Potent mechanism-based inactivators can be rationally designed against pyridoxal 5′-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

中文翻译：

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基于机制的灭活剂对吡咯醛5'-磷酸盐依赖性酶的选择性靶向：灭活和替代周转的机制

可以合理设计基于有效机理的灭活剂，以抗依赖吡ido醛5'-磷酸（PLP）的药物靶标，例如鸟氨酸氨基转移酶（OAT）或γ-氨基丁酸氨基转移酶（GABA-AT）。然而，一个重要的挑战是，由于所有PLP依赖性氨基转移酶反应机理相似，因此对其他PLP依赖性脱靶酶缺乏选择性。在复杂的晶体结构的基础上，我们通过（1 R，3 S，4 S）-3-氨基-4-氟环戊烷-1-羧酸（FCP），一种已知的GABA-AT灭活剂。OAT和FCP的晶体结构表明形成了三元加合物。该加合物可以合理地认为是通过烯胺失活机理发生的，类似于针对GABA-AT的报道。但是，脱靶，依赖PLP的酶，天冬氨酸转氨酶（Asp-AT）的晶体结构与FCP结合，以及尝试的抑制试验结果表明，FCP不是Asp-AT的灭活剂，而是另一种基材。高分辨率质谱也支持Asp-AT进行FCP的周转。在实现众多PLP依赖性酶之间灭活的选择性方面存在困难的情况下，获得的结果提供了证据，表明可以实现理想的选择性，