Mao, E.Y.Nguyen, W.Jana, G.P.Maity, B.C.Pazicky, S.Giannangelo, C.Reader, J.Famodimu, M.T.Birkholtz, L.-M.Delves, M.J.Creek, D.J.Bozdech, Z.Laleu, B.Burrows, J.N.Sleebs, B.E.Gancheva, M.R.Wilson, D.W.Odom John, A.2025-10-142025-10-142025Antimicrobial Agents and Chemotherapy, 2025; 69(9):e0178324-1-e0178324-270066-48041098-6596https://hdl.handle.net/2440/147759Drug resistance is steadily undermining the efficacy of frontline anti-malarials, highlighting the urgent need for novel therapies with alternative mechanisms of action. The chemical addition of different moieties to azithromycin yields compounds with improved quick-killing potency against malaria parasites, with the most active analogs typically containing a chloroquinoline group. Here, we investigated the quick-killing mechanism of five azithromycin analogs, two of which contain differentially oriented chloroquinoline moieties. The improvement in quick-killing activity over azithromycin for non-chloroquinoline analogs was around 10 -to 42-fold, with chloroquinoline-containing analogs showing a further 2- to 17-fold improvement over non-chloroquinoline compounds. Chemical inhibition of hemoglobin digestion and chloroquine’s inhibitory effect against heme polymerization linked analogs with both chloroquinoline and non-chloroquinoline modifications to a chloroquine-like mechanism of action. However, none of the analogs showed a significant reduction in efficacy against chloroquine-resistant asexual blood-stage parasites. Multiple attempts at selecting for azithromycin analog-resistant parasites to elucidate the mechanism of quick-killing were unsuccessful. Application of cellular thermal shift proteomics revealed that azithromycin analogs significantly stabilized 34–155 different proteins in trophozoites, a high number that showed minimal overlap with chloroquine. Additionally, our most potent chloroquinoline-containing analog demonstrated a significant improvement in gametocytocidal activity over azithromycin and further maintained moderate inhibition of chloroquine-insensitive late-stage gametocytes. These findings support that this class of azithromycin analogs kills malaria parasites through a broad range of potential mechanisms, making them promising candidates for optimization as fast and broad-acting anti-malarials.en© 2025 Mao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.antimalarial agentsazithromycincellular thermal shift assaymalariaPlasmodiumJournal article10.1128/aac.01783-24745913Mao, E.Y. [0000-0001-8709-1897] [0000-0002-4040-1381]Gancheva, M.R. [0000-0001-7428-6791]Wilson, D.W. [0000-0002-5073-1405]