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Updated: Apr 1, 2026

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

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Published on: January 16, 2020

Disulfide tethering reveals cryptic pockets in oncogenic KRAS.

Trent E Balius¹, Marcin Dyba¹, Vandana Kumari¹

¹NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.

Communications Chemistry

|March 30, 2026

Summary

Disulfide tethering successfully identified novel binding pockets on oncogenic KRAS (Kirsten rat sarcoma viral oncogene homolog). This drug discovery approach revealed druggable "hot spots" for targeting this previously undruggable cancer-associated protein.

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Published on: July 3, 2013

Area of Science:

Oncology
Chemical Biology
Structural Biology

Background:

Oncogenic KRAS is a challenging cancer target due to high nucleotide affinity.
Disulfide tethering is a fragment-based drug discovery method for difficult targets.
KRAS G12D is a common oncogenic mutation in various cancers.

Purpose of the Study:

To apply disulfide tethering to identify novel ligands and binding sites on oncogenic KRAS.
To explore the druggability of KRAS by mapping potential ligand-binding pockets.
To validate identified hits using biophysical and computational methods.

Main Methods:

Screening a library of 2160 disulfide-containing fragments against 83 engineered KRAS G12D cysteine mutants.
Performing 2-mercaptoethanol competition assays (βME-50) to prioritize ligands.
Utilizing computational chemistry and NMR spectroscopy for hit validation.

Main Results:

Identification of known KRAS binding sites, including the Switch-II / α-helix 3 pocket.
Discovery of previously undescribed cryptic pockets on KRAS.
Validation of select fragment hits, confirming their interaction with identified pockets.

Conclusions:

Disulfide tethering is effective for discovering ligands against challenging targets like KRAS.
Identified cryptic pockets represent promising new opportunities for KRAS-targeted drug discovery.
This approach enhances the understanding of KRAS ligandability and druggable regions.