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

被引：15

作者：

Donohue, Elizabeth ^{[1
,2
]}

Khorsand, Sina ^{[1
,2
]}

Mercado, Gabriel ^{[2
]}

Varney, Kristen M. ^{[3
,4
,5
]}

Wilder, Paul T. ^{[3
,4
,5
]}

Yu, Wenbo ^{[3
,4
,6
]}

MacKerell, Alexander D., Jr. ^{[3
,4
,5
,6
]}

Alexander, Patrick ^{[7
]}

Van, Que N. ^{[7
]}

Moree, Ben ^{[2
]}

Stephen, Andrew G. ^{[7
]}

Weber, David J. ^{[3
,4
,5
]}

Salafsky, Joshua ^{[8
]}

McCormick, Frank ^{[1
,7
]}

机构：

[1] Univ Calif San Francisco, Helen Diller Family Comprehens Canc Ctr, San Francisco, CA 94158 USA

[2] Biodesy Inc, San Francisco, CA 94080 USA

[3] Univ Maryland, Sch Med, Ctr Biomol Therapeut, Baltimore, MD 21201 USA

[4] Univ Maryland, Sch Med, Dept Biochem & Mol Biol, Baltimore, MD 21201 USA

[5] Univ Maryland, Marlene & Stewart Greenebaum Comprehens Canc Ctr, Baltimore, MD 21201 USA

[6] Univ Maryland, Sch Pharm, Dept Pharmaceut Sci, Baltimore, MD 21201 USA

[7] NCI, RAS Initiat, Canc Res Technol Program, Frederick Natl Lab Canc Res,Leidos Biomed Res Inc, Frederick, MD 21702 USA

[8] Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2019年 / 116卷 / 35期

关键词：

second harmonic generation; KRAS; small G protein; cancer; small molecule inhibitors; COMPETITIVE SATURATION SILCS; SITE-IDENTIFICATION; K-RAS; TARGETING RAS; ACTIVATION; KRAS; INHIBITION; P21; SHG; GTP;

D O I：

10.1073/pnas.1905516116

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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 H-1 N-15 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.

引用

页码：17290 / 17297

页数：8

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