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A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement
Robert A D'Ippolito1, Dana Rabara1, Maria Abreu Blanco1
1NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States.
Analytical Chemistry
|March 18, 2024
Summary
A new top-down proteomic assay evaluates KRAS4B-compound engagement and binding site mapping. This method offers improved insights into targeted inhibitor development for KRAS mutations.
Area of Science:
- Biochemistry
- Proteomics
- Oncology
Background:
- Targeted inhibitors for oncogenic KRAS mutants are crucial in cancer therapy.
- Current proteomic methods for validating KRAS inhibitors may lack precision in assessing binding affinity and specificity.
- Understanding KRAS-compound interactions at the protein residue level is essential for drug development.
Purpose of the Study:
- To develop a novel top-down proteomic assay for evaluating in vitro KRAS4B-compound engagement.
- To assess relative quantitation of KRAS4B-compound interactions in parallel.
- To demonstrate the assay's capability in mapping compound binding sites on intact KRAS4B proteins.
Main Methods:
- Development of a novel top-down proteomic assay.
- Application 1: Maleimide-biotin labeling of a KRAS4B G12D cysteine mutant panel.
- Application 2: Treatment of wild-type, KRAS4B G12C, and KRAS4B G13C proteins with small molecule compounds.
Main Results:
- The assay successfully evaluated in vitro KRAS4B-compound engagement.
- Demonstrated time- and concentration-dependence of KRAS4B-compound interactions.
- Directly mapped compound binding sites on the intact KRAS4B protein molecule.
Conclusions:
- The novel top-down proteomic assay provides enhanced insights into KRAS4B-compound interactions.
- This assay can accurately assess binding affinity, specificity, and binding site localization.
- The developed method is valuable for validating targeted inhibitors in KRAS-driven cancer research.
