A Novel Strategy for Identifying Non-covalent KRas Inhibitors Design and Biochemical Characterization of KRas(G12C) Double Mutants for Compound Screening
Main Article Content
Abstract
By analyzing KRas and HRas X-ray structures, we developed a novel strategy that involves the design of a series of “synthetic” or “artificial” KRas mutations, namely T20I, D57E, D57F, T58A, T58V, and G60A, individually introduced into KRas with the G12C natural pathogenic mutation to create double mutants that we would expect to enhance compound binding to the switch II pocket. The goal of using these mutants is to induce greater overall flexibility of the KRas structure to allow the switch II pocket (S-IIP) to open more frequently in the absence of a C12-covalently bound ligand. We developed sensitive assays for the Raf:KRas(GTP) interaction and SOS-driven GDP/GTP exchange to assess these KRas proteins, including the wild-type form, a mutant frequently found in human cancers (G12C), and the “artificial” G12C double mutants. By characterizing these KRas mutants, we hoped to identify at least one mutant that may provide enough flexibility for non-covalent binding to the switch II pocket, thus facilitating future non-covalent compound screening. The results of these assays provide preliminary support that some of the studied mutants demonstrate increased protein flexibility relative to that of KRas(G12C). This strategy of slightly increasing protein flexibility or destabilization through the introduction of selected mutations may be applied to other proteins for which low assay sensitivity is due to transient, high-energy, open-form binding sites.
Keywords:
KRas mutations, non-covalent KRas inhibitors, switch II pocket, X-ray crystallography, SOS-driven GDP/GTP exchange, Raf binding activity
Article Details
How to Cite
ZHAO, Haoshuang et al.
A Novel Strategy for Identifying Non-covalent KRas Inhibitors.
Medical Research Archives, [S.l.], v. 8, n. 6, june 2020.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/2137>. Date accessed: 26 dec. 2024.
doi: https://doi.org/10.18103/mra.v8i6.2137.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
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3. Zenonos K, Kyprianou K. RAS signaling pathways, mutations and their role in colorectal cancer. World J Gastrointest Oncol. 2013; 5(5):97–101. doi:10.4251/wjgo.v5.i5.97.
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6. Maurer T, Garrenton LS, Oh A, et al. Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc Natl Acad Sci USA. 2012;109(14):5299–5304. doi:10.1073/pnas.1116510109.
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9. Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J. 2000;351(2);289–305. doi:10.1042/bj3510289.
10. Glaysher S, Bolton LM, Johnson P, Torrance C, Cree IA. Activity of EGFR, mTOR and PI3K inhibitors in an isogenic breast cell line model. BMC Res Notes. 2014;7(397):1–7. doi:10.1186/1756-0500-7-397.
11. Cosmic, v88, https://cancer.sanger.ac.uk/cosmic; last accessed on October 28, 2019.
12. Hunter JC, Manandhar A, Carrasco MA, Gurbani D, Gondi S, Westover KD. Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations. Mol Cancer Res. 2015;13(9):1325–1335. doi:10.1158/1541-7786.MCR-15-0203.
13. Scheffzek K, Ahmadian MR, Kabsch W, et al. The Ras-RasGAP complex: Structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science. 1997;277(5324):333–338. doi:10.1126/science.277.5324.333.
14. Kotting C, Kallenbach A, Suveyzdis Y, Wittinghofer A, Gerwert K. The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy. Proc Natl Acad Sci USA. 2008;105(17):6260–6265. doi:10.1073/pnas.0712095105.
15. Papke B, Der CJ. Drugging RAS: Know the enemy. Science. 2017;355(6330):1158–1163. doi:10.1126/science.aam7622.
16. Maurer T, Garrenton LS, Oh A, et al. Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc Natl Acad Sci USA. 2012;109(14):5299–5304. doi:10.1073/pnas.1116510109.
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36. Ganguly AK, Wang Y-S, Pramanik BN, et al. Interaction of a novel GDP exchange inhibitor with the Ras protein. Biochemistry. 1998;37(45):15631–15637. doi:10.1021/bi9805691.
37. Shima F, Ijiri Y, Muraoka S, et al. Structural basis for conformational dynamics of GTP-bound Ras protein. J Biol Chem. 2010;285(29):22696–22705. doi:10.1074/jbc.M110.125161.
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39. Ford B, Skowronek K, Boykevisch S, Bar-Sagi D, Nassar N. Structure of the G60A Mutant of Ras: Implications for The Dominant Negative Effect. J Biol Chem. 2005;280(27):25697–25705. doi:10.1074/jbc.M502240200.
40. Lito P, Solomon M, Li L-S, Hansen R, Rosen N. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science. 2016;351(6273):604–608. doi:10.1126/science.aad6204.
41. Hansen R, Peters U, Babbar A, et al. The reactivity-driven biochemical mechanism of covalent KRASG12C inhibitors. Nat Struct Mol Biol. 2018;25(6):454–462. doi:10.1038/s41594-018-0061-5.
42. Statsyuk AV. Let K-Ras activate its own inhibitor. Nat Struct Mol Biol. 2018;25(6):435–437. doi:10.1038/s41594-018-0066-0.