Functionalized Scout Fragments for Site-Specific Covalent Ligand Discovery and Optimization

Covalent ligands
are a versatile class of chemical probes and drugs
that can target noncanonical sites on proteins and display differentiated
pharmacodynamic properties. Chemical proteomic methods have been introduced
that leverage electrophilic fragments to globally profile the covalent
ligandability of nucleophilic residues, such as cysteine and lysine,
in native biological systems. Further optimization of these initial
ligandability events without resorting to the time-consuming process
of individualized protein purification and functional assay development,
however, presents a persistent technical challenge. Here, we show
that broadly reactive electrophilic fragments, or “scouts”,
can be converted into site-specific target engagement probes for screening
small molecules against a wide array of proteins in convenient gel-
and ELISA-based assay formats. We use these assays to expediently
optimize a weak potency fragment hit into a sub-μM inhibitor
that selectively engages an active-site cysteine in the retinaldehyde
reductase AKR1B10. Our findings provide a road map to optimize covalent
fragments into more advanced chemical probes without requiring protein
purification or structural analysis.