A ligand selection strategy to customize small molecule probes for activity-based protein profiling (LS-ABPP)

Area of Science:

• Chemical Biology
• Enzymology
• Drug Discovery

Background:

• Activity-based protein profiling (ABPP) relies on activity-based probes (ABPs).
• Designing specific and selective ABPs is synthetically challenging and time-consuming.
• Existing methods often require extensive probe synthesis for target optimization.

Purpose of the Study:

• To present a novel ligand selection (LS) approach for rapid diversification of ABP molecules.
• To enable efficient optimization of probes for specific enzyme binding site demands.
• To facilitate enzyme characterization and inhibitor discovery.

Main Methods:

• A trifunctional LS probe synthesized from tyrosine was developed.
• The probe incorporates a pentafluorophenyl ester, chloroacetamide warhead, and alkyne reporter.
• A library of probes was generated by reacting amine ligands with the PFP ester and screened against target enzymes.

Main Results:

• The LS approach rapidly diversifies probe molecules for optimal enzyme binding.
• The method was successfully applied to the cysteine protease 3CLpro of SARS-CoV-2.
• A compound library screened by competitive profiling identified potential enzyme inhibitors.

Conclusions:

• The ligand selection approach is a valuable tool for enzyme characterization and inhibitor discovery.
• This protocol is highly adaptable to various target proteins.
• The method streamlines the development of targeted activity-based probes.