The Role of the Human B-Raf Kinase in Plasmodium falciparum Blood-Stage Infection

Malaria, a disease caused by the infection with parasites from the genus Plasmodium, remains a primary global health concern. The selection of mutations under drug pressure has resulted in the emergence of strains resistant to treatment, contributing to resurgences and waning efficacy of antimalarial medications. Hence, there is an urgent need to develop effective alternatives to traditional therapeutics. All available malaria chemotherapeutics target parasite-encoded enzymes or parasite-controlled processes. For example, chloroquine prevents heme polymerization by inhibiting heme polymerase, which converts heme to hemozoin, and artemisinin works by generating toxic free radicals through the cleavage of its endoperoxide bridge, which interacts with heme-iron. However, resistance caused by the single nucleotide polymorphisms (SNPs) on the PfCrt (chloroquine transporter gene), and mutations in the PfKelch13 gene respectively, have reduced the effectiveness of these drugs. This challenge underlies the need for new antimalarials with new mechanisms of action. Recent studies show that several host erythrocytes signalling kinases are required for parasite growth and survival, suggesting that an approach known as Host-Directed Therapy can be implemented. This approach is refractory to the most direct pathway to resistance (selection of parasites encoding a mutated target) since the target proteins are not under the parasite’s genetic control. Possible targets for this approach include host cell B-Raf, c-MET, and MEK kinases, all of which are implicated in the infection of erythrocytes by malaria parasites. The B-Raf kinase is an essential component of the MAPK pathway that regulates signal transduction for cell growth, division, proliferation and survival. Moreover, the dysregulation of the B-Raf gene has been implicated in many cancer cell types. Recent phosphoproteomics studies have suggested sustained activity of the B-Raf kinase in the three asexual stages of erythrocyte malaria infection, but its role in parasite development is unknown. In the work reported in this thesis, we draw insights on the role of the B-Raf kinase in erythrocytic stage P. falciparum infection through the analysis of data generated from dose-response assays of parasite growth inhibition with the B-Raf inhibitors SB590885, Dabrafenib, and PLX8394. In this study, we aimed to validate the hypotheses that the B-Raf kinase is indeed essential for successful malaria pathogenesis, and that targeted B-Raf inhibition impairs parasite proliferation and survival. As expected, wildtype 3D7 P. falciparum did not develop resistance to both lethal and sub-lethal concentrations of most B-Raf inhibitors under varying drug selection pressure, which is exciting because it emphasizes the efficacy of these compounds as potential re-purposed antimalarial compounds, as well as the advantage of a reduced risk of resistance to host-target inhibition by parasites. Surprisingly, resistance was developed to PLX8394, a paradox breaker of the Raf pathway. Paradox breakers are compounds that prevent the re-activation of the downstream pathway (see the main text for a full description). Dose escalation studies allowed the selection of parasites exhibiting resistance to PLX-8394. Further investigations revealed this resistance is unstable in the absence of drug pressure, which reveals the need for a consistent drug dose for maintenance of a resistant population. Other phenotypic assessments show high efficacy of the B-Raf compounds at the early ring stage of infection compared to other parasite blood life stages, which opens up new hypotheses for the function of the B-Raf kinase during infection. Overall, this work demonstrated the use of parasite growth and inhibition assays to understand B-Raf function in erythrocyte P. falciparum infection, and its potential as a drug target for Host Directed Antimalarial Therapy.<p></p>