Inhibition of cholesteryl ester transfer protein by substituted dithiobisnicotinic acid dimethyl ester: involvement of a critical cysteine

SC-71952, a substituted analog of dithiobisnico- tinic acid dimethyl ester, was identified as a potent inhibitor of cholesteryl ester transfer protein (CETP). When tested in an in vitro assay, the concentration of SC-71952 required for half-maximal inhibition was 1 m M. The potency of SC- 71952 was enhanced 200-fold by preincubation of the inhib- itor with CETP, and was decreased 50-fold by treatment with dithiothreitol. Analogs of SC-71952 that did not con- tain a disulfide linkage were less potent, did not display time dependency, and were not affected by dithiothreitol treatment. Kinetic and biochemical characterization of the inhibitory process of CETP by SC-71952 suggested that the inhibitor initially binds rapidly and reversibly to a hydro- phobic site on CETP. With time, the bound inhibitor irre- versibly inactivates CETP, presumably by reacting with one of the free cysteines of CETP. Liquid chromatography/ mass spectroscopy (LC/MS) analyses of tryptic digests of untreated or SC-71952-inactivated CETP was used to iden- tify which cysteine(s) were potentially involved in the time- dependent, irreversible component of inactivation by the inhibitor. One disulfide bond, Cys143-Cys184, was unaf- fected by treatment with the inhibitor. Inactivation of CETP by SC-71952 correlated with a progressive decrease in the abundance of free Cys-13 and Cys-333. Conversion of Cys- 13 to alanine had no effect on the rapid reversible compo- nent of inactivation by SC-71952. However, it abolished the time-dependent enhancement in potency seen with the in- hibitor when using wild-type CETP. These data indicate that Cys-13 is critical for the irreversible inactivation of CETP by SC-71952 and provides support for the structural model that places Cys-13 near the neutral lipid-binding site of CETP. —H. R. Hope, D. Heuvelman, K. Duffin, C. Smith, J. Zablocki, R. Schilling, S. Hegde, L. Lee, B. Witherbee, M. Baganoff, C. Bruce, A. R. Tall, E. Krul, K. Glenn, and D. T. Connolly. Inhibition of cholesteryl ester transfer protein by substituted dithiobisnicotinic acid dimethyl ester: in- volvement of a critical cysteine. J. Lipid Res. 2000. 41: 1604- 1614.