Related Experiment Video
Updated: Aug 16, 2025
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Development of a high-throughput TR-FRET screening assay for a fast-cycling KRAS mutant
Jacob E Larson1, P Brian Hardy1, Noah K Schomburg2
1UNC Eshelman School of Pharmacy, Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
Abstract:
Mutations in the small GTPase protein KRAS are one of the leading drivers of cancers including lung, pancreatic, and colorectal, as well as a group of developmental disorders termed "Rasopathies". Recent breakthroughs in the development of mutant-specific KRAS inhibitors include the FDA approved drug Lumakras (Sotorasib, AMG510) for KRAS G12C-mutated non-small cell lung cancer (NSCLC), and MRTX1133, a promising clinical candidate for the treatment of KRAS G12D-mutated cancers. However, there are currently no FDA approved inhibitors that target KRAS mutations occurring at non-codon 12 positions. Herein, we focused on the KRAS mutant A146T, found in colorectal cancers, that exhibits a "fast-cycling" nucleotide mechanism as a driver for oncogenic activation. We developed a novel high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay that leverages the reduced nucleotide affinity of KRAS A146T. As designed, the assay is capable of detecting small molecules that act to allosterically modulate GDP affinity or directly compete with the bound nucleotide. A pilot screen was completed to demonstrate robust statistics and reproducibility followed by a primary screen using a diversity library totaling over 83,000 compounds. Compounds yielding >50% inhibition of TR-FRET signal were selected as hits for testing in dose-response format. The most promising hit, UNC10104889, was further investigated through a structure activity relationship (SAR)-by-catalog approach in an attempt to improve potency and circumvent solubility liabilities. Overall, we present the TR-FRET platform as a robust assay to screen fast-cycling KRAS mutants enabling future discovery efforts for novel chemical probes and drug candidates.
Insights
Researchers developed a novel assay to find drugs targeting KRAS A146T mutations, common in colorectal cancers. This TR-FRET assay screens for compounds that modulate KRAS activity, paving the way for new cancer therapies.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Mutant KRAS proteins drive various cancers and Rasopathies.
- Existing KRAS inhibitors target specific mutations like G12C and G12D.
- There's a need for inhibitors targeting KRAS mutations beyond codon 12, such as A146T.
Purpose of the Study:
- To develop a high-throughput screening assay for KRAS A146T mutants.
- To identify small molecules that modulate the activity of the fast-cycling KRAS A146T mutant.
- To enable the discovery of novel drug candidates for KRAS A146T-driven cancers.
Main Methods:
- Developed a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay.
- The assay leverages the reduced nucleotide affinity of KRAS A146T.
- Screened over 83,000 compounds from a diversity library.
Main Results:
- The TR-FRET assay demonstrated robust statistics and reproducibility.
- Identified hit compounds that inhibit the TR-FRET signal by >50%.
- The lead compound UNC10104889 showed potential for further optimization.
Conclusions:
- The developed TR-FRET platform is a robust tool for screening fast-cycling KRAS mutants.
- This assay facilitates the discovery of novel chemical probes and drug candidates for KRAS-mutated cancers.
- The study highlights a promising approach for targeting previously undruggable KRAS mutations.
