Among the various components of the protozoan Plasmodium mitochondrial respiratory chain, only Complex III is a validated cellular target for antimalarial drugs. The compound CK-2-68 was developed to specifically target the alternate NADH dehydrogenase of the malaria parasite respiratory chain, but the true target for its antimalarial activity has been controversial. Here, we report the cryo-EM structure of mammalian mitochondrial Complex III bound with CK-2-68 and examine the structure–function relationships of the inhibitor's selective action on Plasmodium. We show that CK-2-68 binds specifically to the quinol oxidation site of Complex III, arresting the motion of the iron-sulfur protein subunit, which suggests an inhibition mechanism similar to that of P_f-type Complex III inhibitors such as atovaquone, stigmatellin, and UHDBT. Our results shed light on the mechanisms of observed resistance conferred by mutations, elucidate the molecular basis of the wide therapeutic window of CK-2-68 for selective action of Plasmodium vs. host cytochrome bc₁, and provide guidance for future development of antimalarials targeting Complex III.

在原生动物疟原虫线粒体呼吸链的各种成分中，只有复合物 III 是抗疟药物经过验证的细胞靶标。化合物 CK-2-68 专门针对疟疾寄生虫呼吸链的替代 NADH 脱氢酶而开发，但其抗疟活性的真正目标一直存在争议。在这里，我们报告了与 CK-2-68 结合的哺乳动物线粒体复合物 III 的冷冻电镜结构，并检查了抑制剂对疟原虫选择性作用的结构-功能关系。我们发现 CK-2-68 特异性结合复合物 III 的对苯二酚氧化位点，阻止铁硫蛋白亚基的运动，这表明其抑制机制类似于 P f 型复合物 III 抑制剂（例如阿托伐醌）的抑制_机制。豆黄素和UHDBT。我们的结果揭示了观察到的突变赋予耐药性的机制，阐明了 CK-2-68 对疟原虫与宿主细胞色素bc ₁选择性作用的宽治疗窗的分子基础，并为未来开发靶向抗疟药提供指导复杂三。