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, 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 microm. The potency of SC-71952 was enhanced 200-fold by preincubation of the inhibitor with CETP, and was decreased 50-fold by treatment with dithiothreitol. Analogs of SC-71952 that did not contain 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 hydrophobic site on CETP. With time, the bound inhibitor irreversibly 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 identify which cysteine(s) were potentially involved in the time-dependent, irreversible component of inactivation by the inhibitor. One disulfide bond, Cys143-Cys184, was unaffected 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 component of inactivation by SC-71952. However, it abolished the time-dependent enhancement in potency seen with the inhibitor 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.
Hope
H R
HR
Biochemistry and Molecular Biology, Pharmacia, St. Louis, MO 63198, USA.
Heuvelman
D
D
Duffin
K
K
Smith
C
C
Zablocki
J
J
Schilling
R
R
Hegde
S
S
Lee
L
L
Witherbee
B
B
Baganoff
M
M
Bruce
C
C
Tall
A R
AR
Krul
E
E
Glenn
K
K
Connolly
D T
DT
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D053480:Cholesterol Ester Transfer Proteins; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009539:Nicotinic Acids / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D013440:Sulfides / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors
C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D004220:Disulfides; D006023:Glycoproteins; D009539:Nicotinic Acids; D010446:Peptide Fragments; C417579:SC 71952; D013440:Sulfides; D003545:Cysteine; D004229:Dithiothreitol