Mutations on the Ras-family of small GTPases are among the most common molecular oncogenic drivers, with the HRas isoform being primarily associated with head-and-neck and genito-urinary cancers. Although once considered "undruggable," recent efforts have identified a structurally conserved surface pocket in the Ras family, designated the SI/II pocket, situated near the binding site of the guanidine exchange factor (GEF) SOS1. The SI/II pocket may represent a potential target site for a pan-Ras drug. A crystal structure representing the native state of GDP-bound HRasG12V was generated to characterize the topology of the SI/II pocket. This native-state structure was employed, together with the published structure of GppNHp-bound HRasG12V in state 1 (PDB ID: 4EFM), as a base for further molecular dynamics simulations exploring the conformational dynamics of the SI/II pocket via four generated synthetic HRas model structures. Our results show that the SI/II pocket is natively inaccessible in GDP-bound HRas yet becomes accessible in state 1 GppNHp-bound HRas systems, an effect that seems to be more evident in the mutated enzyme. This points to the GTP-bound state as a most promising target for Ras inhibitors directed at the SI/II pocket. Occlusion of the SI/II pocket is dictated by the spatial position of the α2 α helix in relation to the protein core, with α2 residue Y71 acting as a "tyrosine toggle" capable of restricting the pocket access.

Center for Basic and Applied Research Faculty of Informatics and Management University of Hradec Kralove 50003 Hradec Kralove Czech Republic

INRS Centre Armand Frappier Santé Biotechnologie 531 des Prairies Boulevard Laval Quebec H7 5 1B7 Canada

Institut National de la Recherche Scientifique Centre Armand Frappier Sante Biotechnologie Universite du Quebec Institut Pasteur International Network Laval QC H7V 1B7 Canada

Laboratory of Molecular Modeling Applied to the Chemical and Biological Defense Praça General Tibúrcio 80 22290 270 Rio de Janeiro Brazil

NMX Research and Solutions Inc Laval Québec H7 5 5B7 Canada

PROTEO the Quebec Network for Research on Protein Function Engineering and Applications Universite Laval Quebec QC G1V 0A6 Canada

Zobrazit více v PubMed

Zenonos K.; Kyprianou K. RAS signaling pathways, mutations and their role in colorectal cancer. World J. Gastrointest. Oncol. 2013, 5 (5), 97. 10.4251/wjgo.v5.i5.97. PubMed DOI PMC

Downward J. Ras signalling and apoptosis. Curr. Opin. Genet. Dev. 1998, 8 (1), 49–54. 10.1016/S0959-437X(98)80061-0. PubMed DOI

Downward J. Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 2003, 3 (1), 11–22. 10.1038/nrc969. PubMed DOI

Pylayeva-Gupta Y.; Grabocka E.; Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat. Rev. Cancer 2011, 11 (11), 761–774. 10.1038/nrc3106. PubMed DOI PMC

Prior I. A.; Hood F. E.; Hartley J. L. The Frequency of Ras Mutations in CancerRas Cancer Statistics. Cancer Res. 2020, 80 (14), 2969–2974. 10.1158/0008-5472.CAN-19-3682. PubMed DOI PMC

Prior I. A.; Lewis P. D.; Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012, 72 (10), 2457–2467. 10.1158/0008-5472.CAN-11-2612. PubMed DOI PMC

Larda S. T.; Ayotte Y.; Denk M. M.; Coote P.; Heffron G.; Bendahan D.; Shahout F.; Girard N.; Iddir M.; Bouchard P.; Bilodeau F.; Woo S.; Farmer L. J.; LaPlante S. R. A Robust Strategy for Hit-to-Lead Discovery: NMR for SAR. J. Med. Chem. 2023, 66, 13427. 10.1021/acs.jmedchem.3c00656. PubMed DOI PMC

Bendahan D.; Franca T. C. C.; Amiens K.; Ayotte Y.; Forgione P.; LaPlante S. Synthesis of a thiophene-based fluorinated library applied to fragment-based drug discovery via 19F NMR with confirmed binding to mutant HRASG12V. New J. Chem. 2024, 48, 17872–17877. 10.1039/d4nj00727a. DOI

Gasper R.; Wittinghofer F. The Ras switch in structural and historical perspective. Biol. Chem. 2019, 401 (1), 143–163. 10.1515/hsz-2019-0330. PubMed DOI

Karouzaki S.; Peta C.; Tsirimonaki E.; Mangoura D. PKCε-dependent H-Ras activation encompasses the recruitment of the RasGEF SOS1 and of the RasGAP neurofibromin in the lipid rafts of embryonic neurons. Neurochem. Int. 2019, 131, 104582. 10.1016/j.neuint.2019.104582. PubMed DOI

Malumbres M.; Barbacid M. RAS oncogenes: the first 30 years. Nat. Rev. Cancer 2003, 3 (6), 459–465. 10.1038/nrc1097. PubMed DOI

Wey M.; Lee J.; Jeong S. S.; Kim J.; Heo J. Kinetic mechanisms of mutation-dependent Harvey Ras activation and their relevance for the development of Costello syndrome. Biochemistry 2013, 52 (47), 8465–8479. 10.1021/bi400679q. PubMed DOI PMC

Gremer L.; Gilsbach B.; Reza Ahmadian M.; Wittinghofer A. Fluoride complexes of oncogenic Ras mutants to study the Ras-RasGap interaction. Biol. Chem. 2008, 389, 1163–1171. 10.1515/bc.2008.132. PubMed DOI

Smith M. J.; Neel B. G.; Ikura M. NMR-based functional profiling of RASopathies and oncogenic RAS mutations. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (12), 4574–4579. 10.1073/pnas.1218173110. PubMed DOI PMC

Muraoka S.; Shima F.; Araki M.; Inoue T.; Yoshimoto A.; Ijiri Y.; Seki N.; Tamura A.; Kumasaka T.; Yamamoto M.; et al. Crystal structures of the state 1 conformations of the GTP-bound H-Ras protein and its oncogenic G12V and Q61L mutants. FEBS Lett. 2012, 586 (12), 1715–1718. 10.1016/j.febslet.2012.04.058. PubMed DOI

Shima F.; Ijiri Y.; Muraoka S.; Liao J.; Ye M.; Araki M.; Matsumoto K.; Yamamoto N.; Sugimoto T.; Yoshikawa Y.; et al. Structural basis for conformational dynamics of GTP-bound Ras protein. J. Biol. Chem. 2010, 285 (29), 22696–22705. 10.1074/jbc.m110.125161. PubMed DOI PMC

Geyer M.; Schweins T.; Herrmann C.; Prisner T.; Wittinghofer A.; Kalbitzer H. R. Conformational transitions in p21 ras and in its complexes with the effector protein Raf-RBD and the GTPase activating protein GAP. Biochemistry 1996, 35 (32), 10308–10320. 10.1021/bi952858k. PubMed DOI

Cherfils J.; Zeghouf M. Regulation of small gtpases by gefs, gaps, and gdis. Physiol. Rev. 2013, 93 (1), 269–309. 10.1152/physrev.00003.2012. PubMed DOI

Zhang M.; Jang H.; Nussinov R. The structural basis for Ras activation of PI3Kα lipid kinase. Phys. Chem. Chem. Phys. 2019, 21 (22), 12021–12028. 10.1039/C9CP00101H. PubMed DOI PMC

Vo U.; Vajpai N.; Embrey K. J.; Golovanov A. P. Dynamic studies of H-Ras• GTPγS interactions with nucleotide exchange factor Sos reveal a transient ternary complex formation in solution. Sci. Rep. 2016, 6 (1), 29706. 10.1038/srep29706. PubMed DOI PMC

Spoerner M.; Herrmann C.; Vetter I. R.; Kalbitzer H. R.; Wittinghofer A. Dynamic properties of the Ras switch I region and its importance for binding to effectors. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (9), 4944–4949. 10.1073/pnas.081441398. PubMed DOI PMC

Kapoor A.; Travesset A. Differential dynamics of RAS isoforms in GDP-and GTP-bound states. Proteins:Struct., Funct., Bioinf. 2015, 83 (6), 1091–1106. 10.1002/prot.24805. PubMed DOI

Buhrman G.; O′Connor C.; Zerbe B.; Kearney B. M.; Napoleon R.; Kovrigina E. A.; Vajda S.; Kozakov D.; Kovrigin E. L.; Mattos C. Analysis of binding site hot spots on the surface of Ras GTPase. J. Mol. Biol. 2011, 413 (4), 773–789. 10.1016/j.jmb.2011.09.011. PubMed DOI PMC

O’Connor C.; Kovrigin E. L. Global conformational dynamics in ras. Biochemistry 2008, 47 (39), 10244–10246. 10.1021/bi801076c. PubMed DOI

Lanfredini S.; Thapa A.; O’Neill E. RAS in pancreatic cancer. Biochem. Soc. Trans. 2019, 47 (4), 961–972. 10.1042/BST20170521. PubMed DOI

Rojas A. M.; Fuentes G.; Rausell A.; Valencia A. The Ras protein superfamily: evolutionary tree and role of conserved amino acids. J. Cell Biol. 2012, 196 (2), 189–201. 10.1083/jcb.201103008. PubMed DOI PMC

Kessler D.; Bergner A.; Böttcher J.; Fischer G.; Döbel S.; Hinkel M.; Müllauer B.; Weiss-Puxbaum A.; McConnell D. B. Drugging all RAS isoforms with one pocket. Future Med. Chem. 2020, 12 (21), 1911–1923. 10.4155/fmc-2020-0221. PubMed DOI

Haza K. Z.; Martin H. L.; Rao A.; Turner A. L.; Saunders S. E.; Petersen B.; Tiede C.; Tipping K.; Tang A. A.; Ajayi M.; et al. RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site. Nat. Commun. 2021, 12 (1), 4045. 10.1038/s41467-021-24316-0. PubMed DOI PMC

Pierre S.; bats A. S.; Coumoul X. Understanding SOS (son of sevenless). Biochem. Pharmacol. 2011, 82 (9), 1049–1056. 10.1016/j.bcp.2011.07.072. PubMed DOI

Kessler D.; Gmachl M.; Mantoulidis A.; Martin L. J.; Zoephel A.; Mayer M.; Gollner A.; Covini D.; Fischer S.; Gerstberger T.; et al. Drugging an undruggable pocket on KRAS. Proc. Natl. Acad. Sci. U.S.A. 2019, 116 (32), 15823–15829. 10.1073/pnas.1904529116. PubMed DOI PMC

Maurer T.; Garrenton L. S.; Oh A.; Pitts K.; Anderson D. J.; Skelton N. J.; Fauber B. P.; Pan B.; Malek S.; Stokoe D.; et al. Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (14), 5299–5304. 10.1073/pnas.1116510109. PubMed DOI PMC

Sun Q.; Burke J. P.; Phan J.; Burns M. C.; Olejniczak E. T.; Waterson A. G.; Lee T.; Rossanese O. W.; Fesik S. W. Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angew. Chem., Int. Ed. 2012, 51 (25), 6140–6143. 10.1002/anie.201201358. PubMed DOI PMC

Chen F.-Y.; Li X.; Zhu H.-P.; Huang W. Regulation of the ras-related signaling pathway by small molecules containing an indole core scaffold: A potential antitumor therapy. Front. Pharmacol 2020, 11, 280. 10.3389/fphar.2020.00280. PubMed DOI PMC

Larda S. T.; Ayotte Y.; Denk M. M.; Coote P.; Heffron G.; Bendahan D.; Shahout F.; Girard N.; Iddir M.; Bouchard P.; et al. Robust Strategy for Hit-to-Lead Discovery: NMR for SAR. J. Med. Chem. 2023, 66, 13416–13427. 10.1021/acs.jmedchem.3c00656. PubMed DOI PMC

Vonrhein C.; Flensburg C.; Keller P.; Sharff A.; Smart O.; Paciorek W.; Womack T.; Bricogne G. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr., Sect. D:Biol. Crystallogr. 2011, 67 (4), 293–302. 10.1107/S0907444911007773. PubMed DOI PMC

Kabsch W. xds. Acta Crystallogr., Sect. D:Biol. Crystallogr. 2010, 66 (2), 125–132. 10.1107/S0907444909047337. PubMed DOI PMC

Evans P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr., Sect. D:Biol. Crystallogr. 2011, 67 (4), 282–292. 10.1107/S090744491003982X. PubMed DOI PMC

Zwart P. H.; Afonine P. V.; Grosse-Kunstleve R. W.; Hung L.-W.; Ioerger T. R.; McCoy A. J.; McKee E.; Moriarty N. W.; Read R. J.; Sacchettini J. C.. Automated structure solution with the PHENIX suite; Springer, 2008. PubMed

Emsley P.; Lohkamp B.; Scott W. G.; Cowtan K. Features and development of Coot. Acta Crystallogr., Sect. D:Biol. Crystallogr. 2010, 66 (4), 486–501. 10.1107/S0907444910007493. PubMed DOI PMC

Afonine P. V.; Grosse-Kunstleve R. W.; Echols N.; Headd J. J.; Moriarty N. W.; Mustyakimov M.; Terwilliger T. C.; Urzhumtsev A.; Zwart P. H.; Adams P. D. Towards automated crystallographic structure refinement with phenix. refine. Acta Crystallogr., Sect. D:Biol. Crystallogr. 2012, 68 (4), 352–367. 10.1107/S0907444912001308. PubMed DOI PMC

Williams C. J.; Headd J. J.; Moriarty N. W.; Prisant M. G.; Videau L. L.; Deis L. N.; Verma V.; Keedy D. A.; Hintze B. J.; Chen V. B.; et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. 2018, 27 (1), 293–315. 10.1002/pro.3330. PubMed DOI PMC

Case D. A.; Darden T.; Cheatham T.; Simmerling C. L.; Wang J.; Duke R. E.; Luo R.; Crowley M.; Walker R. C.; Zhang W.. Amber 10; University of California, 2008.

Nelson M. T.; Humphrey W.; Gursoy A.; Dalke A.; Kalé L. V.; Skeel R. D.; Schulten K. NAMD: a parallel, object-oriented molecular dynamics program. International Journal of Supercomputer Applications and High Performance Computing 1996, 10 (4), 251–268. 10.1177/109434209601000401. DOI

França T. C.; Botelho F. D.; Drummond M. L.; LaPlante S. R. Theoretical Investigation of Repurposed Drugs Potentially Capable of Binding to the Catalytic Site and the Secondary Binding Pocket of Subunit A of Ricin. Acs Omega 2022, 7 (36), 32805–32815. 10.1021/acsomega.2c04819. PubMed DOI PMC

Franca T. C. C.; Goncalves A. d. S.; Bérubé C.; Voyer N.; Aubry N.; LaPlante S. R.. Determining the Predominant Conformations of Mortiamides A–D in Solution Using NMR Data and Molecular Modeling Tools. Acs Omega. 2023 doi: 10.1021/acsomega.3c01206. PubMed DOI PMC

Vieira L. A.; Almeida J. S.; De Koning M. C.; LaPlante S. R.; Borges I. Jr; França T. C. Molecular modeling of Mannich phenols as reactivators of human acetylcholinesterase inhibited by A-series nerve agents. Chem.-Biol. Interact. 2023, 382, 110622. 10.1016/j.cbi.2023.110622. PubMed DOI

França T. C.; Saïdi F.; Ajamian A.; Islam S. T.; LaPlante S. R. Molecular Dynamics of Outer Membrane-Embedded Polysaccharide Secretion Porins Reveals Closed Resting-State Surface Gates Targetable by Virtual Fragment Screening for Drug Hotspot Identification. Acs Omega 2024, 9, 13226. 10.1021/acsomega.3c09970. PubMed DOI PMC

Ono S.; Naylor M. R.; Townsend C. E.; Okumura C.; Okada O.; Lee H.-W.; Lokey R. S. Cyclosporin A: conformational complexity and chameleonicity. J. Chem. Inf. Model. 2021, 61 (11), 5601–5613. 10.1021/acs.jcim.1c00771. PubMed DOI PMC

Hirano M.; Toyota K.; Ishibashi H.; Tominaga N.; Sato T.; Tatarazako N.; Iguchi T. Molecular insights into structural and ligand binding features of methoprene-tolerant in daphnids. Chem. Res. Toxicol. 2020, 33 (11), 2785–2792. 10.1021/acs.chemrestox.0c00179. PubMed DOI

Ali Z.; Cardoza J. V.; Basak S.; Narsaria U.; Singh V. P.; Isaac S. P.; França T. C. C.; LaPlante S. R.; George S. S. Computational design of candidate multi-epitope vaccine against SARS-CoV-2 targeting structural (S and N) and non-structural (NSP3 and NSP12) proteins. J. Biomol. Struct. Dyn. 2023, 41, 13348–13367. 10.1080/07391102.2023.2173297. PubMed DOI

Goga N.; Rzepiela A.; De Vries A.; Marrink S.; Berendsen H. Efficient algorithms for Langevin and DPD dynamics. J. Chem. Theory Comput. 2012, 8 (10), 3637–3649. 10.1021/ct3000876. PubMed DOI

Izaguirre J. A.; Catarello D. P.; Wozniak J. M.; Skeel R. D. Langevin stabilization of molecular dynamics. J. Chem. Phys. 2001, 114 (5), 2090–2098. 10.1063/1.1332996. DOI

Lange O. F.Grubmüller H.Collective Langevin dynamics of conformational motions in proteins J. Chem. Phys. 2006124 (21), PubMed

Stella L.; Lorenz C. D.; Kantorovich L. Generalized Langevin equation: An efficient approach to nonequilibrium molecular dynamics of open systems. Phys. Rev. B 2014, 89 (13), 134303. 10.1103/PhysRevB.89.134303. DOI

Wu X.; Brooks B. R. Self-guided Langevin dynamics simulation method. Chem. Phys. Lett. 2003, 381 (3–4), 512–518. 10.1016/j.cplett.2003.10.013. DOI

Humphrey W.; Dalke A.; Schulten K. VMD: visual molecular dynamics. Graphics 1996, 14, 33–38. 10.1016/0263-7855(96)00018-5. PubMed DOI

Smith R. H.; Dar A. C.; Schlessinger A. PyVOL: a PyMOL plugin for visualization, comparison, and volume calculation of drug-binding sites. BioRxiv 2019, 816702.

DeLano W. L.Pymol: An open-source molecular graphics tool. CCP4 Newsl. Protein Crystallogr.2002, 40( (1), ), 82–92.

DeLano W. L.; Bromberg S.. PyMOL user’s guide; DeLano Scientific LLC; Vol. 2004, p 629.

Kalbitzer H. R.; Spoerner M. State 1 (T) inhibitors of activated Ras. enzymes 2013, 33, 69–94. 10.1016/B978-0-12-416749-0.00004-X. PubMed DOI

Moore A. R.; Rosenberg S. C.; McCormick F.; Malek S. RAS-targeted therapies: is the undruggable drugged?. Nat. Rev. Drug Discovery 2020, 19 (8), 533–552. 10.1038/s41573-020-0068-6. PubMed DOI PMC

Kessler D.; Gollner A.; Gmachl M.; Mantoulidis A.; Martin L. J.; Zoephel A.; Mayer M.; Covini D.; Fischer S.; Gerstberger T.; Gmaschitz T.; et al. Reply to Tran et al.: Dimeric KRAS protein–protein interaction stabilizers. Proc. Natl. Acad. Sci. U.S.A. 2020, 117 (7), 3365–3367. 10.1073/pnas.1921236117. PubMed DOI PMC

Ingólfsson H. I.; Neale C.; Carpenter T. S.; Shrestha R.; López C. A.; Tran T. H.; Oppelstrup T.; Bhatia H.; Stanton L. G.; Zhang X.; et al. Machine learning–driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins. Proc. Natl. Acad. Sci. U.S.A. 2022, 119 (1), e2113297119 10.1073/pnas.2113297119. PubMed DOI PMC

Rudack T.; Teuber C.; Scherlo M.; Güldenhaupt J.; Schartner J.; Lübben M.; Klare J.; Gerwert K.; Kötting C. The Ras dimer structure. Chem. Sci. 2021, 12 (23), 8178–8189. 10.1039/D1SC00957E. PubMed DOI PMC

Simanshu D. K.; Philips M. R.; Hancock J. F. Consensus on the RAS dimerization hypothesis: Strong evidence for lipid-mediated clustering but not for G-domain-mediated interactions. Mol. Cell 2023, 83 (8), 1210–1215. 10.1016/j.molcel.2023.03.008. PubMed DOI PMC

Parker M. I.; Meyer J. E.; Golemis E. A.; Dunbrack R. L. Delineating the RAS Conformational Landscape. Cancer Res. 2022, 82 (13), 2485–2498. 10.1158/0008-5472.Can-22-0804. PubMed DOI PMC

Wang H.; Liu D.; Yu Y.; Fang M.; Gu X.; Long D. Exploring the state- and allele-specific conformational landscapes of Ras: understanding their respective druggabilities. Phys. Chem. Chem. Phys. 2023, 25 (2), 1045–1053. 10.1039/D2CP04964C. PubMed DOI