Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2

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.