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


ABSTRACT: 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.

SUBMITTER: Donohue E 

PROVIDER: S-EPMC6717309 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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

Donohue Elizabeth E   Khorsand Sina S   Mercado Gabriel G   Varney Kristen M KM   Wilder Paul T PT   Yu Wenbo W   MacKerell Alexander D AD   Alexander Patrick P   Van Que N QN   Moree Ben B   Stephen Andrew G AG   Weber David J DJ   Salafsky Joshua J   McCormick Frank F  

Proceedings of the National Academy of Sciences of the United States of America 20190809 35


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  ...[more]

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