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

SC-71952, a substituted analog of dithiobisnicotinic acid dimethyl ester, w as identified as a potent inhibitor of cholesteryl ester transfer protein ( CETP). When tested in an in vitro assay, the concentration of SC-71952 requ ired for half-maximal inhibition was 1 mu M. The potency of SC-71952 was en hanced 200-fold by preincubation of the inhibitor with CETP, and was decrea sed 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 dep endency, and were not affected by dithiothreitol treatment. Kinetic and bio chemical characterization of the inhibitory process of CETP by SC-71952 sug gested that the inhibitor initially binds rapidly and reversibly to a hydro phobic site on CETP, With time, the bound inhibitor irreversibly inactivate s CETP, presumably by reacting with one of the free cysteines of CETP, Liqu id chromatography/mass spectroscopy (LC/MS) analyses of tryptic digests of untreated or SC-71952-inactivated CETP was used to identify which cysteine( s) were potentially involved in the time-dependent, irreversible component of inactivation by the inhibitor. One disulfide bond, Cys143-Cys184, was un affected 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 reversi ble component of inactivation by SC-71952, However, it abolished the time-d ependent enhancement in potency seen with the inhibitor when using wild-typ e CETP. These data indicate that Cys-13 is critical for the irreversible in activation of CETP by SC-71952 and provides support for the structural mode l that places Cys-13 near the neutral lipid-binding site of CETP.