Stapled peptides are a fast growing class of bioactive helical peptides modelled after the alfa-helix domain of proteins involved in intracellular protein-protein interactions (PPIs). Their potential to penetrate cells and bind tightly to shallow PPI interfaces has opening up new opportunities to pharmacologically modulate a range of "undruggable" intracellular biological targets, exceptionally those implicated in oncogenesis. Here, we aim to improve the potency of stapled peptides by applying the concept of covalent inhibition. Covalent targeting has long been exploited to increase therapeutic efficacy of some small organic drugs, particularly those targeting enzymes. Despite some controversy, some covalent drugs have been therapeutically very successful (omeprazole, cloridogrel and aspirin are blockbuster examples) making a major impact on human health.
To demonstrate the feasibility of the covalent approach applied to stapled peptides acting on intracellular PPIs, we incorporated an acrylamide warhead into a hydrocarbon-stapled peptide ligand BIM to target the oncogenic protein Bcl2A1. Upon binding, the warhead is positioned in close proximity to a reactive cysteine at the protein binding site allowing exclusive covalent bonding between the peptide inhibitor and its target Bcl2A1. We demonstrated that the warhead-bearing stapled peptide was capable of entering live cells and binding covalently to cytosolic endogenous Bcl2A1. This innovative approach demonstrates the potential to selectively silence a PPI inside cells with stapled peptide inhibitors, allowing longer duration of action, non-competition with other endogenous ligands and avoiding peptide clearance once bound to target protein.