Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder
| Autor: | Gabriel Mercado, Paul T. Wilder, Joshua Salafsky, Sina Khorsand, Alexander D. MacKerell, Kristen M. Varney, Que N. Van, Andrew G. Stephen, Frank McCormick, Ben Moree, Wenbo Yu, David J. Weber, Patrick Alexander, Elizabeth Donohue |
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| Rok vydání: | 2019 |
| Předmět: |
Conformational change
animal structures Nuclear Magnetic Resonance Mutation Missense Small G Protein 01 natural sciences small G protein Proto-Oncogene Proteins p21(ras) 03 medical and health sciences Rare Diseases KRAS cancer Humans Transverse relaxation-optimized spectroscopy Binding site Nuclear Magnetic Resonance Biomolecular Protein Kinase Inhibitors 030304 developmental biology 0303 health sciences Multidisciplinary Binding Sites 010405 organic chemistry Chemistry second harmonic generation Prevention Ligand (biochemistry) Small molecule small molecule inhibitors 0104 chemical sciences Heteronuclear molecule Amino Acid Substitution PNAS Plus Mutation Biophysics Missense Digestive Diseases Heteronuclear single quantum coherence spectroscopy Biomolecular |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America, vol 116, iss 35 |
| ISSN: | 1091-6490 |
| Popis: | 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 KRas(G12D) and used (1)H (15)N 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. |
| Databáze: | OpenAIRE |
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