Abstract A02: Probing amino acids residues chemical reactivity of KRAS 4b using N-hydroxysuccinimide esters

RAS oncoproteins are frequently mutated in pancreatic, colon and lung cancers and play a causal role in human carcinogenesis. Although it is a potentially good target for anticancer drugs development, it has been challenging to specifically target different forms of RAS using small molecules because of absence of evident target binding sites. Chemical reactivity of protein functional groups depends on the environment of a particular residue and reagent accessibility due to protein structure. We wanted to explore chemical reactivity of KRAS 4b functional groups using chemical reagents to identify novel sites for covalent and noncovalent drug development. NHS esters have been used for years for chemical modification of proteins. They are believed to selectively react with primary amino groups. However, there are reports in the literature that other nucleophilic groups such as sulfhydryl group of Cys can react with N-hydroxysuccinimide esters. It is possible that other functional groups may also react. Sulfo-NHS-acetate, sulfo-NHS-biotin and NHS-biotin were used for modification of K, HRAS proteins in GDP and GppNHp-loaded forms. To follow the extent of protein modification, MALDI-TOF MS was used. Generally, the reaction was performed until almost no unmodified protein was left. After modification the protein was digested by Glu-C and peptides were analyzed by MALDI-TOF MS and ES-MS/MS. A “modification index” (ratio of intensities of modified and unmodified peptide forms in MALDI-TOF MS spectra) was calculated to enable relative quantitation for all modified peptides. The comparison of the modification indexes for peptides derived from NHS-biotin modified KRAS and HRAS in GDP or GppNHp form suggested that GppNHp-loaded protein in general allows higher degree of modification. This suggests that there are inducible and expandable binding sites for small organic molecules that could be exploited for design of covalent and noncovalent drug leads. Acknowledgments: Supported by NCI contract HHSN261200800001E. Citation Format: Oleg Chertov, Xiaoying Ye, Timothy Waybright, Andrew G. Stephen. Probing amino acids residues chemical reactivity of KRAS 4b using N-hydroxysuccinimide esters [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A02.