Malaria is a mosquito-borne disease caused by Plasmodium spp. parasites. In 2021 alone, 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 quick-killing 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, a structurally similar antimalarial that targets the parasite’s digestion of haemoglobin and is now associated with widespread drug resistance. Using synergy and beta-haematin formation assays, we show that chloroquinoline-modified analogues have properties similar to chloroquine, whereas non-chloroquinoline analogues do not. However, our non-chloroquinoline analogues are still able to achieve substantial improvements in quick-killing over azithromycin by up to 40-fold without contribution of this chloroquine-like activity, with the addition of the chloroquinoline moiety further potentiating quick-killing by another 2.5 to 4-fold through a more 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 transmission blocking potential over chloroquine and the maintenance of quick-killing potency in multiple chloroquine-resistant Plasmodium lines. Further investigation of these non-chloroquine related mechanisms by in vitro resistance selection and cellular thermal shift assays (CETSA) highlight that this readily accessible family of compounds, with potential for optimisation as fast and broad acting antimalarials, induce parasite death by targeting a multitude of important parasite pathways.