NVP-DPP728 (1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)- pyrrolidine), a slow-binding inhibitor of dipeptidyl peptidase IV.

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 )-p yrrolidine), 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 K(i) of 11 nM, a k(on) value of 1.3 x 10(5) M(-)(1) s(-)(1), and a k(off) of 1.3 x 10(-)(3) s(-)(1). Purified bovine kidney DPP-IV bound 1 mol/mol [(14)C]-NVP-DPP728 with high affinity (12 nM K(d)). The dissociation constant, k(off), was 1.0 x 10(-)(3) and 1.6 x 10(-)(3) s(-)(1) in the presence of 0 and 200 microM H-Gly-Pro-AMC, respectively (dissociation t(1/2) approximately 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 (K(i) of 11 nM vs K(i) values of 5.6 to >300 microM for the other analogues tested). Surprisingly, it was found that the D-antipode, despite being approximately 500-fold less potent than NVP-DPP728, displayed identical dissociation kinetics (k(off) of 1.5 x 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.

Modified cation activation of the (Na+K)-ATPase following treatment with thimerosal.

Treatment of the Na,K-ATPase enzyme, isolated from canine renal outer medulla, with thimerosal (ethylmercurithiosalicylate) resulted in significant inhibition of the overall Na,K-ATPase with only slight, if any, inhibition of the Na-ATPase and ATP:ADP exchange activities. The K-stimulated PNPPase activity was stimulated [see G. R. Henderson and A. Askari (1977) Arch. Biochem. Biophys. 182, 221-226]. Examination of the Na dependence of the ATPase and ATP:ADP exchange activities revealed an Na-independent, ouabain-sensitive activity that was inhibited by Na in the range 0-10 mM. At greater than or equal to 10 mM concentration of Na the treated and modified enzymes showed similar activities. The apparent affinity of the modified enzyme for ATP in the presence of 100 mM Na was the same as that of the untreated enzyme (0.2-0.3 microM). In the absence of Na, the modified enzyme hydrolyzed ATP with a relatively low affinity (about 120 microM). The enhancement of p-nitrophenylphosphatase (PNPPase) activity measured in the presence of K ions was due to the appearance of K-independent, ouabain-sensitive PNPP activity. The modification was without major affect on the apparent affinity of the enzyme for K ions in the PNPPase activity. Treatment of the thimerosal-modified enzyme with dithiothreitol removed (or greatly reduced) the cation-independent, ouabain-sensitive activities and the Na,K-ATPase activity returned. Modification of a set of enzyme -SH groups in the Na,K-ATPase enzyme made it able to hydrolyze ATP in the absence of Na ions and PNPP in the absence of K ions. The -SH groups modified by thimerosal are evidently critical to the major dephosphoenzyme conformational changes but are not involved in the major transport conformational change between phosphoenzymes.

Inactivation of membrane-bound (Na+ +K+)-ATPase of Yoshida sarcoma cells and cobra venom cytotoxin complex with the glycolipid components of the enzyme system.

The molecular mechanisms involved in the inactivation of (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells by a cytotoxic protein (P6) from cobra venom have been examined. The overall data obtained using purified (Na+ + K+)-stimulated ATPase of Yoshida sarcoma cells suggest that cytotoxin P6 combines with phosphatidyl serine and a glycolipid which are closely associated with (Na+ + K+)-stimulated ATPase which in turn may lead to the inactivation of the enzyme in this cell system.