Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase

Abstract

There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum. Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC 50 ] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED 90 ], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [ 14 C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

New developments in anti-malarial target candidate and product profiles

“…The initial momentum was provided by phenotypic screening of diverse compound collections. New chemical diversity has been screened in the last 4 years, including microbial metabolites from Japan, and molecules with novel chemistries such as boron [168, 169] and increased sp3 types [170]. These sources are rapidly being mined out and it is only through access to novel chemical diversity via new pharmaceutical compound libraries that new relevant compounds can be screened.…”

Identification of a potent benzoxaborole drug candidate for treating cryptosporidiosis

“…Plasmodium parasites, which cause malaria, are members of the phylum Apicomplexa, and are therefore genetically related to Cryptosporidium parasites. Given this and the recent identification of benzoxaboroles with potent antimalarial activity 28,30,31 , we screened a library of benzoxaborole compounds for anticryptosporidial activity. Here, we report identification of a novel drug candidate, AN7973, for treatment of cryptosporidiosis.…”

Altered Plasmodium falciparum Sensitivity to the Antiretroviral Protease Inhibitor Lopinavir Associated with Polymorphisms in pfmdr1

“…At each step of selection, parasites were initially undetectable on Giemsa-stained smears followed by regrowth, suggesting selection of mutations allowing growth under drug pressure. After each step of selection, we assessed parasite sensitivity by counting fluorescently stained parasites incubated with serial dilutions of lopinavir, as previously reported for other compounds (19), and we cloned parasites by limiting dilution. We then characterized wild-type and resistant parasites by wholegenome sequencing.…”

“…Wild-type W2 strain parasites and those with decreased lopinavir sensitivity were cloned by limiting dilution and then characterized by whole-genome sequencing, as reported previously (19). In brief, genomic DNA libraries were prepared, libraries were barcoded with unique sets of indices, fragments of 360 to 560 bp were extracted, the fragments were amplified by limited-cycle PCR, libraries were pooled, and sequencing was performed at the UCSF Center for Advanced Technology on a HiSeq 2000 system (Illumina).…”