Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Kim, Young B.; Kopcho, Lisa M.; Kirby, Mark; Hamann, Lawrence G.; Weigelt, Carolyn A.; Metzler, William J.; Marcinkeviciene, Jovita

doi:10.1016/j.abb.2005.11.010

Cited by 69 publications

(52 citation statements)

References 36 publications

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“…Ideally an increased K d of saxagliptin in DPP-IV Y547F , but not in DPP-IV Y547Q , would indicate the importance of this residue in stabilization of the enzyme-inhibitor complex. We found both mutant proteins bound saxagliptin very tightly (<5 nM), agreeing well with its potent K i of 0.5 nM for wild-type protein (Kim et al 2006). Thus, replacing this residue either by phenylalanine or glutamine did not alter the binding potency significantly.…”

Section: Y547fsupporting

confidence: 62%

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

et al. 2008

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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

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Section: Y547fsupporting

confidence: 62%

“…Mechanism-based enzyme-inhibitor complex formation between DPP-IV and saxagliptin ( Fig. 1) has been extensively described in our previous publication (Kim et al 2006). We suggested initial inhibitor binding is followed by serine addition to the inhibitor nitrile carbon, catalyzed by histidine.…”

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

et al. 2008

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“…The a-keto-ester 14 was isolated in 42% yield after purification. Hydroxylation of 14 in 50% HNO 3 and 98% H 2 SO 4 followed by hydrolysis in 1 N NaOH gave hydroxyadamantyl keto-acid 16, the substrate for bioconversion. PDH/FDH-catalyzed reductive amination, followed by Boc protection provided 2b in 87% yield.…”

Section: Synthesis Of (S)-mentioning

confidence: 99%

Carbon‐14 labeling of Saxagliptin (BMS‐477118)

Cao

Bonacorsi

Balasubramanian

et al. 2007

Labelled Comp Radiopharmac

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“…The docking calculations were performed under the assumption of reversible noncovalent binding. The possibility of reversible covalent binding through interaction of Ser630 and 6-imino group of the compounds considered cannot be neglected (Kim et al, 2006). However, the polar 6-imino 2-thioxo pyrimidinone heterocycle is quite unlikely to enter the S1 pocket, and, Isopropyl (iso-C 3 H 7 ) 4 5 6…”

Section: In-vitro Inhibition Of Dpp IV Activity and Enzyme Kinetic Exmentioning

confidence: 99%

6-Imino-2-thioxo-pyrimidinones as a new class of dipeptidyl peptidase IV inhibitors

et al. 2010

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Dipeptidyl peptidase IV is a glycoprotein which removes N-terminal dipeptides from physiologically relevant polypeptides. An homologous series of 6-imino-2-thioxo-5-{[3,4,5-tris(methyloxy)phenyl]methyl}-2,5-dihydro-4(3H)-pyrimidinones has been tested for inhibition of DPP IV activity. The inhibitory effects at 0.1 mM were observed. Enzyme kinetic studies revealed that compounds inhibit DPP IV activity competitively. According to the molecular docking analysis, the inhibitors are anchored into the DPP IV hydrolytic site by interactions of the pyrimidinone core with Glu206, Tyr662, and Tyr547, with the alkyl chain entering the S1 pocket. We conclude that pyrimidinone-like compounds are a promising new scaffold for reversible inhibition of DPP IV.

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Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Cited by 69 publications

References 36 publications

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

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

Carbon‐14 labeling of Saxagliptin (BMS‐477118)

6-Imino-2-thioxo-pyrimidinones as a new class of dipeptidyl peptidase IV inhibitors

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