Authors

Discussion

Substantial advancements have been made in the discovery of novel antileishmanial leads and clinical candidates by phenotypic evaluation on intramacrophage amastigotes of the visceral Leishmania species. Aminopyrazoles have emerged as a promising series and hit-to-lead optimization by the Drugs for Neglected Diseases initiative (DNDi) resulted in compounds with highly potent activity in animal models of leishmaniasis.

Molecular target deconvolution for the most potent aminopyrazoles has proven to be a major challenge because successive drug exposure failed to select for stably resistant phenotypes. Chemical mutagenesis with either ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU) combined with drug selective pressure and whole genome sequencing was used as an alternative approach. From the obtained panel of 28 resistant lines an association between >10-fold resistance and multiple independent heterozygous mutations adjacent to the Zn²⁺ binding site of the zinc finger containing protein LINF_180011100 was discovered. Overexpression of the mutated gene increased resistance up to 10-fold, whereas susceptibility could be restored in mutant lines by transfection of a wildtype copy. Gene editing by CRISPR-Cas9 independently confirmed the contribution of the EMS and ENU mutations, resulting in H594Y and H594P substitutions respectively, to 10-32-fold resistance exhibited both at the extracellular promastigote and intracellular amastigote stage. Prediction of the molecular function of LINF_180011100 suggests a role in nucleocytoplasmic transport through the nuclear pore complex and cell cycle control.

Collectively, our data provide a sequential validation of LINF_180011100 as a drug target or resistance determinant for several aminopyrazole leads.