Determining the Function of Plasmodium Hemolysin III and Discovery of Novel Antimalarial Drugs
Robinett, Natalie G.
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Elimination of the malaria parasite from endemic areas requires a multi-faceted approach, including development of novel antimalarial drugs and a deeper understanding of parasite-host interactions. Here we describe functional characterization of a Plasmodium hemolysin III (PfHlyIII) along with various approaches to determine whether hemolysin is a virulence factor in malaria, contributing to severe malaria anemia. In addition we also describe two antimalarial drug discovery projects including characterization of novel quinine and quinidine derivatives as efficacious, non-toxic antimalarials, as well as the development of a robust high throughput assay to screen for gametocytocidal compounds. Regarding characterization of Plasmodium hemolysin III, we have evidence for heterologous pore formation of recombinant PfHlyIII in Xenopus and also show expression of soluble native PfHlyIII in asexual blood stage parasites. Together these data support our hypothesis that PfHlyIII may be available upon schizont egress as a cytolytic protein that could damage and increase clearance of bystander erythrocytes. Unexpectedly, genetic disruption of P. berghei HlyIII (PbHlyIII KO) resulted in greater virulence in Balb/c mice leading to an early death phenotype and altered parasitophorous vacuole morphology in the asexual blood stages. We hypothesize that early death in mice infected with the PbHlyIII KO parasite may be a result of altered deformability of infected erythrocytes and increased sequestration leading to brainstem hemorrhage. Though we did not prove or disprove our hypothesis that PfHlyIII may damage uninfected erythrocytes and contribute to severe malaria anemia, our knockout phenotype of severe membrane defects suggests PfHlyIII may play a role in membrane homeostasis or remodeling, either directly or indirectly through functioning as a receptor, similar to yeast homolog Izh2p. Synthesis and characterization of hydroxyethylapoquinine and derivatives involved revisiting an old quinine derivative with promising historical data supporting greatly reduced toxicity in humans and comparable efficacy against bird malaria compared to quinine. The modifications to quinine included hydroxyethylation at the methoxy side chain and isomerization of a vinyl group. Our studies included a novel synthetic approach to hydroxyethylapoquinine in addition to synthesis of three novel compounds: hydroxyethylapoquinidine and quinine and quinidine derivatives with only the hydroxyethyl substitution. We demonstrate antimalarial efficacy of all four derivatives against three strains of P. falciparum in vitro, with nanomolar IC50s against a quinine-sensitive strain 3D7, and elevated IC50s against quinine tolerant strains INDO and Dd2. In a murine malaria model the quinidine intermediate, hydroxyethylquinidine (HEQD) showed the greatest potency, similar to quinine and also performed the best in the in vitro assays. Furthermore the hydroxyethyl modifications greatly reduced the hERG channel inhibitory properties of all derivatives compared to the parent drugs, and further derivation of HEQD may yield a safer alternative to quinine or quinidine and be a potential long-half life partner drug in artemisinin-based combination therapies. SYBR Green I and a green fluorescence background suppressor from CyQUANT were used in conjunction with exflagellation media to develop a novel transmission blocking assay that can be used to screen for gametocytocidal compounds. Following optimization of the assay, we screened the Johns Hopkins Clinical Compound Library version 1.3 as well as the Medicines for Malaria Venture malaria box, a total of over 2,000 compounds, resulting in 43 hits with good efficacy against late stage gametocytes. Quaternary ammonium compounds and acridine-like compounds were novel drug classes revealed in our screen. Transmission blocking activity of top hits was confirmed using membrane feeding assays and correlation with other assays strengthened the validity of our assay. Overall our data supports use of the SYBR Green assay to screen for novel transmission blocking compounds for use in malaria elimination strategies.