Chemical probes and proteomics

Activity-based protein profiling (ABPP) uses probes whose warhead reacts covalently with a catalytic residue, so labelling reports enzyme activity, not just abundance. Fluorophosphonates label the active-site serine of serine hydrolases; epoxyketones and vinyl sulfones target proteasome and cysteine protease nucleophiles. Probes usually carry a small alkyne, and the reporter (rhodamine azide for in-gel fluorescence, biotin azide for streptavidin enrichment) is attached after lysis by CuAAC click chemistry, so the probe itself stays cell-permeable. In competitive ABPP, a drug is added first and loss of probe signal reveals its targets and off-targets across a whole enzyme family at once.

Residue-centred platforms go further. isoTOP-ABPP and its TMT-multiplexed successors use iodoacetamide-alkyne to map reactive cysteines proteome-wide, then ask which ones a library of electrophilic fragments engages. This has found ligandable cysteines on proteins once considered undruggable and supports the wider revival of covalent drugs, whose best-known clinical success is the KRAS G12C inhibitor sotorasib.

For non-covalent ligands, photoaffinity labelling installs a diazirine or benzophenone; UV light generates a carbene or diradical that crosslinks to whatever is nearby. Minimal "fully functionalised" diazirine-alkyne tags let fragment libraries be screened directly in cells. Label-free alternatives, such as thermal proteome profiling (ligand-induced shifts in melting temperature) and limited proteolysis, avoid modifying the compound at all.

Targeted protein degradation turned target identification into a therapeutic idea. A PROTAC links a target ligand to an E3 ligase ligand (usually for cereblon or VHL), forming a ternary complex that ubiquitinates the target for proteasomal destruction; the molecule is released and acts catalytically. Too much PROTAC gives binary complexes and less degradation, the hook effect. Molecular glues such as lenalidomide reprogramme cereblon to degrade new substrates, a mechanism uncovered by proteomics. Open problems include using the hundreds of unexploited E3 ligases, rational glue discovery, oral bioavailability of large degraders and quantifying occupancy versus degradation in tissue.