Disulfide tethering reveals cryptic pockets in oncogenic KRAS.

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.