Characterizing the quick-killing mechanism of action of azithromycin analogs against malaria parasites

Published in: Antimicrobial Agents and Chemotherapy, Vol. 69, No. 9

DOI: 10.1128/aac.01783-24

Authors: Emma Y. Mao, William Nguyen, GourangaP. Jana[TCGLS MEMBER], BikashC. Maity[TCGLS MEMBER], 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 

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.