Integrative Antiparasitic & Erythrotoxic Characterization of a Red Blood Cell Host-Biased Compound Library

Abstract

Malaria is a deadly mosquito-transmitted disease caused by Apicomplexan Plasmodium parasites that infect millions of people every year: In 2020 alone, 241 million people were infected by the parasites and ~627,000 people were killed. Despite the arsenal of antimalarial compounds that have been successfully developed to combat malaria, every antimalarial deployed to date has been counteracted by cases of drug resistance, highlighting a pressing need for novel drug discovery strategies & targets. Targeting host factors is an underexplored alternative approach to increasing barriers to drug resistance in malaria that has the potential to uncover targets required for multiple Plasmodium species due to overlaps in host requirements. Additionally, many Plasmodium host factors are already FDA approved drug targets or clinical probes, paving the way for drug repurposing.

Despite the abundance of “druggable” Plasmodium host factors in the red blood cell (RBC) amenable to small molecule inhibition, Imatinib is the only putatively host-targeted antimalarial candidate that has made it into the clinic. Additionally, while Plasmodium host factors have been identified through individual studies, no studies have evaluated compounds targeting these factors against Apicomplexan parasites or the RBC host in an integrative manner. Such studies can assist in prioritizing host targets and compounds for antimalarial host-directed therapy (HDT).

My dissertation spotlights host pathways that could potentially be targeted during the Plasmodium intraerythrocytic development cycle (IDC) to combat blood stage malaria (Chapter 2; Chapter 3); investigates whether preclinical, clinical, and FDA approved compounds targeting druggable host factors within these pathways have broad spectrum antiparasitic activity (Chapter 3); and counterscreens against erythrotoxic antiparasitic compounds that induce RBC phenotypes predicted to result in anemia in vivo (i.e. osmotic fragility, hemolysis, or eryptosis), worsening the disease pathology of malaria (Chapter 4).

I discovered that most curated compounds (25/31) in my RBC host-biased library were active against P. falciparum with a multistage IC50 value of 10µM or less, with a fifth of these compound hits possessing submicromolar inhibitory activity. Remarkably, roughly 80% of active compound hits exhibited broad spectrum antimalarial activity against drug susceptible and resistant P. falciparum (3D7 & Dd2) and P. knowlesi species, with almost half of these compounds also restricting phylogenetically distant Babesia divergens parasites. Most compounds (16/25) were also found to restrict multiple stages of the P. falciparum intraerythrocytic development cycle, a mechanism predicted to further increase barriers to drug resistance. Antiparasitic IC50 values were consistent with cellular IC50 values for many compounds, suggestive of host targeting; and four compounds were found to retain their antiparasitic activity following RBC preincubation and compound washout, confirming an irreversible host targeted mechanism of action. Notably, roughly one third of compounds hits had not previously been evaluated against human Plasmodium species.

While the majority (16/25) of compounds were found to be non-erythrotoxic—including Trametinib (FDA approved MEK1 inhibitor), the most potent antimalarial compound in my library—all irreversibly host-targeted compounds were among the 9/25 erythrotoxic hits, as were all compound hits annotated to target RBC channels. Strikingly, all compounds targeting RBC kinases that phosphorylate the RBC cytoskeleton did not induce significant erythrotoxicity, supporting this mechanism as a viable target for antimalarial HDT. These hits included Imatinib, Ruboxistaurin (Phase III Clinical probe; Protein Kinase C beta inhibitor; not previously tested), and TG100-115 (kinase-specific TRPM7 chanzyme inhibitor). The ability to link entire mechanisms of action to erythrotoxicity or lack thereof underscores the value of integratively evaluating prospective antimalarial HDTs.

Overall, these studies highlight the value of considering prospective antimalarials in parallel, emphasize the significance of counterscreening for erythrotoxicity prior to their translation, and provide a rich foundation for compound prioritization in RBC host-directed antimalarial drug discovery.