González Pablo M, Lagos Carlos F, Ward Weslyn C, Polli James E
Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile , Av Vicuña Mackenna 4860, Santiago, Chile.
Mol Pharm. 2014 Feb 3;11(2):588-98. doi: 10.1021/mp400575t. Epub 2013 Dec 26.
Bile acids (BAs) are the end products of cholesterol metabolism. One of the critical steps in their biosynthesis involves the isomerization of the 3β-hydroxyl (-OH) group on the cholestane ring to the common 3α-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 3β-hydroxylated BAs were not examined. The aim of this study was to elucidate the role of the 3α-OH group on BAs binding and translocation by hASBT. Ten 3β-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 3α-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 3α-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 3α-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 3α-OH group. Results have implications for BA prodrug design.
胆汁酸（BAs）是胆固醇代谢的终产物。其生物合成的关键步骤之一涉及胆甾烷环上的3β-羟基（-OH）异构化为胆汁酸上常见的3α-构型。人顶端钠依赖性胆汁酸转运蛋白（hASBT）以高亲和力和容量从小肠中主动重摄取胆汁酸。先前的研究表明，胆汁酸上没有特定的羟基对于hASBT的结合或转运至关重要，尽管未检测3β-羟基化的胆汁酸。本研究的目的是阐明3α-OH基团在胆汁酸与hASBT结合及转运中的作用。合成、表征了十种3β-羟基化胆汁酸（异胆汁酸，iBAs），并对其进行了hASBT抑制和摄取研究。将iBAs的hASBT抑制和摄取动力学与天然3α-OH胆汁酸进行了比较。对天然和异构胆汁酸的甘氨酸缀合物进行分子动力学模拟，以确定与hASBT结合和转运相关的拓扑描述符。异胆汁酸与hASBT的结合亲和力较低，且其转运较各自的3α-差向异构体减少。动力学数据表明，与天然胆汁酸中hASBT结合是限速步骤不同，iBAs的转运限速步骤是转运而非结合。值得注意的是，7-脱羟基化的iBAs不是hASBT的底物，突出了7-OH基团在hASBT介导的胆汁酸转运中的关键作用，尤其是对于iBAs。甘氨酰-iBAs和天然胆汁酸的构象分析确定了最佳结合的拓扑特征为：凹面甾体核、3-OH在甾体平面“之上”或之下、7-OH在平面之下以及12-OH部分朝向平面。我们的结果强调了胆汁酸上3α-OH基团对于hASBT正确结合和转运的相关性，并揭示了7-OH基团在胆汁酸转运中的关键作用，特别是在没有3α-OH基团的情况下。研究结果对胆汁酸前药设计具有启示意义。
