Abstract

Bile acids (BAs) are the end products of cholesterol metabolism. One of the critical steps in their biosynthesis involves the isomerization of the 3 beta-hydroxyl (-OH) group on the cholestane ring to the common 3 alpha-configuration on BAs. BAs are actively recaptured from the small intestine by the human Apical Sodium-dependent Bile Acid Transporter (hASBT) with high affinity and capacity. Previous studies have suggested that no particular hydroxyl group on BAs is critical for binding or transport by hASBT, even though 3 beta-hydroxylated BAs were not examined. The aim of this study was to elucidate the role of the 3 alpha-OH group on BAs binding and translocation by hASBT. Ten 3 beta-hydroxylated BAs (Iso-bile acids, iBAs) were synthesized, characterized, and subjected to hASBT inhibition and uptake studies. hASBT inhibition and uptake kinetics of iBAs were compared to that of native 3 alpha-OH BAs. Glycine conjugates of native and isomeric BAs were subjected to molecular dynamics simulations to identify topological descriptors related to binding and translocation by hASBT: Iso-BAs bound to hASBT with lower affinity and exhibited reduced translocation than their respective 3 alpha-epimers. Kinetic data suggests that, in contrast to native BAs where hASBT binding is the rate-limiting step, iBAs transport was rate-limited by translocation and not binding. Remarkably, 7-dehydroxylated iBAs were not hASBT substrates, highlighting the critical role of 7-OH group on BA translocation by hASBT, especially for iBAs. Conformational analysis of gly-iBAs and native BAs identified topological features for optimal binding as: concave steroidal nucleus, 3-OH on- or below-steroidal plane, 7-OH below-plane, and 12-OH moiety toward-plane. Our results emphasize the relevance of the 3 alpha-OH group on BAs for proper hASBT binding and transport and revealed the critical role of 7-OH group on BA translocation, particularly in the absence of a 3 alpha-OH group. Results have implications for BA prodrug design.