O'Connell, T. N. | bioRxiv Science

bioRxiv2023

Chemical Tools for the Gid4 Subunit of the Human E3 Ligase C-terminal to LisH (CTLH) Degradation Complex

We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.

biophysics↗

bioRxiv2023

A chemical probe to modulate human GID4 Pro/N-degron interactions

The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to this complex as a potential mediator of targeted protein degradation, but the role GID4-mediated substrate ubiquitylation and proteasomal degradation plays in humans has thus far remained unclear. Here, we report PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences. GID4 interactors are enriched for nuclear and nucleolar proteins including RNA helicases. GID4 antagonism by PFI-7 altered protein levels of several proteins including RNA helicases as measured by label-free quantitative proteomics, defining proteins that are regulated by GID4 and the CTLH complex in humans. Interactions with GID4 via Pro/N-degron pathway did not result in proteasomal degradation, demonstrating that CTLH interactors are regulated through a combination of degradative and non-degradative functions. The lack of degradation of GID4 interactors highlights potential challenges in utilizing GID4-recruiting bifunctional molecules for targeted protein degradation. Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.

molecular biology↗