Identification of α-azacyclic acetamide-based inhibitors of P. falciparum Na ⁺ pump ( Pf ATP4) with fast-killing asexual blood-stage antimalarial activity by phenotypic screening

Malaria treatments are compromised by drug resistance, creating an urgent need to discover new drugs. We used a phenotypic high-throughput screening (HTS) platform to identify new antimalarials, uncovering three related pyrrole-, indole-, and indoline-based series with a shared α-azacyclic acetamide core. These compounds showed fast-killing activity on asexual blood-stage Plasmodium falciparum parasites, were not cytotoxic, and disrupted parasite intracellular pH and Na ⁺ regulation similarly to cipargamin (KAE609), a clinically advanced inhibitor of the P. falciparum Na ⁺ pump ( Pf ATP4). Pf ATP4 is localized to the parasite plasma membrane and is essential for maintaining a low cytosolic Na ⁺ concentration. Resistance selections on P. falciparum parasites with two α-azacyclic acetamide analogs identified mutations in Pf ATP4, and cross-resistance was observed across the α-azacyclic acetamides and KAE609, confirming Pf ATP4 as the target. Pf ATP4 is a well-established antimalarial target, and identification of additional Pf ATP4 inhibitors provides alternative avenues to disrupt its function.