In vitro and in vivo characterization and protein-bound structural elucidation of three microbial choline TMA lyase inhibitors

Microbial choline TMA lyases (CutC, catalytic polypeptide) are glycyl radical-dependent enzymes that catalyze the biochemical transformation of choline into both trimethylamine (TMA) and acetaldehyde. Present in intestinal microbiota, this gut microbial enzyme is a major contributor to TMA production, the precursor for host-generated trimethylamine N-oxide (TMAO). Both clinical and mechanistic studies reveal TMAO as a contributor to host cardiometabolic diseases, making CutC a target for the development of small molecule inhibitors. Here, we characterize the inhibitory potency of three choline analog-based CutC inhibitor tool drugs (cyclopropylcholine, CpC; fluoromethylcholine, FMC; and difluoromethylcholine, FMF) against phylogenetically distant cloned and expressed microbial CutC, whole cell microbial monocultures, both human and mouse polymicrobial fecal cultures, and in mice following an oral [d9]-choline challenge. Finally, we resolved the crystal structures of CutC from Klebsiella pneumoniae with each of the three inhibitors bound to the enzyme and demonstrate the molecular basis for their interaction with active site amino acid residues critical for enzyme activity.微生物胆碱TMA裂解酶（CutC，催化多肽）是一类甘氨酰自由基依赖型酶，可催化胆碱转化为三甲胺（TMA）与乙醛。该肠道微生物酶存在于肠道菌群中，是TMA生成的主要贡献者，而TMA是宿主源性三甲胺N-氧化物（TMAO）的前体物质。临床与机制研究均证实，TMAO可促进宿主心血管代谢疾病的发生发展，因此CutC成为小分子抑制剂开发的重要靶点。 本研究对三种基于胆碱类似物的CutC抑制剂工具化合物——环丙基胆碱（CpC）、氟甲基胆碱（FMC）与二氟甲基胆碱（FMF）——的抑制活性进行了系统表征，测试对象涵盖系统发育亲缘关系较远的克隆表达型微生物CutC、全细胞微生物单培养体系、人源与鼠源多菌群粪便培养体系，以及经口服[d9]-胆碱攻毒的小鼠体内实验。 此外，本研究解析了肺炎克雷伯菌（Klebsiella pneumoniae）CutC分别结合三种抑制剂的晶体结构，阐明了其与酶活关键活性位点氨基酸残基的相互作用分子机制。