Awalt, J.K.Ooi, Z.K.Ashton, T.D.Mansouri, M.Calic, P.P.S.Zhou, Q.Vasanthan, S.Lee, S.Loi, K.Jarman, K.E.Penington, J.S.Qiu, D.Zhang, X.Lehane, A.M.Mao, E.Y.Gancheva, M.R.Wilson, D.W.Giannangelo, C.MacRaild, C.A.Creek, D.J.et al.2025-09-092025-09-092025Journal of Medicinal Chemistry, 2025; 68(8):8933-89660022-26231520-4804https://hdl.handle.net/2440/147203To discover new antimalarials, a screen of the Janssen Jumpstarter library against Plasmodium falciparum uncovered the N-acetamide indole hit class. The structure−activity relationship of this chemotype was defined and culminated in the optimized frontrunner analog WJM664, which exhibited potent asexual stage activity and high metabolic stability. Resistant selection and whole-genome sequencing revealed mutations in PfATP4, which was validated as the target by showing that analogs exhibited reduced potency against parasites with resistanceconferring mutations in PfATP4, a metabolomic signature similar to that of the PfATP4 inhibitor KAE609, and inhibition of Na⁺- dependent ATPase activity consistent with on-target inhibition of PfATP4. WJM664 inhibited gamete development and blocked parasite transmission to mosquitoes but exhibited low efficacy in aPlasmodium berghei mouse model, which was attributed to ATP4 species differentiation and its moderate systemic exposure. Optimization of these attributes is required for N-acetamide indoles to be pursued for development as a curative and transmission-blocking therapy.en© 2025 The Authors. Published by American Chemical Society. This article is licensed under CC-BY-NC-ND 4.0Alkyls; Antimicrobial agents; Assays; Indoles; ParasitesAnimalsHumansMicePlasmodium bergheiPlasmodium falciparumAcetamidesIndolesAntimalarialsStructure-Activity RelationshipJournal article10.1021/acs.jmedchem.5c00614737023Mao, E.Y. [0000-0001-8709-1897] [0000-0002-4040-1381]Gancheva, M.R. [0000-0001-7428-6791]Wilson, D.W. [0000-0002-5073-1405]