Targeting Artemisinin-Resistant Malaria by Repurposing the Anti-Hepatitis C Virus Drug Alisporivir

Abstract

The emergence of Plasmodium falciparum resistance raises an urgent need to find new antimalarial drugs. Here, we report the rational repurposing of the anti-hepatitis C virus drug, alisporivir, a nonimmunosuppressive analog of cyclosporin A, against artemisinin-resistant strains of P. falciparum. <i>In silico</i> docking studies and molecular dynamic simulation predicted strong interaction of alisporivir with <i>Pf</i>Cyclophilin 19B, confirmed through biophysical assays with a <i>K_d</i> value of 354.3 nM. Alisporivir showed potent antimalarial activity against chloroquine-resistant (<i>Pf</i>RKL-9 with resistance index [Ri] 2.14 ± 0.23) and artemisinin-resistant (<i>Pf</i>Kelch13^R539T with Ri 1.15 ± 0.04) parasites. The Ri is defined as the ratio between the IC₅₀ values of the resistant line to that of the sensitive line. To further investigate the mechanism involved, we analyzed the expression level of <i>Pf</i>Cyclophilin 19B in artemisinin-resistant P. falciparum (<i>Pf</i>Kelch13^R539T). Semiquantitative real-time transcript, Western blot, and immunofluorescence analyses confirmed the overexpression of <i>Pf</i>Cyclophilin 19B in <i>Pf</i>Kelch13^R539T. A 50% inhibitory concentration in the nanomolar range, together with the targeting of <i>Pf</i>Cyclophilin 19B, suggests that alisporivir can be used in combination with artemisinin. Since artemisinin resistance slows the clearance of ring-stage parasites, we performed a ring survival assay on artemisinin-resistant strain <i>Pf</i>Kelch13^R539T and found significant decrease in parasite survival with alisporivir. Alisporivir was found to act synergistically with dihydroartemisinin and increase its efficacy. Furthermore, alisporivir exhibited antimalarial activity <i>in vivo</i>. Altogether, with the rational target-based Repurposing of alisporivir against malaria, our results support the hypothesis that targeting resistance mechanisms is a viable approach toward dealing with drug-resistant parasite.