Oral Presentation 15th Lorne Infection and Immunity 2025

Azithromycin analogues with improved quick-killing activity have broad mechanisms of action against malaria parasites (#29)

Emma Y Mao 1 2 , William Nguyen 3 4 , Gouranga P Jana 5 , Bikash C Maity 5 , Samuel Pazicky 6 , Carlo Giannangelo 7 , Janette Reader 8 , Mufuliat T Famodimu 9 , Lyn-Marie Birkholtz 8 , Michael J Delves 9 , Darren J Creek 7 , Zbynek Bozdech 6 , Benoit Laleu 10 , Jeremy N Burrows 10 , Brad E Sleebs 3 4 , Maria R Gancheva 1 2 , Danny W Wilson 1 2 11
  1. School of Biological Sciences, The University of Adelaide, Adelaide, South Australia, Australia
  2. ARC Training Centre for Environmental and Agricultural Solutions to Antimicrobial Resistance , (CEAStAR)
  3. WEHI - Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia
  4. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  5. TCG Lifesciences Private Limited, Kolkata, India
  6. School of Biological Sciences, Nanyang Technology University, Singapore
  7. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia
  8. Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria, South Africa
  9. London School of Hygiene and Tropical Medicine, London, United Kingdom
  10. MMV Medicines for Malaria Venture, Geneva, Switzerland
  11. Burnet Institute, Melbourne , Victoria, Australia

Malaria remains a significant global health and economic burden, causing ~249 million cases and ~608,000 deaths in 2022 alone. Resistance has now developed to nearly all clinically available anti-malarials, highlighting the urgent need for new drugs with novel mechanisms of action (MoAs). Azithromycin is a safe and long-acting antibiotic that targets an essential parasite organelle of bacterial origin, the apicoplast, resulting in a delayed-death phenotype whereby parasite death is manifested only in the lifecycle after treatment initiation (~5 days). At higher treatment concentrations, azithromycin also has quick-killing activity independent of apicoplast targeting, leading to parasite death within the first replication cycle (~2 days). Chemical modification of azithromycin greatly enhances this quick-killing activity, however, the mechanism by which this occurs remains elusive. Here, we investigated the quick-killing mechanism of five azithromycin analogues, two of which contain chloroquinoline groups, structurally similar to chloroquine, an anti-malarial that targets the parasite’s digestion of haemoglobin. The non-chloroquinoline analogues demonstrated ~40-fold improvement in quick-killing across multidrug-sensitive and resistant lines, with chloroquinoline analogues being ~4-fold more potent. Experiments to test for evidence of chloroquine-like activity yielded conflicting results. Treating malaria parasites with the chloroquine resistance reversing agent, verapamil, and disrupting in vitro β-haematin formation linked chloroquinoline, but not non-chloroquinoline analogues to a chloroquine-like MoA. However, chloroquine-resistant parasites remained susceptible, and all analogues, not just the chloroquinoline ones, were antagonised when haemoglobin digestion was chemically inhibited. Furthermore, our most potent chloroquinoline analogue demonstrated activity against chloroquine-insensitive transmission stages of malaria parasites, the late-stage gametocytes. Application of thermal-shift proteomics to probe for alternative MoAs detected up to 291 impacted proteins, a remarkably high number with minimal overlap to chloroquine. These findings support that the bulk of the azithromycin-analogue quick-killing activity is likely driven by other non-chloroquine-like MoAs, broadly disrupting multiple cellular pathways in the parasite to rapidly induce death.