Classifying covalent protein binders by their targeted binding site

[Display omitted] Covalent protein targeting represents a powerful tool for protein characterization, identification, and activity modulation. The safety of covalent therapeutics was questioned for many years due to the possibility of off-target binding and subsequent potential toxicity. Researchers have recently, however, demonstrated many covalent binders as safe, potent, and long-acting therapeutics. Moreover, they have achieved selective targeting among proteins with high structural similarities, overcome mutation-induced resistance, and obtained higher potency compared to non-covalent binders. In this review, we highlight the different classes of binding sites on a target protein that could be addressed by a covalent binder. Upon folding, proteins generate various concavities available for covalent modifications. Selective targeting to a specific site is driven by differences in the geometry and physicochemical properties of the binding pocket residues as well as the geometry and reactivity of the covalent modifier “warhead”. According to the warhead reactivity and the nature of the binding region, covalent binders can alter or lock a targeted protein conformation and inhibit or enhance its activity. We survey these various modification sites using case studies of recently discovered covalent binders, bringing to the fore the versatile application of covalent protein binders with respect to drug discovery approaches.