The rapid and ever-growing advancements from within the field of proteolysis-targeting chimeras (PROTAC)-induced protein degradation

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined "functional hotspots". Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here, we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

Targeted protein degradation is a new pharmacologic paradigm established by drugs that recruit target proteins to E3 ubiquitin ligases via a ternary ligase-degrader-target complex. Based on the structure of the degrader and the neosubstrate, different E3 ligase interfaces are critically involved in this process, thus forming defined functional hotspots. Understanding disruptive mutations in functional hotspots informs on the architecture of the underlying assembly, and highlights residues prone to cause drug resistance. Until now, their identification was driven by structural methods with limited scalability. Here, we employ haploid genetics to show that hotspot mutations cluster in the substrate receptors of the hijacked ligases and find that type and frequency of mutations are shaped by the essentiality of the harnessed ligase. Intersection with deep mutational scanning data revealed hotspots that are either conserved, or specific for chemically distinct degraders or recruited neosubstrates. Biophysical and structural validation suggest that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we identified and validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and experimentally widely accessible methodology that empowers the characterization of small-molecule degraders and informs on associated resistance mechanisms.

The Src homology 2 (SH2) domain is present in many proteins that bind to phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signalling. Since pY-driven protein-protein interactions can sustain disease, SH2 domains have long been the focus of small-molecule ligand discovery efforts, but have been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we have used structure-based design to target the SH2 domain of the suppressor of cytokine signalling 2 (SOCS2) – the substrate recognition subunit of a Cullin5 RING E3 ligase. Starting from the highly ligand-efficient pY amino acid, a fragment growing approach improved binding affinities in the nanomolar range. During the course optimization campaign, in one of our co-crystal structures we observed serendipitous modification of Cys111 at a flexible variable loop distal from the phosphate binding site. This inspired the rational design of a cysteine-directed electrophilic covalent inhibitor MN551. A prodrug strategy using a pivaloyloxymethyl (POM) protecting group aided cell permeability and its rapid unmasking inside the cell enabled efficient and selective covalent engagement of SOCS2. We qualify MN551 and its masked analogue MN714 as covalent inhibitors of SOCS2, that could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as covalent E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation. More broadly, this work provides a blueprint for developing and exploiting pY-based small molecules to target other SH2 domains.

Hypoxia-inducible factor-1alpha (HIF-1alpha) constitutes the principal mediator of cellular adaptation to hypoxia in humans. Hydroxylation of proline residues on HIF-1alpha is recognized by the E3 ligase von Hippel-Lindau (VHL), leading to ubiquitin-dependent proteasomal degradation of HIF-1alpha. In this study, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors with improved cellular activity at blocking HIF-1alpha degradation. With the prototypical ligand VH298 as starting point, our iterative and combinatorial optimization strategy focused on introducing chemical variability into the phenylene unit of the VHL ligand structure and encompassed further points of diversity. The exploitation of tailored phenylene fragments combined with the stereoselective installation of the (S)-configured methyl group at the benzylic position provided VHL ligands with superior binding affinity compared to VH298. Three high-resolution structures of VHL in complex with three new ligands were determined and bioactive conformations of these ligands were explored. The most potent inhibitor (compound 30) exhibited dissociation constants (Kd) lower than 40 nM, independently determined by fluorescence polarization (FP) and surface plasmon resonance (SPR). The improved binding affinity to VHL was accompanied by enhanced cellular potency of 30, considerably exceeding the stabilization of HIF-1alpha by established VHL inhibitors. Our work is anticipated to inspire future efforts towards HIF-1alpha stabilizers and our optimized ligands to serve as an excellent starting point for proteolysis-targeting chimeras (PROTACs) hijacking VHL.