NVP-DPP728 (1-[[[2-[(5-Cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine), a Slow-Binding Inhibitor of Dipeptidyl Peptidase IV

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Abstract
Inhibition of dipeptidyl peptidase IV (DPP-IV) has been proposed recently as a therapeutic approach to the treatment of type 2 diabetes. N-Substituted-glycyl-2-cyanopyrrolidide compounds, typified by NVP-DPP728 (1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine), inhibit degradation of glucagon-like peptide-1 (GLP-1) and thereby potentiate insulin release in response to glucose-containing meals. In the present study NVP-DPP728 was found to inhibit human DPP-IV amidolytic activity with a Ki of 11 nM, a kon value of 1.3 × 105 M-1 s-1, and a koff of 1.3 × 10-3 s-1. Purified bovine kidney DPP-IV bound 1 mol/mol [14C]-NVP-DPP728 with high affinity (12 nM Kd). The dissociation constant, koff, was 1.0 × 10-3 and 1.6 × 10-3 s-1 in the presence of 0 and 200 μM H-Gly-Pro-AMC, respectively (dissociation t1/2 ∼10 min). Through kinetic evaluation of DPP-IV inhibition by the d-antipode, des-cyano, and amide analogues of NVP-DPP728, it was determined that the nitrile functionality at the 2-pyrrolidine position is required, in the l-configuration, for maximal activity (Ki of 11 nM vs Ki values of 5.6 to >300 μM for the other analogues tested). Surprisingly, it was found that the d-antipode, despite being ∼500-fold less potent than NVP-DPP728, displayed identical dissociation kinetics (koff of 1.5 × 10-3 s-1). NVP-DPP728 inhibited DPP-IV in a manner consistent with a two-step inhibition mechanism. Taken together, these data suggest that NVP-DPP728 inhibits DPP-IV through formation of a novel, reversible, nitrile-dependent complex with transition state characteristics.