Covalent Inhibitors of an Immune Checkpoint Protein-Protein Interaction

Immune-checkpoint inhibitors (ICIs) are drugs that prompt T cells to mount attacks against cancer cells. The ICI proteins are among the most challenging biomedical targets owing to their flat binding interfaces and their tightly colocalized multimeric interaction at the contact point between cancer and T cells. By necessity, all first-generation ICI drugs have been monoclonal antibodies that bind with exceptional affinity. Yet, for many patients the antibody drugs have limitations. Covalent inhibitors could offer an ideal mix of properties to expand the scope, efficacy, and tolerability of ICIs, including bioavailability and brain penetration. However, we lack a blueprint for covalent inhibition of protein-protein interactions without privileged reactive residues like those in catalytic sites. We sought to exploit the exposed tyrosine residues on the binding interface of a key ICI protein, despite their similar reactivity to water. The combination of ligand–binding orientation and transition-state stabilization by a neighboring positively charged sidechain enabled us to identify the first series of covalent inhibition with mild, biocompatible, water-stable warheads. Protein 2D-NMR and mass-spectroscopy revealed the reaction site and biochemical experiments demonstrated a desirable reaction rate and excellent selectivity against a close paralog. The lessons derived from this work will inform the discovery of covalent inhibitors for many other unaddressed biomedical problems.