Synthesis of new (-)-Bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase.

The essential malarial PfA-M1 metalloaminopeptidase is a validated drug target that functions in the terminal stages of hemoglobin digestion. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1 and provides an excellent scaffold for the development of novel research tools as well as more effective PfA-M1 inhibitors. Here we present a new, efficient and high yielding protocol for the synthesis of bestatin-derivatives from commercially available natural and unnatural N-Boc-D-amino acids. We developed a diverse library of bestatin derivatives with variants at the sidechain of either the α-hydroxy-β-amino acid or the adjacent natural α-amino acid. Surprisingly we found that large aromatic rings at the P1 position resulted in potent inhibition against PfA-M1, while small hydrophobic sidechains were favored at the P1’ position. These data contrast previous studies that suggested the primary substrate specificity (S1) pocket of the PfA-M1 enzyme is unable to accommodate side-chains much larger than a P1 phenylalanine. To understand these apparently contradictory data, we determined the X-ray crystal structure of the PfA-M1 / bestatin-Tyr(OBzl) complex. The structure revealed a substantial inhibitor-induced rearrangement of the primary loop that forms the S1 pocket that permits accommodation of the bestatin-Tyr(OBzl) inhibitor. These findings are in contrast to most proteases where the S1 pocket is considered to define primary enzyme specificity through substantial rigidity. Taken together, our data provide important insights for the rational design of more potent and selective inhibitors of this enzyme, which may eventually be of therapeutic value for the treatment of malaria.

不可或缺的疟原虫源性PfA-M1金属氨基肽酶（malarial PfA-M1 metalloaminopeptidase）是一类经过验证的药物靶点，其参与血红蛋白消化的终末阶段。天然产物二肽模拟物乌苯美司（bestatin）是PfA-M1的强效抑制剂，可为新型研究工具及更高效PfA-M1抑制剂的开发提供优异的母核骨架。本研究报道了一种以商业化天然及非天然N-叔丁氧羰基-D-氨基酸（N-Boc-D-amino acids）为原料合成bestatin衍生物的高效、高产率新方法。我们构建了一个多样化的bestatin衍生物库，其修饰位点位于α-羟基-β-氨基酸侧链或邻近的天然α-氨基酸侧链。令人意外的是，我们发现P1位带有大体积芳香环的衍生物对PfA-M1具有强效抑制活性，而P1'位则偏好小体积疏水侧链。上述结果与此前研究结论相悖，此前研究认为PfA-M1酶的主要底物特异性S1口袋（S1 pocket）无法容纳比P1位苯丙氨酸更大的侧链基团。为解析这一看似矛盾的实验结果，我们解析了PfA-M1与bestatin-Tyr(OBzl)复合物的X射线晶体结构。该结构显示，构成S1口袋的主要环区发生了抑制剂诱导的显著构象重排，从而得以容纳bestatin-Tyr(OBzl)抑制剂。这一发现与大多数蛋白酶的特性相悖，通常认为这类蛋白酶的S1口袋凭借高度刚性来决定其主要底物特异性。综上，本研究数据为合理设计该酶的更强效、高选择性抑制剂提供了重要理论依据，此类抑制剂未来有望用于疟疾的临床治疗。