Investigation of azithromycin analogues and proteasome-like inhibitors as quick-killing antimalarials

Abstract

Malaria is caused by mosquito-borne parasites of the genus Plasmodium which were responsible for ~435,000 of deaths annually, with >90% caused by the deadliest species, P. falciparum. Over the last two decades, global implementation of vector control and artemisinin combination therapies have resulted in significant reductions in the global burden of malaria. Of current concern is the spread of multi-drug resistant parasites that have severely limited the efficacy of antimalarials, including front-line artemisinins, highlighting the urgent need to identify new antimalarials for use as treatments. The aim of this thesis was to investigate novel antimalarial development avenues and identify new chemotypes that could be used in the near future as treatments. The macrolide antibiotic azithromycin is known to target the malaria parasites remnant plastid organelle (the apicoplast’s) bacterial-like ribosome and causes slow-killing ‘delayed death’, where the parasite dies in the second replication cycle (4 days). Azithromycin has also been shown to inhibit invading merozoites and kill blood stages within the first replication cycle (2 days) via an unidentified mechanism, proposed to be independent of delayed death. Thus, we hypothesised that azithromycin could be redeveloped into an antimalarial with two different mechanisms of action against parasites: delayed death and quick-killing. Over 100 azithromycin analogues that featured a high proportion of different structural profiles were obtained, leading to improved quick-killing activities over azithromycin. Quick-killing was also confirmed to be completely unrelated to delayed death, as blood stage parasites lacking the apicoplast were equally susceptible to quick-killing of azithromycin and analogues. Two different avenues were also confirmed for azithromycin’s antimalarial re-development: delayed death and quick-killing or quick-killing only, which could be modulated depending on the location of added functional groups. Azithromycin and analogues were found to be active across blood stage development, with only short treatments required to kill parasites. The metabolomics signatures of parasites treated with azithromycin and analogues suggested that quick-killing acts multi-factorially, with the parasite’s food vacuole and mitochondria being likely targets. Finally, in vitro activities of two subtypes of tri-peptide proteasome-like inhibitors, vinyl sulfone and aldehydes, were addressed against P. falciparum and the zoonotic malaria parasite P. knowlesi. All compounds exhibited low-nanomolar activities against both Plasmodium spp. and showed excellent selectivity for parasites over human cells, suggesting these inhibitors provide viable chemical scaffolds for optimisation. There was no evidence of increased protein ubiquitination upon treating parasites with these compounds, suggesting they do not target the proteasome. We also investigated whether hypoxia inducible pro-drug proteasome-like inhibitors could be used to reduce host toxicity of antimalarials. However, these pro-drugs could be not activated in in vitro culture conditions and there was limited evidence suggesting this strategy would be applicable in malaria. These studies build on previous findings on the drug-killing efficacy, mechanism of action and possible application of redeveloping azithromycin analogues as new and improved antimalarials. I also identified new proteasome inhibitor-like scaffolds as starting points for further development. This body of work provides thorough biological characterisation of a panel of compounds that could lead to new avenues for antimalarial development.