Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Kirsten rat sarcoma (KRas) is a critical oncogene frequently mutated in pancreatic, colon, and lung cancers.
• Developing small molecule ligands for KRas is challenging due to its lack of deep binding pockets.
• Fragment-based drug discovery offers a promising avenue for targeting KRas.

Purpose of the Study:

• To implement and validate second harmonic generation (SHG) as a primary screening platform for identifying KRas fragment ligands.
• To characterize the binding site and conformational effects of identified KRas ligands.
• To compare SHG-derived conformational data with existing KRas ligands.

Main Methods:

• Utilized second harmonic generation (SHG) for high-throughput screening of fragment libraries against KRas.
• Employed ¹H ¹⁵N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR for binding site characterization.
• Analyzed conformational changes induced by fragment binders using SHG.

Main Results:

• Successfully identified a fragment binder to the oncogenic KRas^G12D mutation using SHG screening.
• Characterized the fragment's binding site adjacent to the KRas switch 2 region via NMR.
• SHG revealed distinct conformational changes induced by the identified fragment compared to a known Ras ligand (DCAI).

Conclusions:

• Second harmonic generation (SHG) is a sensitive and effective high-throughput screening platform for identifying KRas fragment binders.
• SHG provides valuable structural insights into ligand-induced biomolecular conformational changes.
• This approach advances the development of novel therapeutics targeting KRas-driven cancers.