Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry
John C Tran, Thomas Hunsaker, Christina Bell1
1CellCarta, Montreal, Quebec H2X 3Y7, Canada.
Abstract:
The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.
Insights
Developing automated mass spectrometry methods for KRAS G12C inhibitors is crucial for drug discovery. This study presents a streamlined sample preparation technique for measuring drug activity in tumors, improving efficiency for pharmacodynamic studies.
Area of Science:
- Biochemistry
- Pharmacology
- Analytical Chemistry
Background:
- Covalent KRAS G12C inhibitors offer therapeutic opportunities but require robust methods for in vivo activity assessment.
- Current sample preparation for tumor proteins is labor-intensive, limiting throughput for pharmacodynamic (PD) studies.
- Developing efficient assays is essential for drug discovery and development of targeted therapies.
Purpose of the Study:
- To describe an automated, integrated sample preparation method for measuring KRAS G12C inhibitor alkylation in complex tumor samples.
- To enable high-throughput quantitation of drug activity using mass spectrometry.
- To correlate target occupancy with pharmacodynamic effects in preclinical models.
Main Methods:
- Automated high-throughput detergent removal and preconcentration of proteins from tumor samples.
- Quantitation of KRAS G12C drug inhibitor alkylation using mass spectrometry.
- Assay validation demonstrating low intra-assay (4% CV) and inter-assay (6% CV) variability.
Main Results:
- The developed method accurately measures KRAS G12C alkylation in mouse tumor samples.
- The assay exhibits high reproducibility across multiple studies.
- Demonstrated dose-dependent target inhibition and MAPK pathway inhibition by the KRAS G12C inhibitor GDC-6036.
- Correlated target engagement with antitumor potency in a pancreatic xenograft model.
Conclusions:
- An automated sample preparation method significantly enhances the efficiency of measuring covalent KRAS G12C inhibitor activity in tumors.
- The validated mass spectrometry assay provides a robust tool for PD studies in drug discovery.
- GDC-6036 exhibits promising dose-dependent efficacy and target inhibition, supporting its potential as an antitumor therapeutic.
