# KRAS G12C: Kinetic and Redox Characterization of Covalent Inhibition

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2024 · $540,620

## Abstract

ABSTRACT
Inhibition of oncogenic KRAS is a highly pursued goal in drug discovery efforts, as RAS mutations are found in
~25% of human cancers. One key oncogenic KRAS mutation (KRASG12C) contains a reactive cysteine at a
hotspot location, is the fourth most prevalent mutation in KRAS-driven tumors and is found at particularly high
frequency (40% of RAS mutations, 13% overall) in non-small cell lung cancer (NSCLC). Excitement has
accelerated rapidly around the discovery and application of covalent, Cys12-specific inhibitors of KRASG12C in
recent years as these compounds have shown efficacy in advanced clinical trials, with Sotorasib (AMG510,
LUMAKRAS™) recently receiving FDA approval for treatment of locally advanced or metastatic NSCLC.
However, the kinetic mechanisms underlying the activity of this class of inhibitors remain poorly understood
impeding rational drug design efforts. To address this gap in knowledge, we developed a new fluorescence-
based approach to kinetically characterize the reactions of the proteins with these acrylamide-based inhibitors.
Intriguingly, we find that the two clinical compounds, AMG510 (Amgen) and MRTX849/Adagrasib (Mirati
Therapeutics) possess distinctly different kinetic properties, which we propose to investigate in further detail
here. Recognizing the oxidative environment promoted by oncogenic KRAS signaling and tumorigenesis, we
also evaluated the redox sensitivity and oxidative status in cells and found that Cys12 of KRASG12C is prone to
oxidation. Moreover, oxidation at this site prevents inhibitor attachment, suggesting that redox modification of
KRASG12C may constitute a mechanism of resistance to AMG510 and other covalent inhibitors. Other
unanswered questions remain in the field, including the propensity of the protein-drug adducts to undergo
chemical reversibility of the Michael addition reactions through which they bind, and how this could be affected
by altered acrylamide “warheads”. Aim 1 proposes structural and kinetic studies combined with computational
modeling and molecular dynamics simulations to elucidate the differential mechanisms of KRASG12C inhibitor
engagement, inactivation and reversal. As development of treatment resistance remains a significant hurdle for
targeted inhibition strategies, Aims 2 and 3 propose to investigate the linkage between the redox sensitivity of
KRASG12C and inhibitor efficacy by measuring functional, signaling-relevant outputs for recombinant proteins and
biological samples (lung cancer cell lines and patient-derived organoids). For Aim 2, lung cancer cells under
variably oxidizing conditions, with and without inhibitor present, will be assessed for KRASG12C modifications and
downstream signaling outputs; use of HyPer-DAAO genetic constructs with targeting to the plasma membrane
will allow spatiotemporal and dosage control over hydrogen peroxide production within cells, near KRASG12C. In
Aim 3, the relationship between tumor redox properties and inhibitor efficac...

## Key facts

- **NIH application ID:** 10773196
- **Project number:** 5R01CA281295-02
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Sharon L Campbell
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $540,620
- **Award type:** 5
- **Project period:** 2023-03-01 → 2028-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10773196

## Citation

> US National Institutes of Health, RePORTER application 10773196, KRAS G12C: Kinetic and Redox Characterization of Covalent Inhibition (5R01CA281295-02). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10773196. Licensed CC0.

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