Deconvoluting the mode of action of antileishmanial aminopyrazoles through chemical mutagenesis

Aminopyrazoles have emerged as a promising series of antileishmanial compounds. Molecular target deconvolution has been a major challenge because successive drug exposure failed to select for resistance. Chemical mutagenesis combined with aminopyrazole selective pressure and whole genome sequencing was used as an alternative approach. From the obtained panel of 28 resistant lines, an association was discovered between >10-49-fold resistance and multiple independent heterozygous mutations in the C-terminal FYVE Zn finger domain of LINF_180011100. LINF_180011100 gene overexpression and CRISPR-Cas9 gene editing has independently confirmed the association with 10- to 30-fold aminopyrazole resistance. Genetic fusion with an N-terminal green fluorescent protein tag and partial colocalization with a C-terminal mCherry-tagged small GTPase Rab5 demonstrated that the functional protein primarily localizes in endocytic vesicles. In situ target engagement assays confirmed a selective effect of aminopyrazoles and its abrogation by introducing a resistance-conferring mutation. Finally, a direct interaction between the lead compound and recombinantly produced LINF_180011100 FYVE Zn finger domain was demonstrated through a biophysical assay. Hence, this work formally validates LINF_180011100 as a drug target and resistance determinant for several aminopyrazole leads and provides insights into the mechanism of action. Aminopyrazoles have emerged as a promising series of compounds against leishmaniasis, yet understanding how aminopyrazoles work has proven to be a major challenge. Authors apply chemical mutagenesis and analyse resistant parasite lines, to provide mechanistic insight on the mode of action of aminopyrazoles.