The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the small-molecule binding site in the human MBOAT Hedgehog acyltransferase (HHAT) based on HHAT inhibitor RUSKI-201. Structure-activity relationship investigation identified the improved enantiomeric inhibitor IMP 1575, which is the most potent HHAT inhibitor reported to-date, and guided rational design of a photocrosslinking probe that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT inhibitor IMP-1575 (Ki = 38 nM) and a strategy for mapping of interaction sites in MBOATs.

中文翻译：

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光化学探针鉴定哺乳动物膜结合的O-酰基转移酶中的小分子结合位点

哺乳动物膜结合O-酰基转移酶（MBOAT）超家族参与生物过程，包括生长，发育和食欲感应。MBOAT是治疗癌症和肥胖的诱人药物。但是，有关小分子抑制的结合位点和分子机制的信息尚不清楚。这项研究报告了开发一种光化学探针以询问基于HHAT抑制剂RUSKI-201的人MBOAT刺猬酰基转移酶（HHAT）中的小分子结合位点。结构-活性关系研究确定了改进的对映体抑制剂IMP 1575，这是迄今为止报道的最有效的HHAT抑制剂，并指导了合理设计的光交联探针，该探针可保持HHAT抑制能力。HHAT的光交联和蛋白质组学测序可鉴定哺乳动物MBOAT中的第一个小分子结合位点。拓扑和同源性数据表明了HHAT抑制的潜在机制，这已通过动力学分析得到了证实。我们的结果提供了一种最佳的HHAT抑制剂IMP-1575（Ki = 38 nM）和一种在MBOATs中定位相互作用位点的策略。