Malaria is a mosquito-borne disease caused by Plasmodium spp. parasites. In 2021, malaria was responsible for approximately 247 million cases and 619 thousand deaths worldwide. While antimalarials have contributed significantly to the decline in global mortality, drug resistance is a looming threat. Azithromycin is a safe and long-acting antibiotic known to target the parasite’s essential plastid organelle, the apicoplast, resulting in a delayed-death phenotype whereby parasite death is manifested only in the lifecycle after treatment initiation. At higher treatment concentrations, azithromycin also has quick-killing activity independent of apicoplast targeting, leading to parasite death within the first replication cycle. Chemical modification of azithromycin can greatly enhance this quick-killing activity, however, the mechanism by which this occurs remains elusive.
We investigated the antimalarial activity of five azithromycin analogues. All analogues rapidly killed multidrug sensitive and resistant Plasmodium parasites within one blood-stage lifecycle at IC50s < 500 nM. Two of our compounds possess chloroquinoline moieties, raising the possibility that they act like chloroquine, an antimalarial that targets haemoglobin digestion and is now associated with widespread drug resistance. Using synergy assays with both inhibitors of haemoglobin digestion and reversers of chloroquine resistance, alongside beta-haematin formation assays, we show that chloroquinoline-modified analogues, but not non-chloroquinoline ones, exhibit a chloroquine-like mechanism of quick-killing. However, non-chloroquinoline analogues are still able to improve quick-killing potency by up to 40-fold over azithromycin independently of chloroquine-like activity, with the addition of a chloroquinoline moiety seen to further enhance this by up to 17-fold through a chloroquine-like mechanism. These data support that the bulk of quick-killing improvement is likely driven by other non-chloroquine related mechanisms, allowing for improved late-stage gametocytocidal activity over chloroquine and maintenance of quick-killing potency in multiple chloroquine-resistant Plasmodium lines. Further elucidation of these non-chloroquine related mechanisms may identify novel drug targets suitable for future drug development efforts.