NHS-Esters As Versatile Reactivity-Based Probes for Mapping Proteome-Wide Ligandable Hotspots

Most
of the proteome is considered undruggable, oftentimes hindering
translational efforts for drug discovery. Identifying previously unknown
druggable hotspots in proteins would enable strategies for pharmacologically
interrogating these sites with small molecules. Activity-based protein
profiling (ABPP) has arisen as a powerful chemoproteomic strategy
that uses reactivity-based chemical probes to map reactive, functional,
and ligandable hotspots in complex proteomes, which has enabled inhibitor
discovery against various therapeutic protein targets. Here, we report
an alkyne-functionalized N-hydroxysuccinimide-ester
(NHS-ester) as a versatile reactivity-based probe for mapping the
reactivity of a wide range of nucleophilic ligandable hotspots, including
lysines, serines, threonines, and tyrosines, encompassing active sites,
allosteric sites, post-translational modification sites, protein interaction
sites, and previously uncharacterized potential binding sites. Surprisingly,
we also show that fragment-based NHS-ester ligands can be made to
confer selectivity for specific lysine hotspots on specific targets
including Dpyd, Aldh2, and Gstt1. We thus put forth NHS-esters as
promising reactivity-based probes and chemical scaffolds for covalent
ligand discovery.