Small molecule mediated inhibition of protein cargo recognition by peroxisomal transport receptor PEX5 is toxic to Trypanosoma

Abstract

Trypanosomiases are life-threatening infections of humans and livestock, and novel effective therapeutic approaches are needed. Trypanosoma compartmentalize glycolysis into specialized organelles termed glycosomes. Most of the trypanosomal glycolytic enzymes harbor a peroxisomal targeting signal-1 (PTS1) which is recognized by the soluble receptor PEX5 to facilitate docking and translocation of the cargo into the glycosomal lumen. Given its pivotal role in the glycosomal protein import, the PEX5–PTS1 interaction represents a potential target to inhibit import of glycolytic enzymes and thus kill the parasite. We developed a fluorescence polarization (FP)-based assay for monitoring the PEX5–PTS1 interaction and performed a High Throughput Screening (HTS) campaign to identify small molecule inhibitors of the interaction. Six of the identified hits passed orthogonal selection criteria and were found to inhibit parasite growth in cell culture. Our results validate PEX5 as a target for small molecule inhibitors and provide scaffolds suitable for further pre-clinical development of novel trypanocidal compounds.

Identification of Leishmania donovani PEX5-PTS1 Interaction Inhibitors through Fluorescence Polarization-Based High-Throughput Screening

“…In this study, fluorescence polarization-based high-throughput screening (FP-HTS) was employed to identify inhibitors of the LdPEX5-PTS1 interaction. Prior to this study, investigations focusing on LdPEX5-PTS1 interaction had not been reported, with only one study on TcPEX5-PTS1 interaction, which identified 48 hits from HTS of a ~30,000 compound library resulting in a 0.16% hit rate [28]. The hit rate from this study was even lower (0.054%) possibly due to the differences in substrate concentrations, Kd values, or the structural features of compounds in the library.…”

“…Recently, the PEX14-PEX5 protein-protein interaction, essential for glycosomal protein import, has been highlighted as an attractive target for Trypanosoma inhibitor discovery [25,26]. Furthermore, hit compounds that inhibit the interaction between TcPEX5 and PTS1 cargo have been shown to effectively kill T. brucei parasites in cell culture [28]. Notably, despite belonging to the same kinetoplastid parasites group as Trypanosoma, no research has yet explored this interaction in Leishmania parasites.…”

“…On the C-terminal side of PEX5, the site where the TPR domain is recognized by PTS1 in glycosomal proteins, is another potential site for inhibitor discovery. Recently, screening of approximately 30,000 compounds has yielded six hits that inhibit the T. cruzi PEX5-PTS1 interaction and have also demonstrated activity against T. brucei growth in vitro [28].…”

PEX1 is essential for glycosome biogenesis and trypanosomatid parasite survival

“…Thus, targeting glycosomal biogenesis has become an attractive drug target. This druggability of the glycosome biogenesis machinery has been genetically validated using RNA interference mediated knockdown of various peroxins ( Moyersoen et al., 2003 ; Barros-Alvarez et al., 2014 ; Banerjee et al., 2019 ), as well as pharmacologically through identification and development of small molecule inhibitors that block peroxin protein-protein interactions (PPIs) ( Dawidowski et al., 2017 ; Kalel et al., 2018 ; Banerjee et al., 2021 ; Napolitano et al., 2022 ).…”

PEX1 is essential for the glycosome biogenesis and trypanosomatid parasite survival

“…Thus, targeting glycosomal biogenesis has become an attractive drug target (Kalel et al, 2018). This has been genetically validated using RNA interference mediated knockdown of various peroxins (Moyersoen et al, 2003;Barros-Alvarez et al, 2014;Banerjee et al, 2019), as well as pharmacologically through identification and development of small molecule inhibitors that block peroxin protein-protein interactions (PPIs) (Dawidowski et al, 2017;Banerjee et al, 2021;Napolitano et al, 2022).…”