Identification and deconvolution of cross-resistance signals from antimalarial compounds using multidrug-resistant Plasmodium falciparum strains

Abstract

Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.

FIG 1

Phenotypic validation of the panel of MDR strains. The relative IC₅₀s of artesunate (ART), atovaquone (ATO), chloroquine (CHQ), cycloguanil (CYC), mefloquine (MEF), and pyrimethamine (PYR) against the panel of P. falciparum laboratory strains are shown. The solid line shows a 10-fold increase in the IC₅₀ relative to that for NF54 (n ≥ 2; error bars are SDs).

FIG 2

Resistance profile of the 1,2,4-oxadiazole series. (A) Structures of the three compounds from the 1,2,4-oxadiazol series. Ph, phenyl; Bn, benzyl; Me, methyl. (B) Relative IC₅₀s of compounds 1, 2, and 3 against a panel of P. falciparum laboratory strains. The solid line shows a 10-fold increase in the IC₅₀ relative to that for NF54 (n ≥ 2; error bars are SDs).

FIG 3

Cross-resistance deconvolution. (A) Venn diagram of the profiles of resistance of the P. falciparum strains to atovaquone (ATO), chloroquine (CHQ), cycloguanil (CYC), and pyrimethamine (PYR). The main genetic determinants of resistance for each compound is indicated, and the strains in boxes with bold borders correspond to the ones evaluated in the assay whose results are presented in panel B. (B) Deconvolution cascade of cross-resistance signals determined using the multidrug-resistant P. falciparum strain panel. R, resistant; S, sensitive; CNV, copy number variation.