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

Proc Natl Acad Sci U S A. 2019 Aug 27;116(35):17290-17297. doi: 10.1073/pnas.1905516116. Epub 2019 Aug 9.


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.

Keywords: KRAS; cancer; second harmonic generation; small G protein; small molecule inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Humans
  • Mutation, Missense
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Kinase Inhibitors / chemistry*
  • Proto-Oncogene Proteins p21(ras) / antagonists & inhibitors*
  • Proto-Oncogene Proteins p21(ras) / chemistry*
  • Proto-Oncogene Proteins p21(ras) / genetics


  • KRAS protein, human
  • Protein Kinase Inhibitors
  • Proto-Oncogene Proteins p21(ras)