Aim: Azithromycin, a safe, long-lasting antibiotic that has been trialled as a partner drug for ACTs, is known to kill malaria parasites after 5 days (so called ‘delayed-death’) by targeting their essential remnant plastid ‘the apicoplast’. We identified that azithromycin also rapidly inhibits merozoite invasion of the host red blood cell (RBC) and sought to retarget azithromycin into a fast-acting antimalarial that retains activity against the apicoplast to broaden the clinical applications of azithromycin analogues against malaria disease.
Methods: The antimalarial activity of 92 azithromycin analogues was characterised against developing blood stage Plasmodium spp., invasion of purified merozoites into RBCs, drug-resistant lines and delayed-death (apicoplast targeting) resistant lines using modified in vitro assays.
Results: More than 60% of the azithromycin analogues tested rapidly killed parasites within one 48hr blood stage lifecycle (termed ‘quick-killing’) at concentrations up to 1500-fold lower than azithromycin. Parasite crisis forms could be clearly visualised within 6 hours of treatment with low nanomolar concentrations of some analogues, highlighting the speed of drug activity. Improved 48hr intracellular blood stage growth inhibitory activity corresponded to improvements in invasion inhibition, resulting in drugs that rapidly inhibit throughout the disease-causing blood stage lifecycle. Quick-killing activity is effective against multiple Plasmodium spp. and occurs via a secondary mechanism of action, independent of the known apicoplast target.
Conclusions: We have identified analogues of azithromycin that achieve significantly increased quick-killing potency (rapid parasite clearance) whilst maintaining activity against the apicoplast (long-term prophylaxis) in the low nanomolar range. Further development of lead analogues as antimalarials with dual mechanisms of action offers a safe, broad acting antimalarial strategy for use in combination therapies that would: (i) rapidly clear disease-causing parasites, (ii) protect against reinfection and (iii) provide ‘resistance proofing’ through dual mechanisms of action.