Chattopadhyay Debasish, Swingle Mark R, Salter Edward A, Wood Eric, D'Arcy Brandon, Zivanov Catherine, Abney Kevin, Musiyenko Alla, Rusin Scott F, Kettenbach Arminja, Yet Larry, Schroeder Chad E, Golden Jennifer E, Dunham Wade H, Gingras Anne-Claude, Banerjee Surajit, Forbes David, Wierzbicki Andrzej, Honkanen Richard E
Department of Medicine, University of Alabama Birmingham, Birmingham, AL 35294, USA.
Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL 36688, USA.
Biochem Pharmacol. 2016 Jun 1;109:14-26. doi: 10.1016/j.bcp.2016.03.011. Epub 2016 Mar 23.
Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.
斑蝥素是一种天然毒素，也是一种用于治疗肿瘤的传统中药中的活性成分。斑蝥素作为PPP家族丝氨酸/苏氨酸蛋白磷酸酶的半选择性抑制剂。尽管与PP1C、PP2AC和PP5C具有共同的催化机制和显著的结构相似性，但发现人PP4C对斑蝥素的抑制活性不敏感。为了探究这种选择性的分子基础，我们合成并测试了基于PP5C与斑蝥素共晶体结构中揭示的相互作用的量子模型设计的新型C5/C6衍生物。结构-活性关系研究以及高分辨率（1.25Å）的PP5C-抑制剂共晶体结构分析表明，抑制剂桥头氧与催化金属离子和非催化苯丙氨酸残基之间存在紧密接触，后者在PP4C中被色氨酸取代。量子化学计算预测，与PP4C中体积更大的色氨酸侧链的空间冲突会迫使所有基于斑蝥素的抑制剂进入不利的结合模式，破坏在PP5C共晶体结构中观察到的活性位点金属离子的强配位，从而使PP4C对抑制剂不敏感。采用天然人PP4C的抑制研究证实了这一预测。将PP5C（F446W）和PP1C（F257W）突变以模拟PP4C活性位点，导致对斑蝥素的敏感性明显降低。这些观察结果为斑蝥素的天然选择性提供了结构基础，并为PP4C的筛选提供了途径。这些新型晶体结构还揭示了与其他PPP家族磷酸酶相比，C5/C6修饰对PP5C具有更高选择性的相互作用。
