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 approach (E-ASMS).
• To demonstrate the capability of E-ASMS in identifying weak, enantioselective binders for challenging human protein targets.
• To confirm ligand-target interactions and elucidate mechanisms of enantioselectivity.

Main Methods:

• Method development using control proteins.
• Screening of 8,217 chiral compounds against 31 human proteins using E-ASMS.
• Orthogonal biophysical assays for interaction confirmation.
• X-ray crystallography for in-depth characterization of selected binders.

Main Results:

• Identification of 16 binders for 12 human protein targets, including challenging ones.
• Seven binders exhibited enantioselective binding with K_D values from 3 to 20 µM.
• Structural elucidation of enantioselectivity mechanisms for four target-binder pairs.

Conclusions:

• E-ASMS is a powerful, high-throughput, and sensitive method for identifying weak and selective ligands.
• The approach enables the discovery of ligands for previously intractable protein targets.
• E-ASMS provides orthogonal confirmation and mechanistic insights into enantioselective binding.