Characterizing the quick-killing mechanism of action of azithromycin analogs against malaria parasites
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(Published version)
Date
2025
Authors
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.
Editors
Odom John, A.
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Journal article
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Antimicrobial Agents and Chemotherapy, 2025; 69(9):e0178324-1-e0178324-27
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Emma Y. Mao, William Nguyen, Gouranga P. Jana, Bikash C. Maity, Samuel Pazicky, Carlo Giannangelo, Janette Reader, Mufuliat T. Famodimu, Lyn-Marie Birkholtz, Michael J. Delves, Darren J. Creek, Zbynek Bozdech, Benoît Laleu, Jeremy N.Burrows, Brad E. Sleebs, Maria R. Gancheva, Danny W. Wilson
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Abstract
Drug 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.
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© 2025 Mao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.