LY295427, a novel hypocholesterolemic agent, enhances [3H]25-hydroxycholesterol binding to liver cytosolic proteins

Abstract

LY295427, (3 alpha,4 alpha,5 alpha)-4-(2-propenylcholestan-3-ol), acts through an unknown mechanism to derepress the transcription of the low density lipoprotein (LDL) receptor in the presence of 25-hydroxycholesterol (25-OH chol). Preincubation with LY295427 in Chinese hamster ovary (CHO) cells increased uptake of 25-OH chol in a time-dependent manner, suggesting that the drug interfered with the negative feedback mechanism of 25-OH chol on LDL receptor expression. To explore the mechanism by which LY295427 inhibited the suppressive actions of 25-OH chol, the radioactive ligand [3H]25-OH chol and specific antibodies to the oxysterol binding protein (OSBP) were used to identify possible drug:protein interactions. After separation by anion exchange chromatography, protein fractions from hamster liver cytosol were found to selectively bind [3H]25-OH chol with high affinity. In fractions in which 25-OH chol binding was evident, and in other distinct fractions that lacked specific binding, addition of LY295427 increased [3H]25-OH chol binding 2- to 5-fold. LY306039, the 3 beta-isomer of LY295427, failed to derepress the LDL receptor in CHO cells, and it had no effect on [3H]25-OH chol binding. Analysis of Western blots using polyclonal antibodies to OSBP showed that specific [3H]25-OH chol binding in the absence of LY295427 was present only in fractions containing OSBP. However, enhanced [3H]25-OH chol binding in the presence of LY295427 was evident in distinct fractions after immunodepletion of both the 90-100 kDa form of OSBP and a 170 kDa protein; and specific binding of a radioiodinated analog of LY295427 was detected in select fractions lacking [3H]25-OH chol binding in the absence of LY295427. Moreover, LY295427 did not displace or enhance [3H]25-OH chol binding to OSBP purified to near homogeneity. These data suggest that LY295427, while not dependent on the presence of oxysterol binding protein, binds to cytosolic protein(s) that interact with 25-hydroxycholesterol and other oxystcrols, thus preventing the repression of the LDL receptor.