Influence of membrane partitioning on inhibitors of membrane-bound enzymes

Membrane-water partitioning of inhibitors of acyl-coenzyme A:chalesterol ac yltransferase (ACAT) governs the concentration of inhibitor that ACAT: is e xposed to and determines the corresponding extent of cholesterol esterifica tion inhibition. Partitioning of the ACAT inhibitors CI-976, CL 277,082, an d SaH 58-035 into rat liver microsomes containing ACAT was detected by shif ts in the level of inhibition that were independent of inhibitor concentrat ion but inversely dependent on microsome membrane concentration. The equili brium distribution of the ACAT inhibitors between aqueous and membrane phas es was derived directly from these data by application of a previously desc ribed method of linear analysis. The accuracy of membrane partitioning anal ysis based on kinetic data was verified for CI-976 by direct measurements o f [C-14]CI-976 partitioning into phospholipid membranes. The results show t hat the ACAT inhibitors are highly partitioned into membranes by factors ex ceeding 1 x 10(6). This result is consistent with the far greater influence of membrane content over aqueous volume on inhibitor activity. The results demonstrate that the size of the membrane phase in aqueous suspension must be taken into account to obtain accurate and reproducible kinetic characte rizations of membrane-active molecules. Analyses of the membrane-dependent shifts in activity can be used to calculate the membrane-water partitioning coefficient of membrane-active molecules such as ACAT inhibitors. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association.