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

      Varney, K.M.; Wilder, P.T.; Yu, W.; MacKerell, A.D., Jr. (National Academy of Sciences, 2019)
      Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (KRas). KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a fragment binder to KRasG12D and used 1H 15N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR to characterize its binding site as a pocket adjacent to the switch 2 region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a Ras ligand previously described to bind the same pocket. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.