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

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

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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 Pickled 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 Screws 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.