The Current Toolbox for Covalent Inhibitors: From Hit Identification to Drug Discovery.

Covalent modification of therapeutic targets has emerged as a powerful platform for creating clinical drugs and chemical probes. Covalent drugs have evolved from serendipitous discoveries to rationally designed therapeutics, driven by advances in electrophile-first screening technologies. This perspective takes stock of alternative technologies currently available in laboratories and industry that collectively enable targeted covalent inhibitor development across historically "undruggable" targets. We highlight five such technologies: activity-based protein profiling (ABPP), provides functional proteomic mapping to identify ligandable residues; covalent tethering, exploits dynamic chemistry to capture transient pockets; covalent DNA-encoded libraries, leverages trillion-member libraries for multiresidue targeting; phage/mRNA display, which facilitates evolution of covalent macrocyclic peptides; and sulfur-(VI) fluoride exchange (SuFEx), engages residues beyond cysteine. Integration of these approaches with chemoproteomics and artificial intelligence accelerates the discovery of covalent inhibitors with enhanced selectivity and reduced off-target risks. This technological convergence establishes a new paradigm for precision covalent therapeutics, offering innovative solutions to overcome drug resistance and target challenging protein interfaces.