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Updated: Jan 25, 2026
Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation
Published on: October 4, 2024
K-RasG12D Has a Potential Allosteric Small Molecule Binding Site
Abstract:
KRAS is the most commonly mutated oncogene in human cancer, with particularly high mutation frequencies in pancreatic cancers, colorectal cancers, and lung cancers [Ostrem, J. M., and Shokat, K. M. (2016) Nat. Rev. Drug Discovery 15, 771-785]. The high prevalence of KRAS mutations and its essential role in many cancers make it a potentially attractive drug target; however, it has been difficult to create small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on K-RasG12D using computational analyses of the protein structure and then used a combination of computational and biochemical approaches to discover small molecules that may bind to this pocket, which we have termed the P110 site, due to its adjacency to proline 110. We confirmed that one compound, named K-Ras allosteric ligand KAL-21404358, bound to K-RasG12D, as measured by microscale thermophoresis, a thermal shift assay, and nuclear magnetic resonance spectroscopy. KAL-21404358 did not bind to four mutants in the P110 site, supporting our hypothesis that KAL-21404358 binds to the P110 site of K-RasG12D. This compound impaired the interaction of K-RasG12D with B-Raf and disrupted the RAF-MEK-ERK and PI3K-AKT signaling pathways. We synthesized additional compounds, based on the KAL-21404358 scaffold with more potent binding and greater aqueous solubility. In summary, these findings suggest that the P110 site is a potential site for binding of small molecule allosteric inhibitors of K-RasG12D.
Insights
Researchers identified a novel binding site (P110) on KRAS G12D and discovered a small molecule inhibitor, KAL-21404358, that targets this site. This discovery offers a new strategy for developing KRAS G12D-targeted cancer therapies.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- KRAS is a frequently mutated oncogene in various cancers, including pancreatic, colorectal, and lung cancers.
- Developing small molecule inhibitors for mutant KRAS proteins has been challenging.
- Targeting KRAS mutations presents a significant opportunity for cancer therapy.
Purpose of the Study:
- To identify and characterize a novel small molecule binding site on KRAS G12D.
- To discover and validate small molecules that bind to this site and inhibit KRAS G12D activity.
- To explore the therapeutic potential of targeting the identified KRAS G12D binding site.
Main Methods:
- Computational analyses were used to identify a putative small molecule binding site (P110) on KRAS G12D.
- A combination of computational and biochemical methods was employed to discover and validate small molecule binders.
- Techniques such as microscale thermophoresis, thermal shift assay, and nuclear magnetic resonance spectroscopy were used to confirm compound binding.
Main Results:
- A novel binding site, termed the P110 site, was identified on KRAS G12D.
- The small molecule KAL-21404358 was confirmed to bind specifically to the P110 site on KRAS G12D.
- KAL-21404358 demonstrated inhibition of KRAS G12D downstream signaling pathways, including RAF-MEK-ERK and PI3K-AKT.
- Development of more potent and soluble analogs based on the KAL-21404358 scaffold.
Conclusions:
- The P110 site represents a druggable target for KRAS G12D.
- Small molecule allosteric inhibitors targeting the P110 site hold promise for KRAS G12D-driven cancer treatment.
- Further development of KAL-21404358 analogs could lead to novel cancer therapeutics.
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