The pantothenate analogue hopantenate (HoPan) is widely used as a modulator of coenzyme A (CoA) levels in cell biology and disease models─especially for pantothenate kinase associated neurodegeneration (PKAN), a genetic disease rooted in impaired CoA metabolism. This use of HoPan was based on reports that it inhibits pantothenate kinase (PanK), the first enzyme of CoA biosynthesis. Using a combination of in vitro enzyme kinetic studies, crystal structure analysis, and experiments in a typical PKAN cell biology model, we demonstrate that instead of inhibiting PanK, HoPan relies on it for metabolic activation. Once phosphorylated, HoPan inhibits the next enzyme in the CoA pathway─phosphopantothenoylcysteine synthetase (PPCS)─through formation of a nonproductive substrate complex. Moreover, the obtained structure of the human PPCS in complex with the inhibitor and activating nucleotide analogue provides new insights into the catalytic mechanism of PPCS enzymes─including the elusive binding mode for cysteine─and reveals the functional implications of mutations in the human PPCS that have been linked to severe dilated cardiomyopathy. Taken together, this study demonstrates that the molecular mechanism of action of HoPan is more complex than previously thought, suggesting that the results of studies in which it is used as a tool compound must be interpreted with care. Moreover, our findings provide a clear framework for evaluating the various factors that contribute to the potency of CoA-directed inhibitors, one that will prove useful in the future rational development of potential therapies of both human genetic and infectious diseases.

中文翻译：

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辅酶 A 水平调节剂 Hopantenate (HoPan) 抑制磷酸泛酸酰半胱氨酸合成酶活性

泛酸类似物霍泛酸 (HoPan) 在细胞生物学和疾病模型中被广泛用作辅酶 A (CoA) 水平的调节剂，尤其是泛酸激酶相关神经变性 (PKAN)，这是一种根植于辅酶 A 代谢受损的遗传性疾病。HoPan 的这种用途是基于其抑制泛酸激酶 (PanK) 的报道，泛酸激酶是辅酶 A 生物合成的第一种酶。使用体外组合通过酶动力学研究、晶体结构分析和典型 PKAN 细胞生物学模型中的实验，我们证明了 HoPan 不是抑制 PanK，而是依赖它进行代谢激活。磷酸化后，HoPan 会通过形成非生产性底物复合物来抑制 CoA 途径中的下一种酶——磷酸泛酰半胱氨酸合成酶 (PPCS)。此外，获得的与抑制剂和活化核苷酸类似物复合的人 PPCS 结构为 PPCS 酶的催化机制提供了新的见解——包括难以捉摸的半胱氨酸结合模式——并揭示了人 PPCS 突变的功能意义，这些突变具有与严重扩张型心肌病有关。综上所述，这项研究表明 HoPan 的分子作用机制比以前认为的更复杂，这表明必须谨慎解释将其用作工具化合物的研究结果。此外，我们的研究结果为评估影响 CoA 定向抑制剂效力的各种因素提供了一个清晰的框架，该框架将在未来合理开发人类遗传病和传染病的潜在疗法中被证明是有用的。