Application and Cross-Validation of a High-Throughput SPR Method for Characterizing Covalent Binding Ligands.

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Irreversible covalent inhibitors are crucial in drug development.
• Accurate determination of inhibition constants (k_inact/K_I) is essential for characterizing these inhibitors.
• Conventional methods for determining k_inact/K_I can be time-consuming and costly.

Purpose of the Study:

• To validate and present a high-throughput surface plasmon resonance (SPR) method for rapid determination of k_inact/K_I.
• To demonstrate the utility of SPR using covalent inhibitors of MK2 and KRAS-G12C as case studies.
• To highlight the advantages of SPR over traditional methods for kinetic profiling.

Main Methods:

• Utilized a high-throughput surface plasmon resonance (SPR) system with a regenerable sensor chip.
• Employed covalent inhibitors targeting MK2 and KRAS-G12C for method validation.
• Compared SPR-derived k_inact/K_I values with those obtained from mass spectrometry and enzymatic assays.

Main Results:

• SPR method demonstrated excellent correlation with conventional methods (mass spectrometry, enzymatic assays) for k_inact/K_I determination.
• The use of regenerable biotin chips significantly enhanced throughput and reduced costs.
• SPR provides accurate and reliable kinetic data for irreversible covalent inhibitors.

Conclusions:

• SPR is an efficient, accurate, and cost-effective method for determining k_inact/K_I.
• SPR offers significant advantages over traditional enzymatic and MS-based methods for kinetic analysis.
• This validated SPR approach facilitates rapid characterization of covalent inhibitors in drug discovery programs.