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Studies to elucidate the molecular targets of two potent antimalarial benzoxaborole compounds in Plasmodium falciparum

Abstract

With increasing resistance of malaria parasites to available drugs, there is a great need for new antimalarials, ideally with novel mechanisms of action. We are investigating the antimalarial activity and mechanisms of action of benzoxaboroles, a novel class of boron-containing compounds. Compounds 3661, 1467 and related compounds demonstrated good activity in vitro against chloroquine-resistant Plasmodium falciparum and in vivo against murine Plasmodium berghei infection (compound 3661: IC50 37 nM against W2-strain P. falciparum, ED90 0.3 mg/kg against murine P.berghei; compound 1467: IC50 196 nM, ED90 7.4 mg/kg). In an attempt to characterize mechanisms of action, we selected for parasites with decreased sensitivity to 3661 and 1467 by culturing with step-wise increases in concentration of the compounds followed by whole genome sequencing. Sequencing of parasites selected for resistance to 1467 showed several SNPs in the editing domain of a predicted leucyl tRNA synthetase (LeuRS) gene (PF3D7_0622800) and in another gene of unknown function (PF3D7_1218100). Additionally, both compounds were tested for stage-specificity by incubation with test compounds for 8 hour intervals across the parasite erythrocytic life cycle. Both compounds were most active against trophozoite stage parasites. To further understand the mechanism of action of 1467 and the related compound 1474, the incorporation of 14[C] leucine in parasite cultures or parasite extracts including exogenous tRNA was assessed in the presence or absence of the compounds. Dose-dependent inhibition of both protein synthesis and LeuRS activity was observed for 1467 and 1474, but not 3661 or the control artemisinin, supporting different mechanisms for the different benzoxaboroles. For 3661, in vitro resistance selection was also achieved by culturing parasites in step-wise increasing concentration and in a single high concentration of the compound. Cross-resistance was not seen between parasites selected with 3661 and those selected with 1467. Whole genome sequencing of multiple clones selected for resistance to 3661 revealed several SNPs in a gene that codes for a homolog of mammalian cleavage and polyadenylation specificity factor (CPSF; PF14_0364). In summary, we offer strong evidence for unique antimalarial mechanisms of action for two benzoxaboroles, identifying two potential novel antimalarial drug targets. Further investigation of these novel benzoxaborole mechanisms is underway.

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