV inhibitor with modest potency (DPP-IV IC50=1.9 microM). Optimization of 3 led to compound 37, which is among the most potent and selective DPP-IV inhibitors discovered to date.     

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.     

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.     

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.     

(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.     

Synthesis and structure-activity relationships of potent 4-fluoro-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors

Dipeptidyl peptidase IV (DPP-IV) inhibitors are promising antidiabetic drugs, and several drugs are in the developmental stage. We previously reported that the introduction of fluorine to the 4-position of 2-cyanopyrrolidine enhanced the DPP-IV inhibitory effect. In the present report, we examined the structure-activity relationship (SAR) of 2-cyano-4-fluoropyrrolidine with N-substituted glycine at the 1-position. We report the identification of a potent and stable DPP-IV inhibitor (TS-021) with a long-term persistent plasma drug concentration and a potent antihyperglycemic activity.     

Novel trans-2-aryl-cyclopropylamine analogues as potent and selective dipeptidyl peptidase IV inhibitors

A series of trans-2-aryl-cyclopropylamine derived compounds were synthesized and evaluated their biological activities against DPP-IV. The structure-activity relationships (SAR) led to the discovery of novel series of DPP-IV inhibitors, having IC₅₀ values of <100 nM with excellent selectivity over the closely related enzymes, DPP8, DPP-II and FAP. The studies identified a potent and selective DPP-IV inhibitor 24b, which exhibited the ability to both significantly inhibit plasma DPP-IV activity in rats and improve glucose tolerance in lean mice and diet induced obese mice.     

2-Cyano-4-fluoro-1-thiovalylpyrrolidine analogues as potent inhibitors of DPP-IV

We report the synthesis and biological activity of a series of 2-cyano-4-fluoro-1-thiovalylpyrrolidine inhibitors of DPP-IV. Within this series, compound 19 provided a potent, selective, and orally active DPP-IV inhibitor which demonstrated a very long duration of action in both rat and dog.     

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.     

Discovery of long-acting N-(cyanomethyl)-N-alkyl-L-prolinamide inhibitors of dipeptidyl peptidase IV

Details of structure-activity relationships (SAR) for P2 moiety of a P1 2-cyanopyrrolidine dipeptidyl peptidase IV (DPP-IV) inhibitor 4a including stereochemistry are presented. Based on this information, a series of P1 (N-alkyl)aminoacetonitrile analogs 9-20 possessing optimal P2 structure were synthesized and evaluated as inhibitors of DPP-IV. Among them, a representative compound 11, N-(cyanomethyl)-N-ethyl-L-prolinamide, was further evaluated to determine its effect on the plasma glucose level. Also 4a, 10, and 11 were evaluated for their isozyme selectivity to predict their safety problems.     

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC₅₀ 216 μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC₅₀ values of 3.5 and 44.2 μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.     

Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha)

Dipeptidyl peptidase IV (DPP-IV) and seprase belong to a small group of membrane-bound, proline-specific serine proteases, the serine integral membrane proteases (SIMPs). Whilst DPP-IV is the most exhaustively studied peptidase in this class, relatively less is known about the inhibitor/substrate specificity of its close homolog seprase. Additionally, whereas, DPP-IV expression is largely ubiquitous, seprase expression is restricted to tumour and tissue remodelling sites in vivo. Consequently, the highly restricted expression and distribution of seprase potentially make it an excellent therapeutic target for the modulation of neoplastic invasion and metastasis. Against this background, we now wish to report on the design, synthesis, and kinetic testing of a series of dipeptide proline diphenyl phosphonates, against DPP-IV and seprase. The most potent inhibitor of DPP-IV and seprase was found to be Gly-ProP(OPh)2, which exhibited overall second-order rate constants of inactivation of 5.24 x 105 M-1 min-1 and 1.06 x 104 M-1 min-1 against DPP-IV and seprase, respectively. Both proteases displayed differing profiles of susceptibility towards the other members of the series of inhibitors synthesised. In addition, Gly-ProP(OPh)2 and Tyr-ProP(OPh)2 were found to exert a considerable, dose-dependent anti-invasive effect on the LOX melanoma cell line, in vitro.     

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.     

Discovery of non-covalent dipeptidyl peptidase IV inhibitors which induce a conformational change in the active site

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.     

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.     

Type 2 diabetes--therapy with dipeptidyl peptidase IV inhibitors

The sole application of an inhibitor of the dipeptidyl peptidase DP IV (also DP 4, CD26, DPP-IV or DPP-4) to a mammal subsequently leading to improved glucose tolerance marks a major breakthrough in metabolic research bearing the potential of a new revolutionary diabetes therapy. This was demonstrated in rat applying the specific DP IV inhibitor isoleucyl thiazolidine. It was published in 1996 for the first time that a specific DP IV inhibitor in a given dose was able to completely block glucagon-like peptide-1 (GLP-1) degradation in vivo resulting in improved insulin response accompanied, by accelerated peripheral glucose disposal. Later on, these results were confirmed by several research teams applying DP IV inhibitors intravenously or orally. Today, the DP IV inhibition for the treatment of metabolic disorders is a validated principle. Now, more than 10 years after the initial animal experiments, first DP IV inhibitors as investigational drugs are tested in phase 3 clinical trials.     

Non-competitive and selective dipeptidyl peptidase IV inhibitors with phenethylphenylphthalimide skeleton derived from thalidomide-related α-glucosidase inhibitors and liver X receptor antagonists

Novel dipeptidyl peptidase IV (DPP-IV) inhibitors with a phenethylphenylphthalimide skeleton were prepared based on α-glucosidase inhibitors and liver X receptor (LXR) antagonists derived from thalidomide. Representative compounds showed non-competitive inhibition of DPP-IV and 28a exhibited 10-fold selectivity for DPP-IV over DPP-8. Compound 28a is the first non-competitive, selective DPP-IV inhibitor.     

Isolation of rugosin A,B and related compounds as dipeptidyl peptidase-IV inhibitors from rose bud extract powder

Dipeptidyl peptidase-IV (DPP-IV) is a protease responsible for the degradation of the incretin hormone. A number of DPP-IV inhibitors have been approved for use in the treatment of type 2 diabetes. While these inhibitors are effective for this treatment, methods for the prevention of this disease are also required as diabetes patient numbers are currently increasing rapidly worldwide. We screened the DPP-IV inhibitory activities of edible plant extracts with the intention of using these extracts in a functional food supplement for the prevention of diabetes. Rose (Rosa gallica) bud extract powder was a promising material with high inhibitory activity. In this study, seven ellagitannins were isolated as active compounds through activity-guided fractionations, and their DPP-IV inhibitory activities were measured. Among them, rugosin A and B showed the highest inhibitory activities and rugosin B was shown as the major contributing compound in rose bud extract powder.     

A new screening method based on yeast-expressed human dipeptidyl peptidase IV and discovery of novel inhibitors

Dipeptidyl peptidase (DPP) IV inhibitors provide a new strategy for the treatment of type 2 diabetes. Human DPP-IV gene was cloned from differentiated Caco-2 cells and expressed in Pichia pastoris. The recombinant enzyme was used in a new system for screening of DPP-IV inhibitors. By high throughput screening, a novel compound (W5188) was identified from 75,000 compounds with an IC₅₀ of 6.5 μM. This method is highly reproducible and reliable for discovery of DPP-IV inhibitors as shown by Z′ value of 0.73 and S/N ratio of 6.89.     

Long-acting peptidomimetics based DPP-IV inhibitors

Pyrrolidine based peptidomimetics are reported as potent and selective DPP-IV inhibitors for the treatment of T2DM. Compounds 16c and 16d showed excellent in vitro potency and selectivity towards DPP-IV and the lead compound 16c showed sustained antihyperglycemic effects, along with improved pharmacokinetic profile.