Involvement of DPP‐IV catalytic residues in enzyme–saxagliptin complex formation

Abstract

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 Å ). 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 ;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 ;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.Keywords: DPP-IV; X-ray crystal structure; mutant; ITC; proton NMR; short, strong hydrogen bond; serine protease; saxagliptin Dipeptidyl peptidase IV is a serine protease that modulates the biological activity of specific circulating peptide hormones, chemokines, cytokines, and neuropeptides Abbreviations: ANS, 1-anilino-8-naphthalene sulfonate; DPP-IV, dipeptidyl peptidase IV; GLP-1, glucagon-like peptide-1; ITC, isothermal titration calorimetry; pNA, p-nitroaniline; P n -P n9 , amino acid residues of the substrate which numerically indicate the position relatively to the scissile bond, n being the residues toward the N terminus and n9 being the residues toward the C terminus (this nomenclature was first defined by Schechter and Berger [1967]); SEC-MALS, size-exclusion chromatography-multiple angle light scattering; SKIE, solvent kinetic isotope effect; SSHB, short strong hydrogen bond; SIHB, short ionic hydrogen bond; TSE, thermal stability enhancement.Article and publication are at http://www.proteinscience.org/cgi

Anagliptin, a potent dipeptidyl peptidase IV inhibitor: its single-crystal structure and enzyme interactions

“…Interestingly, a dipole interaction of the cyano group was observed in the S 1 subsite in contrast to the covalent bonds observed in the analyses of vildagliptin 27 and saxagliptin 20 . This novel non-covalent bond interaction of the cyano group was considered to be a transition state before formation of a covalent imidate, which was supported by the facts that both anagliptin and vildagliptin were highly potent in this analysis, and a major metabolite of anagliptin was a hydrolysed compound of the cyano group.…”

“…The covalent imidate intermediates with the Og of Ser630 in the S 1 subsite are observed in the X-ray analyses of the inhibitors having a cyano group, vildagliptin 27 and saxagliptin 20 . However, the cyano group of anagliptin underwent dipole interactions in the S 1 subsite.…”

“…The structure of the DPP-4-anagliptin complex was solved by the molecular replacement method with the program MOLREP 19 using the structure of DPP-4-saxagliptin complex 20 refined to 2.85 Å resolution (PDB code 3BJM) as a model structure. The rigid body refinement was performed with the program REFMAC 21 .…”

Pharmacokinetics of the Dipeptidyl Peptidase 4 Inhibitor Saxagliptin in Rats, Dogs, and Monkeys and Clinical Projections

“…This metabolite, which is also a potent and specific inhibitor of DPP4, circulates in significant concentrations in human plasma (Table 3). Second, both saxagliptin and M2 display prolonged binding to the catalytic site of DPP4 (Kirby et al, 2008;Metzler et al, 2008). The extended rate of dissociation of saxagliptin and M2 from the DPP4 active site would be expected to give hysteresis between plasma concentration and DPP4 inhibition, which has been observed in animal models and humans, providing a longer pharmacodynamic half-life than pharmacokinetic half-life for the compound.…”

“…1), is a potent and selective inhibitor of DPP4 with an inhibition constant (K i ) value of 1.3 nM and is currently under development for the treatment of type 2 diabetes (Augeri et al, 2005;Metzler et al, 2008;Rosenstock et al, 2008). Saxagliptin was designed to provide high potency and selectivity and extended inhibition of DPP4.…”