Exploration of chemical probes and conformational flexibility of GID4 - the substrate receptor of human CTLH E3 ligase complex

ABSTRACT The application of targeted protein degradation (TPD) is currently constrained by the limited availability of low-molecular-weight molecules that can recruit E3 ligases other than CRBN (Cereblon) or VHL (Von Hippel-Lindau ligase). In this study, we present the structure-based drug design (SBDD) of high-affinity ligands that engage E3 ligase GID4 (Glucose-induced degradation protein 4) in biophysical and cellular experiments. Through structural studies and molecular modeling, we identified three clusters of compounds that induce distinct conformations of GID4. We characterized potential exit vectors and used the most promising ligand as a building block to prepare bifunctional degraders in the form of proteolysis-targeting chimeras (PROTACs). Although ternary complex formation was successful in vitro, degradation of BRD4 was not observed, highlighting the need for further optimization of the degraders. Finally, we theoretically investigated the likelihood of the identified GID4 conformations participating in protein-protein interactions mediated by molecular glue mechanisms. We believe the expanded ligand diversity discovered in this study may pave the way for tuning the selectivity and efficacy of interactions involving GID4 and its neosubstrates. Competing Interest Statement The authors have declared no competing interest. Footnotes This version has been revised to reflect changes made during peer review process. ABBREVIATIONS - TPD - targeted protein degradation - SBDD - Structure-based drug design - UPS - ubiquitin-proteasome system - CTLH - C-terminal to LisH complex - GID4 - Glucose-Induced Degradation protein 4 homolog - hGID4 - human GID4 - HMGCS1 - 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 - FBS - Fragment-Based Screening - DSF - Differential Scanning Fluorimetry - SPR - surface plasmon resonance - c - concentration - PDB - Protein Data Bank - PCA - principal component analysis - LMW - Low Molecular Weight - PPI - protein-protein interface