A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Autor:	D'Ippolito RA; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Rabara D; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Blanco MA; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Alberico E; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Drew MR; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Ramakrishnan N; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Sontan D; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Widmeyer SRT; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Scheidemantle GM; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Messing S; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Turner D; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Arkin M; Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States.; Small Molecule Discovery Center, University of California, San Francisco, California 94143, United States., Maciag AE; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Stephen AG; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., Esposito D; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., McCormick F; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States.; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California 94158, United States., Nissley DV; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States., DeHart CJ; NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States.
Jazyk:	angličtina
Zdroj:	Analytical chemistry [Anal Chem] 2024 Apr 02; Vol. 96 (13), pp. 5223-5231. Date of Electronic Publication: 2024 Mar 18.
DOI:	10.1021/acs.analchem.3c05626
Abstrakt:	Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4B G12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu