Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

Area of Science:

• Chemical biology
• Drug discovery
• Fragment-based screening

Background:

• Covalent probes offer superior potency and duration but are difficult to discover.
• Electrophilic fragments, though potentially powerful, often lack selectivity.
• Previous fragment screening methods are limited by low affinity of reversible binders.

Purpose of the Study:

• To develop a practical and efficient method for discovering selective covalent ligands.
• To overcome the selectivity challenge associated with electrophilic fragments.
• To identify novel covalent probes for challenging protein targets.

Main Methods:

• Construction and characterization of a 993-member library of mildly electrophilic fragments.
• Development of a high-throughput thiol-reactivity assay for fragment characterization.
• Screening of the library against 10 cysteine-containing proteins, followed by high-throughput crystallography.

Main Results:

• Mild electrophiles proved to be selective, with highly reactive fragments being rare.
• The screening approach yielded hits for most of the tested protein targets.
• Potent and selective covalent probes were rapidly developed for OTUB2 and NUDT7, enzymes with no prior known inhibitors.

Conclusions:

• Electrophile-fragment screening is a viable and efficient strategy for covalent ligand discovery.
• Mild electrophiles can be successfully employed to achieve target selectivity.
• This approach enables rapid identification of novel covalent probes for enzyme targets.