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

Area of Science:

• Chemical Biology
• Drug Discovery
• Proteomics

Background:

• Covalent ligands offer unique advantages for targeting proteins and modulating their function.
• Current methods for optimizing covalent ligandability often require laborious protein purification and assay development.
• Profiling nucleophilic residues like cysteine and lysine with electrophilic fragments is a key strategy in chemical proteomics.

Purpose of the Study:

• To develop a streamlined method for optimizing covalent fragments into site-specific target engagement probes.
• To enable rapid screening of small molecules against diverse protein targets using convenient assay formats.
• To demonstrate the utility of this approach by optimizing a fragment against a specific enzyme.

Main Methods:

• Utilizing broadly reactive electrophilic fragments, termed "scouts", as starting points.
• Converting "scouts" into site-specific probes for screening.
• Employing gel- and ELISA-based assay formats for high-throughput screening and optimization.
• Applying the method to optimize a fragment targeting the AKR1B10 enzyme.

Main Results:

• Demonstrated the conversion of "scout" fragments into site-specific probes.
• Successfully screened small molecules against a wide array of proteins in gel- and ELISA-based assays.
• Optimized a weak fragment hit into a sub-micromolar inhibitor of AKR1B10.
• Achieved selective engagement of an active-site cysteine in AKR1B10.

Conclusions:

• The developed method provides a roadmap for optimizing covalent fragments into advanced chemical probes.
• This approach significantly reduces the need for protein purification and structural analysis.
• Facilitates efficient drug discovery and chemical biology research by streamlining probe optimization.