Identification of small molecule inhibitors of Trypanosoma PEX15– PEX6 interaction

Abstract Trypanosomatid parasites that cause life threatening tropical diseases harbor specialized essential organelles, called glycosomes. Like other peroxisome-related organelles, the biogenesis of glycosomes is mediated by proteins known as peroxins (PEX). A cascade of PEX protein-protein interactions (PPIs) is essential for glycosome function and parasite survival. Accordingly, small molecule inhibitors of PEX proteins that disrupt glycosomal matrix or membrane protein import have been reported as potential therapies for trypanosomiasis. We recently identified the long sought-after Trypanosoma PEX15 (TbPEX15), which anchors the PEX1-PEX6 complex to the glycosomal membrane for recycling of the receptor PEX5. Defects in this process cause PEX5 degradation, mislocalization of glycosomal matrix proteins and parasite death. In this study, we targeted the interaction between TbPEX6 and TbPEX15. Recombinant TbPEX6 and TbPEX15 were purified, and their interaction was confirmed by in vitro pull-down assays and size exclusion chromatography. Furthermore, we established an AlphaScreen-based method to identify small molecule inhibitors of this PPI. Screening of a drug-repurposing library identified two inhibitors with trypanocidal activity against T. brucei in vitro and the amastigote stage of T. cruzi. Given its essentiality and low sequence similarity to its human homolog, parasite PEX15 and its interaction with PEX6 represent promising targets for the development of new therapies against trypanosomatid infections.