RESUMEN
A series of novel N,N-disubstituted trifluoro-3-amino-2-propanols has been prepared as potent inhibitors of cholesteryl ester transfer protein (CETP). Modifying the aromatic 3-tetrafluoroethoxy group in the lead molecule 1a with various heteroaryl moieties produced new 2-furyl analogues 2a,b with submicromolar potency in vitro.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Glicoproteínas , Propanoles/química , Propanoles/farmacología , Proteínas de Transferencia de Ésteres de ColesterolAsunto(s)
Proteínas Portadoras/química , Ésteres del Colesterol/sangre , Glicoproteínas , Propanolaminas/síntesis química , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/sangre , Proteínas de Transferencia de Ésteres de Colesterol , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Propanolaminas/sangre , Propanolaminas/química , EstereoisomerismoRESUMEN
Chiral N,N-disubstituted trifluoro-3-amino-2-propanols represent a recently discovered class of compounds that inhibit the neutral lipid transfer activity of cholesteryl ester transfer protein (CETP). These compounds all contain a single chiral center that is essential for inhibitory activity. (R,S)SC-744, which is composed of a mixture of the two enantiomers, inhibits CETP-mediated transfer of [(3)H]cholesteryl ester ([(3)H]CE) from HDL donor particles to LDL acceptor particles with an IC(50) = 200 nM when assayed using a reconstituted system in buffer and with an IC(50) = 6 microM when assayed in plasma. Upon isolation of the enantiomers, it was found that the (R,+) enantiomer, SC-795, was about 10-fold more potent than the mixture, and that the (S,-) enantiomer, SC-794, did not have significant inhibitory activity (IC(50) > 0.8 microM). All of the activity of the (S,-)SC-794 enantiomer could be accounted for by contamination of this sample with a residual 2% of the highly potent (R,+) enantiomer, SC-795. The IC(50) of (R,+)SC-795, 20 nM, approached the concentration of CETP (8 nM) in the buffer assay. These chiral N,N-disubstituted trifluoro-3-amino-2-propanols were found to associate with both LDL and HDL, but did not disrupt overall lipoprotein structure. They did not affect the on or off rates of CETP binding to HDL disk particles. Inhibition was highly specific since the activities of phospholipid transfer protein and lecithin cholesterol acyl transferase were not affected. Competition experiments showed that the more potent enantiomer (R)SC-795 prevented cholesteryl ester binding to CETP, and direct binding experiments demonstrated that this inhibitor bound to CETP with high affinity and specificity. It is estimated, based on the relative concentrations of inhibitor and lipid in the transfer assay, that (R)SC-795 binds approximately 5000-fold more efficiently to CETP than the natural ligand, cholesteryl ester. We conclude that these chiral N,N-disubstituted trifluoro-3-amino-2-propanol compounds do not affect lipoprotein structure or CETP-lipoprotein recognition, but inhibit lipid transfer by binding to CETP reversibly and stereospecifically at a site that competes with neutral lipid binding.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Ésteres del Colesterol/antagonistas & inhibidores , Glicoproteínas , Proteínas de Transferencia de Fosfolípidos , Propanolaminas/farmacología , Triglicéridos/antagonistas & inhibidores , Animales , Unión Competitiva/efectos de los fármacos , Células CHO , Proteínas Portadoras/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Ésteres del Colesterol/metabolismo , Cricetinae , Disulfuros/química , Disulfuros/farmacología , Sinergismo Farmacológico , Electroforesis en Gel de Agar , Humanos , Lipoproteínas HDL/antagonistas & inhibidores , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/antagonistas & inhibidores , Lipoproteínas LDL/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfolípidos/antagonistas & inhibidores , Propanolaminas/química , Estereoisomerismo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
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.