Identification of chemicals breaking the USP8 interaction with its endocytic substrate CHMP1B.

The ubiquitin-specific protease USP8 plays a major role in controlling the stability and intracellular trafficking of numerous cell surface proteins among which the EGF receptor that regulates cell growth and proliferation in many physio-pathological processes. The function of USP8 at the endocytic pathway level partly relies on binding to and deubiquitination of the Endosomal Sorting Complex Required for Transport (ESCRT) protein CHMP1B. In the aim of finding chemical inhibitors of the USP8::CHMP1B interaction, we performed a high-throughput screening campaign using an HTRF® assay to monitor the interaction directly in lysates of cells co-expressing both partners. The assay was carried out in an automated format to screen the academic Fr-PPIChem library (Bosc N et al., 2020), which includes 10,314 compounds dedicated to the targeting of protein-protein interactions (PPIs). Eleven confirmed hits inhibited the USP8::CHMP1B interaction within a range of 30% to 70% inhibition at 50 µM, while they were inactive on a set of other PPI interfaces demonstrating the feasibility of specifically disrupting this particular interface. In parallel, we adapted this HTRF® assay to compare the USP8 interacting capacity of CHMP1B variants. As anticipated from earlier studies, a deletion of the MIM (Microtubule Interacting and Trafficking domain Interacting Motif) domain or mutation of two conserved leucine residues, L192 and L195, in this domain respectively abolished or strongly impeded the USP8::CHMP1B interaction. By contrast, a CHMP1B mutant that displays a highly decreased ubiquitination level following mutation of four lysine residues in arginine interacted at a similar level as the wild-type form with USP8. Therefore, conserved leucine residues within the MIT domain rather than its ubiquitinated status triggers CHMP1B substrate recognition by USP8.