Enantioselective Protein Affinity Selection Mass Spectrometry (E-ASMS).

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Identifying selective ligands for challenging protein targets remains a significant hurdle in drug discovery.
• Weak binders and enantioselectivity are critical factors often overlooked in traditional screening methods.

Purpose of the Study:

• To develop and validate an enantioselective protein affinity selection mass spectrometry screening (EAS-MS) approach.
• To detect weak binders and assess ligand selectivity against a diverse set of human proteins.
• To confirm binding events through orthogonal assays and structural characterization.

Main Methods:

• Method development using control proteins.
• Screening of 8,210 chiral compounds against 31 human proteins using EAS-MS.
• Orthogonal assays and X-ray crystallography for validation and mechanistic studies.

Main Results:

• Discovery of 16 binders targeting 12 human proteins, including 'challenging-to-ligand' targets.
• Identification of 7 enantioselective binders with dissociation constants (KDs) between 3-20 μM.
• Structural elucidation of four target-binder complexes, explaining the mechanism of enantioselective binding.

Conclusions:

• EAS-MS is a sensitive and high-throughput method for identifying weak and enantioselective binders.
• The approach enables the characterization of ligands for novel and challenging protein targets.
• EAS-MS provides a powerful tool for advancing drug discovery and chemical biology research.