Covalent Library Screening by Targeted Mass Spectrometry for Rapid Binding Site Identification

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Covalent drug discovery is gaining traction with recent FDA approvals of BTK and KRAS G12C inhibitors.
• Intact protein mass spectrometry is used for high-throughput screening of covalent fragment libraries but lacks binding site information.
• Identifying binding sites requires time- and resource-intensive follow-up assays, delaying hit confirmation.

Purpose of the Study:

• To develop a novel, targeted mass spectrometry-based covalent screening method (CoMPAS) that provides binding site information during the initial screen.
• To improve the efficiency and reduce the resource requirements of covalent screening.
• To enable higher-throughput screening and simplify structure-activity relationship interpretation.

Main Methods:

• Development of CoMPAS, a targeted mass spectrometry approach for covalent screening.
• Utilizing high sensitivity of targeted detection for potent binder characterization and reduced protein needs.
• Employing an Agilent RapidFire system for high-throughput data acquisition and statistical analysis.
• Assessing target selectivity of screening hits against off-target kinases to demonstrate multiplexing capabilities.

Main Results:

• CoMPAS successfully provides binding site information in the initial covalent screening phase.
• The method demonstrates high sensitivity, enabling characterization of potent binders with reduced protein reagent usage.
• Binding site-specific EC50 values simplify structure-activity relationship interpretation.
• Parallel data acquisition on an Agilent RapidFire system facilitated higher-throughput screening.
• CoMPAS demonstrated multiplexing capabilities by assessing target selectivity against a kinase panel.

Conclusions:

• CoMPAS is a novel, targeted mass spectrometry-based method for covalent screening that integrates binding site identification.
• This approach accelerates hit confirmation and simplifies SAR analysis in covalent drug discovery.
• CoMPAS offers advantages in sensitivity, reagent efficiency, and throughput compared to intact protein mass spectrometry.