A bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent means of malaria control, drug-resistant parasites necessitate the discovery of new antimalarials. Peptidases are a promising class of drug targets and perform several important roles during the P. falciparum erythrocytic life cycle. Herein, we report a multidisciplinary effort combining activity-based protein profiling, biochemical, and peptidomic approaches to functionally analyze two genetically essential P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. Through the synthesis of a suite of activity-based probes (ABPs) based on the general MAP inhibitor scaffold, bestatin, we generated specific ABPs for these two enzymes. Specific inhibition of PfA-M1 caused swelling of the parasite digestive vacuole and prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, likely starving the parasite resulting in death. In contrast, inhibition of Pf-LAP was lethal to parasites early in the lifecycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential role outside of Hb digestion.

疟疾在全球范围内引发大量发病与死亡，尽管化疗仍是疟疾防控的有效策略，但抗药性寄生虫的出现亟需开发新型抗疟药物。肽酶是一类极具潜力的药物靶点，在恶性疟原虫（Plasmodium falciparum, P. falciparum）的红细胞内生命周期中承担多项关键功能。本文报道一项多学科研究工作，整合基于活性的蛋白质谱分析、生化及肽组学方法，对两种遗传必需的恶性疟原虫金属氨肽酶（metallo-aminopeptidases, MAPs）——PfA-M1与Pf-LAP开展功能分析。研究人员基于通用金属氨肽酶抑制剂乌苯美司（bestatin）的骨架合成了一系列基于活性的探针（activity-based probes, ABPs），并获得了针对这两种酶的特异性探针。对PfA-M1的特异性抑制会导致寄生虫消化泡肿胀，并阻断血红蛋白（hemoglobin, Hb）来源寡肽的蛋白水解过程，最终可能通过剥夺寄生虫营养使其死亡。与之相反，对Pf-LAP的抑制会在寄生虫生命周期早期、血红蛋白降解启动前就导致其死亡，这表明Pf-LAP在血红蛋白消化过程之外发挥着不可或缺的生理作用。