Ashton, Trent D.Calic, Petar P.S.Dans, Madeline G.Ooi, Zi KangZhou, QingmiaoPalandri, JosephineLoi, KatieJarman, Kate E.Qiu, DeyunLehane, Adele M.Maity, Bikash ChandraDe, NirupamGiannangelo, CarloMacRaild, Christopher A.Creek, Darren J.Mao, Emma Y.Gancheva, Maria R.Wilson, Danny W.Chowdury, Mrittikade Koning-Ward, Tania F.Famodimu, Mufuliat T.Delves, Michael J.Pollard, HarrySutherland, Colin J.Baud, DelphineBrand, StephenJackson, Paul F.Cowman, Alan F.Sleebs, Brad E.2025-12-212025-12-210022-2623PubMed:39134060https://hdl.handle.net/1885/733796797To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S-WJM992, were shown to inhibit PfATP4-associated Na+-ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S-WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.This work was funded by the National Health and Medical Research Council of Australia (Development grant 1135421 to B.E.S. and A.F.C.; Synergy grant to 1185354 to T.F.dK-W. and D.J.C.), the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. B.E.S. gratefully acknowledges the support from the MMV Medicines for Malaria Venture (RD-18-0003) for the chemistry, in vitro ADME profiling, and the SCID mouse model. This work was supported by a Medical Research Council Career Development Award (MR/V010034/1) awarded to M.J.D. M.T.F. is supported by an MMV grant (RD-21-1003) awarded to M.J.D. Mosquito infection studies at LSHTM are supported by Wellcome Trust Biomedical Resources Grant 221363/Z/20/Z awarded to C.J.S. We thank and acknowledge the Australian Red Cross Lifeblood for the provision of fresh red blood cells. We thank Dr Keith Watson from the Walter and Eliza Hall Institute for the helpful advice. A.F.C. is a Howard Hughes International Scholar and an Australia Fellow of the NHMRC. B.E.S. is a Corin Centenary Fellow. Z.K.O. and Q.Z. were supported by an Alan Harris Scholarship, Q.Z. was supported by the China Scholarship Council, and E.Y.M. was supported by an ARC-RTP Scholarship.31en© 2024 The Authors.2024-08-1210.1021/acs.jmedchem.4c0124185201165629