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

Area of Science:

• Chemical biology
• Proteomics
• Drug discovery

Background:

• Many proteins are considered undruggable, limiting drug discovery efforts.
• Identifying druggable hotspots is crucial for developing new therapeutic strategies.
• Activity-based protein profiling (ABPP) is a key chemoproteomic technique for mapping protein reactivity.

Purpose of the Study:

• To develop a versatile chemical probe for mapping nucleophilic ligandable hotspots across the proteome.
• To explore the potential of N-hydroxysuccinimide-esters (NHS-esters) as reactivity-based probes and scaffolds for covalent ligand discovery.
• To demonstrate the ability of NHS-ester ligands to achieve selectivity for specific protein targets.

Main Methods:

• Utilized an alkyne-functionalized N-hydroxysuccinimide-ester (NHS-ester) as a reactivity-based chemical probe.
• Applied the probe to map a wide range of nucleophilic hotspots, including lysines, serines, threonines, and tyrosines.
• Investigated fragment-based NHS-ester ligands for target selectivity against Dpyd, Aldh2, and Gstt1.

Main Results:

• The NHS-ester probe successfully mapped diverse ligandable hotspots, including active sites, allosteric sites, and post-translational modification sites.
• Demonstrated that NHS-esters can be engineered to confer selectivity for specific lysine hotspots on target proteins.
• Identified potential new binding sites and therapeutic targets through reactivity mapping.

Conclusions:

• N-hydroxysuccinimide-esters (NHS-esters) are versatile and effective reactivity-based probes for mapping protein hotspots.
• NHS-esters serve as promising chemical scaffolds for the discovery of covalent ligands.
• This approach expands the druggable proteome and facilitates the development of novel therapeutics.