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NMR in integrated biophysical drug discovery for RAS: past, present, and future

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Abstract

Mutations in RAS oncogenes occur in ~ 30% of human cancers, with KRAS being the most frequently altered isoform. RAS proteins comprise a conserved GTPase domain and a C-terminal lipid-modified tail that is unique to each isoform. The GTPase domain is a ‘switch’ that regulates multiple signaling cascades that drive cell growth and proliferation when activated by binding GTP, and the signal is terminated by GTP hydrolysis. Oncogenic RAS mutations disrupt the GTPase cycle, leading to accumulation of the activated GTP-bound state and promoting proliferation. RAS is a key target in oncology, however it lacks classic druggable pockets and has been extremely challenging to target. RAS signaling has thus been targeted indirectly, by harnessing key downstream effectors as well as upstream regulators, or disrupting the proper membrane localization required for signaling, by inhibiting either lipid modification or ‘carrier’ proteins. As a small (20 kDa) protein with multiple conformers in dynamic equilibrium, RAS is an excellent candidate for NMR-driven characterization and screening for direct inhibitors. Several molecules have been discovered that bind RAS and stabilize shallow pockets through conformational selection, and recent compounds have achieved substantial improvements in affinity. NMR-derived insight into targeting the RAS-membrane interface has revealed a new strategy to enhance the potency of small molecules, while another approach has been development of peptidyl inhibitors that bind through large interfaces rather than deep pockets. Remarkable progress has been made with mutation-specific covalent inhibitors that target the thiol of a G12C mutant, and these are now in clinical trials. Here we review the history of RAS inhibitor development and highlight the utility of NMR and integrated biophysical approaches in RAS drug discovery.

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References

  • Agazie YM, Hayman MJ (2003) Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Mol Cell Biol 23:7875–7886

    Google Scholar 

  • Ahearn IM, Haigis K, Bar-Sagi D, Philips MR (2011) Regulating the regulator: post-translational modification of RAS. Nat Rev Mol Cell Biol 13:39–51

    Google Scholar 

  • Ahmed TA et al (2019) SHP2 Drives Adaptive Resistance to ERK Signaling Inhibition in Molecularly Defined Subsets of ERK-Dependent Tumors. Cell Rep 26:65–78

    Google Scholar 

  • Alvarez-Moya B, Barcelo C, Tebar F, Jaumot M, Agell N (2011) CaM interaction and Ser181 phosphorylation as new K-Ras signaling modulators. Small GTPases 2:99–103

    Google Scholar 

  • Antic I, Biancucci M, Zhu Y, Gius DR, Satchell KJF (2015) Site-specific processing of Ras and Rap1 Switch I by a MARTX toxin effector domain. Nat Commun 6:7396

    ADS  Google Scholar 

  • Baines AT, Xu D, Der CJ (2011) Inhibition of Ras for cancer treatment: the search continues. Future Med Chem 3:1787–1808

    Google Scholar 

  • Barbacid M (1987) ras genes. Annu Rev Biochem 56:779–827

    Google Scholar 

  • Barr AJ (2010) Protein tyrosine phosphatases as drug targets: strategies and challenges of inhibitor development. Future Med Chem 2:1563–1576

    Google Scholar 

  • Basso AD, Kirschmeier P, Bishop WR (2006) Lipid posttranslational modifications. Farnesyl transferase inhibitors. J Lipid Res 47:15–31

    Google Scholar 

  • Bennett AM, Tang TL, Sugimoto S, Walsh CT, Neel BG (1994) Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptor beta to Ras. Proc Natl Acad Sci USA 91:7335–7339

    ADS  Google Scholar 

  • Bentires-Alj M et al (2004) Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia. Cancer Res 64:8816–8820

    Google Scholar 

  • Berndt N, Hamilton AD, Sebti SM (2011) Targeting protein prenylation for cancer therapy. Nat Rev Cancer 11:775–791

    Google Scholar 

  • Bery N et al (2018) BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions. Elife 7:e37122

    Google Scholar 

  • Bery N et al (2019) KRAS-specific inhibition using a DARPin binding to a site in the allosteric lobe. Nat Commun 10:2607

    ADS  Google Scholar 

  • Bery N, Miller A, Rabbitts T (2020) A potent KRAS macromolecule degrader specifically targeting tumours with mutant KRAS. Nat Commun 11:3233

    ADS  Google Scholar 

  • Bhagatji P, Leventis R, Rich R, Lin CJ, Silvius JR (2010) Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane. Biophys J 99:3327–3335

    Google Scholar 

  • Binz HK, Stumpp MT, Forrer P, Amstutz P, Pluckthun A (2003) Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J Mol Biol 332:489–503

    Google Scholar 

  • Bivona TG et al (2006) PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. Mol Cell 21:481–493

    Google Scholar 

  • Bollag G et al (2012) Vemurafenib: the first drug approved for BRAF-mutant cancer. Nat Rev Drug Discov 11:873–886

    Google Scholar 

  • Bos JL (1989) ras oncogenes in human cancer: a review. Cancer Res 49:4682–4689

    Google Scholar 

  • Brown JS, Banerji U (2017) Maximising the potential of AKT inhibitors as anti-cancer treatments. Pharmacol Ther 172:101–115

    Google Scholar 

  • Brunsveld L et al (2006) Lipidated ras and rab peptides and proteins–synthesis, structure, and function. Angew Chem Int Ed Engl 45:6622–6646

    Google Scholar 

  • Bunda S et al (2014) Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation. Proc Natl Acad Sci USA 111:E3785–E3794

    Google Scholar 

  • Bunda S et al (2015) Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis. Nat Commun 6:8859

    ADS  Google Scholar 

  • Campbell-Burk SL, Domaille PJ, Starovasnik MA, Boucher W, Laue ED (1992) Sequential assignment of the backbone nuclei (1H, 15N and 13C) of c-H-ras p21 (1–166). GDP using a novel 4D NMR strategy. J Biomol NMR 2:639–646

    Google Scholar 

  • Canon J et al (2019) The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575:217–223

    ADS  Google Scholar 

  • Caunt CJ, Sale MJ, Smith PD, Cook SJ (2015) MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road. Nat Rev Cancer 15:577–592

    Google Scholar 

  • Ceska T, Chung CW, Cooke R, Phillips C, Williams PA (2019) Cryo-EM in drug discovery. Biochem Soc Trans 47:281–293

    Google Scholar 

  • Chandra A et al (2011) The GDI-like solubilizing factor PDEdelta sustains the spatial organization and signalling of Ras family proteins. Nat Cell Biol 14:148–158

    Google Scholar 

  • Chen X, Makarewicz JM, Knauf JA, Johnson LK, Fagin JA (2014) Transformation by Hras(G12V) is consistently associated with mutant allele copy gains and is reversed by farnesyl transferase inhibition. Oncogene 33:5442–5449

    Google Scholar 

  • Chen YN et al (2016) Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases. Nature 535:148–152

    ADS  Google Scholar 

  • Chen D et al (2019) Fragment-based drug discovery of triazole inhibitors to block PDEdelta-RAS protein-protein interaction. Eur J Med Chem 163:597–609

    Google Scholar 

  • Cho KJ et al (2016) AMPK and endothelial nitric oxide synthase signaling regulates K-Ras plasma membrane interactions via cyclic GMP-dependent protein kinase 2. Mol Cell Biol 36:3086–3099

    Google Scholar 

  • Cox AD, Fesik SW, Kimmelman AC, Luo J, Der CJ (2014) Drugging the undruggable RAS: Mission possible? Nat Rev Drug Discov 13:828–851

    Google Scholar 

  • Cox AD, Der CJ, Philips MR (2015) Targeting RAS membrane association: back to the future for anti-RAS drug discovery? Clin Cancer Res 21:1819–1827

    Google Scholar 

  • Cruz-Migoni A et al (2019) Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds. Proc Natl Acad Sci USA 116:2545–2550

    Google Scholar 

  • Dalvit C et al (2000) Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water. J Biomol NMR 18:65–68

    Google Scholar 

  • Dance M, Montagner A, Salles JP, Yart A, Raynal P (2008) The molecular functions of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway. Cell Signal 20:453–459

    Google Scholar 

  • Dardaei L et al (2018) SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors. Nat Med 24:512–517

    Google Scholar 

  • De Roock W et al (2010) Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA 304:1812–1820

    Google Scholar 

  • Denisov IG, Grinkova YV, Lazarides AA, Sligar SG (2004) Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J Am Chem Soc 126:3477–3487

    Google Scholar 

  • Dharmaiah S et al (2016) Structural basis of recognition of farnesylated and methylated KRAS4b by PDEdelta. Proc Natl Acad Sci USA 113:E6766–E6775

    Google Scholar 

  • Donohue E et al (2019) Second harmonic generation detection of Ras conformational changes and discovery of a small molecule binder. Proc Natl Acad Sci USA 116:17290–17297

    Google Scholar 

  • Durrant DE, Morrison DK (2018) Targeting the Raf kinases in human cancer: the Raf dimer dilemma. Br J Cancer 118:3–8

    Google Scholar 

  • Eglen RM et al (2008) The use of AlphaScreen technology in HTS: current status. Curr Chem Genomics 1:2–10

    Google Scholar 

  • Fang Z et al (2018) Inhibition of K-RAS4B by a unique mechanism of action: stabilizing membrane-dependent occlusion of the effector-binding site. Cell Chem Biol 25:1327–1336

    Google Scholar 

  • Fang Z et al (2020) Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane. Proc Natl Acad Sci USA 117:12101–12108

    Google Scholar 

  • Feng H et al (2019) K-Ras(G12D) has a potential allosteric small molecule binding site. Biochemistry 58:2542–2554

    Google Scholar 

  • Fivaz M, Meyer T (2005) Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2+/calmodulin. J Cell Biol 170:429–441

    Google Scholar 

  • Ford B, Skowronek K, Boykevisch S, Bar-Sagi D, Nassar N (2005) Structure of the G60A mutant of Ras: implications for the dominant negative effect. J Biol Chem 280:25697–25705

    Google Scholar 

  • Gai SA, Wittrup KD (2007) Yeast surface display for protein engineering and characterization. Curr Opin Struct Biol 17:467–473

    Google Scholar 

  • Gajate P et al (2012) Influence of KRAS p.G13D mutation in patients with metastatic colorectal cancer treated with cetuximab. Clin Colorectal Cancer 11:291–296

    Google Scholar 

  • Gehringer M, Laufer SA (2019) Emerging and re-emerging warheads for targeted covalent inhibitors: applications in medicinal chemistry and chemical biology. J Med Chem 62:5673–5724

    Google Scholar 

  • Geyer M et al (1996) Conformational transitions in p21ras and in its complexes with the effector protein Raf-RBD and the GTPase activating protein GAP. Biochemistry 35:10308–10320

    Google Scholar 

  • Ghosh AK, Samanta I, Mondal A, Liu WR (2019) Covalent inhibition in drug discovery. ChemMedChem 14:889–906

    Google Scholar 

  • Go ML et al (2010) Amino derivatives of indole as potent inhibitors of isoprenylcysteine carboxyl methyltransferase. J Med Chem 53:6838–6850

    Google Scholar 

  • Grant BMM et al (2020) Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety. Sci Signal 13(625):eaaz0344

    Google Scholar 

  • Guillard S et al (2017) Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange. Nat Commun 8:16111

    ADS  Google Scholar 

  • Gupta AK et al (2019) Multi-target, ensemble-based virtual screening yields novel allosteric KRAS inhibitors at high success rate. Chem Biol Drug Des 94:1441–1456

    Google Scholar 

  • Ha JM et al (1989) Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study. Biochemistry 28:8411–8416

    Google Scholar 

  • Hallin J et al (2020) The KRAS(G12C) inhibitor MRTX849 provides insight toward therapeutic susceptibility of KRAS-mutant cancers in mouse models and patients. Cancer Discov 10:54–71

    Google Scholar 

  • Hanafusa H, Torii S, Yasunaga T, Nishida E (2002) Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nat Cell Biol 4:850–858

    Google Scholar 

  • Hancock JF, Magee AI, Childs JE, Marshall CJ (1989) All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 57:1167–1177

    Google Scholar 

  • Hancock JF, Paterson H, Marshall CJ (1990) A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell 63:133–139

    Google Scholar 

  • Hillig RC et al (2019) Discovery of potent SOS1 inhibitors that block RAS activation via disruption of the RAS-SOS1 interaction. Proc Natl Acad Sci USA 116:2551–2560

    Google Scholar 

  • Hof P, Pluskey S, Dhe-Paganon S, Eck MJ, Shoelson SE (1998) Crystal structure of the tyrosine phosphatase SHP-2. Cell 92:441–450

    Google Scholar 

  • Holderfield M (2018) Efforts to develop KRAS inhibitors. Cold Spring Harb Perspect Med 8:a031864

    Google Scholar 

  • Hunter JC et al (2015) Biochemical and structural analysis of common cancer-associated KRAS mutations. Mol Cancer Res 13:1325–1335

    Google Scholar 

  • Ismail SA et al (2011) Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo. Nat Chem Biol 7:942–949

    Google Scholar 

  • Ito Y et al (1997) Regional polysterism in the GTP-bound form of the human c-Ha-Ras protein. Biochemistry 36:9109–9119

    Google Scholar 

  • Jackson JH, Li JW, Buss JE, Der CJ, Cochrane CG (1994) Polylysine domain of K-ras 4B protein is crucial for malignant transformation. Proc Natl Acad Sci USA 91:12730–12734

    ADS  Google Scholar 

  • Jansen JM et al (2017) Inhibition of prenylated KRAS in a lipid environment. PLoS ONE 12:e0174706

    Google Scholar 

  • John J et al (1990) Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry 29:6058–6065

    Google Scholar 

  • Jura N, Scotto-Lavino E, Sobczyk A, Bar-Sagi D (2006) Differential modification of Ras proteins by ubiquitination. Mol Cell 21:679–687

    Google Scholar 

  • Kalbitzer HR, Spoerner M (2013) State 1(T) inhibitors of activated Ras. Enzymes 33(Pt A):69–94

    Google Scholar 

  • Kalbitzer HR, Spoerner M, Ganser P, Hozsa C, Kremer W (2009) Fundamental link between folding states and functional states of proteins. J Am Chem Soc 131:16714–16719

    Google Scholar 

  • Kano Y et al (2019) Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation. Nat Commun 10:224

    ADS  Google Scholar 

  • Kauke MJ et al (2017) An engineered protein antagonist of K-Ras/B-Raf interaction. Sci Rep 7:5831

    ADS  Google Scholar 

  • Kemp DS, Chien SW (1967) A new peptide coupling reagent. J Am Chem Soc 89:2743–2745

    Google Scholar 

  • Kessler D et al (2019) Drugging an undruggable pocket on KRAS. Proc Natl Acad Sci USA 116:15823–15829

    Google Scholar 

  • Kessler D et al (2020) Reply to Tran et al.: Dimeric KRAS protein-protein interaction stabilizers. Proc Natl Acad Sci USA 117:3365–3367

    Google Scholar 

  • Khan I, Spencer-Smith R, O'Bryan JP (2019) Targeting the alpha4-alpha5 dimerization interface of K-RAS inhibits tumor formation in vivo. Oncogene 38:2984–2993

    Google Scholar 

  • Khan I, Rhett JM, O'Bryan JP (2020) Therapeutic targeting of RAS: New hope for drugging the "undruggable". Biochim Biophys Acta Mol Cell Res 1867:118570

    Google Scholar 

  • Kidger AM, Sipthorp J, Cook SJ (2018) ERK1/2 inhibitors: New weapons to inhibit the RAS-regulated RAF-MEK1/2-ERK1/2 pathway. Pharmacol Ther 187:45–60

    Google Scholar 

  • Kondo Y et al (2019) Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases. Science 366:109–115

    ADS  Google Scholar 

  • Laheru D et al (2012) Integrated preclinical and clinical development of S-trans, trans-Farnesylthiosalicylic Acid (FTS, Salirasib) in pancreatic cancer. Invest New Drugs 30:2391–2399

    Google Scholar 

  • Lander HM et al (1997) A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction. J Biol Chem 272:4323–4326

    Google Scholar 

  • Lavoie H, Therrien M (2015) Regulation of RAF protein kinases in ERK signalling. Nat Rev Mol Cell Biol 16:281–298

    Google Scholar 

  • Lee KY et al (2020) Two distinct structures of membrane-associated homodimers of GTP- and GDP-bound KRAS4B revealed by paramagnetic relaxation enhancement. Angew Chem Int Ed Engl 59:11037–11045

    Google Scholar 

  • Leshchiner ES et al (2015) Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. Proc Natl Acad Sci USA 112:1761–1766

    ADS  Google Scholar 

  • Leventis R, Silvius JR (1998) Lipid-binding characteristics of the polybasic carboxy-terminal sequence of K-ras4B. Biochemistry 37:7640–7648

    Google Scholar 

  • Li Z, Buck M (2020) Computational design of myristoylated cell-penetrating peptides targeting oncogenic K-Ras.G12D at the effector-binding membrane interface. J Chem Inf Model 60:306–315

    Google Scholar 

  • Li S, Jang H, Zhang J, Nussinov R (2018a) Raf-1 cysteine-rich domain increases the affinity of K-Ras/Raf at the membrane, promoting MAPK signaling. Structure 26:513–525

    Google Scholar 

  • Li ZL, Prakash P, Buck M (2018b) A "Tug of War" maintains a dynamic protein-membrane complex: molecular dynamics simulations of C-Raf RBD-CRD bound to K-Ras4B at an anionic membrane. ACS Cent Sci 4:298–305

    Google Scholar 

  • Lim SM et al (2014) Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor. Angew Chem Int Ed Engl 53:199–204

    Google Scholar 

  • Lobell RB et al (2002) Preclinical and clinical pharmacodynamic assessment of L-778,123, a dual inhibitor of farnesyl:protein transferase and geranylgeranyl:protein transferase type-I. Mol Cancer Ther 1:747–758

    Google Scholar 

  • Lopez-Alcala C et al (2008) Identification of essential interacting elements in K-Ras/calmodulin binding and its role in K-Ras localization. J Biol Chem 283:10621–10631

    Google Scholar 

  • Lu S, Jang H, Nussinov R, Zhang J (2016) The structural basis of oncogenic mutations G12, G13 and Q61 in small GTPase K-Ras4B. Sci Rep 6:21949

    ADS  Google Scholar 

  • Mainardi S et al (2018) SHP2 is required for growth of KRAS-mutant non-small-cell lung cancer in vivo. Nat Med 24:961–967

    Google Scholar 

  • Markham A (2019) Alpelisib: first global approval. Drugs 79:1249–1253

    Google Scholar 

  • Martin-Gago P et al (2017a) Covalent protein labeling at glutamic acids. Cell Chem Biol 24:589–597

    Google Scholar 

  • Martin-Gago P et al (2017b) A PDE6delta-KRas inhibitor chemotype with up to seven H-bonds and picomolar affinity that prevents efficient inhibitor release by Arl2. Angew Chem Int Ed Engl 56:2423–2428

    Google Scholar 

  • Matsumoto S et al (2018) Molecular basis for allosteric inhibition of GTP-bound H-Ras protein by a small-molecule compound carrying a naphthalene ring. Biochemistry 57:5350–5358

    Google Scholar 

  • Maurer T et al (2012) Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc Natl Acad Sci USA 109:5299–5304

    ADS  Google Scholar 

  • Mayer M, Meyer B (2001) Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J Am Chem Soc 123:6108–6117

    Google Scholar 

  • Mazhab-Jafari MT et al (2015) Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site. Proc Natl Acad Sci USA 112:6625–6630

    ADS  Google Scholar 

  • McCarthy MJ et al (2019) Discovery of high-affinity noncovalent allosteric KRAS inhibitors that disrupt effector binding. ACS Omega 4:2921–2930

    Google Scholar 

  • McGee JH et al (2018) Exceptionally high-affinity Ras binders that remodel its effector domain. J Biol Chem 293:3265–3280

    Google Scholar 

  • McLean MA, Stephen AG, Sligar SG (2019) PIP2 influences the conformational dynamics of membrane-bound KRAS4b. Biochemistry 58:3537–3545

    Google Scholar 

  • Mendola CE, Backer JM (1990) Lovastatin blocks N-ras oncogene-induced neuronal differentiation. Cell Growth Differ 1:499–502

    Google Scholar 

  • Millburn MV et al (1990) Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science 247:939–945

    ADS  Google Scholar 

  • Montagner A et al (2005) A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras activation. J Biol Chem 280:5350–5360

    Google Scholar 

  • Nagasaka M et al (2020) KRAS G12C Game of Thrones, which direct KRAS inhibitor will claim the iron throne? Cancer Treat Rev 84:101974

    Google Scholar 

  • Nan X et al (2015) Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway. Proc Natl Acad Sci USA 112:7996–8001

    ADS  Google Scholar 

  • Nichols RJ et al (2018) RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers. Nat Cell Biol 20:1064–1073

    Google Scholar 

  • Noonan T, Brown N, Dudycz L, Wright G (1991) Interaction of GTP derivatives with cellular and oncogenic ras-p21 proteins. J Med Chem 34:1302–1307

    Google Scholar 

  • Novotny CJ, Hamilton GL, McCormick F, Shokat KM (2017) Farnesyltransferase-mediated delivery of a covalent inhibitor overcomes alternative prenylation to mislocalize K-Ras. ACS Chem Biol 12:1956–1962

    Google Scholar 

  • Ostrem JM, Peters U, Sos ML, Wells JA, Shokat KM (2013) K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503:548–551

    ADS  Google Scholar 

  • Papke B et al (2016) Identification of pyrazolopyridazinones as PDEdelta inhibitors. Nat Commun 7:11360

    ADS  Google Scholar 

  • Park E et al (2019) Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes. Nature 575:545–550

    ADS  Google Scholar 

  • Patgiri A, Jochim AL, Arora PS (2008) A hydrogen bond surrogate approach for stabilization of short peptide sequences in alpha-helical conformation. Acc Chem Res 41:1289–1300

    Google Scholar 

  • Patgiri A, Yadav KK, Arora PS, Bar-Sagi D (2011) An orthosteric inhibitor of the Ras-Sos interaction. Nat Chem Biol 7:585–587

    Google Scholar 

  • Patricelli MP et al (2016) Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state. Cancer Discov 6:316–329

    Google Scholar 

  • Prior IA, Lewis PD, Mattos C (2012) A comprehensive survey of Ras mutations in cancer. Cancer Res 72:2457–2467

    Google Scholar 

  • Prior IA, Hood FE, Hartley JL (2020) The frequency of Ras mutations in cancer. Cancer Res

  • Quambusch L et al (2019) Covalent-allosteric inhibitors to achieve Akt isoform-selectivity. Angew Chem Int Ed Engl 58:18823–18829

    Google Scholar 

  • Quatela SE, Sung PJ, Ahearn IM, Bivona TG, Philips MR (2008) Analysis of K-Ras phosphorylation, translocation, and induction of apoptosis. Methods Enzymol 439:87–102

    Google Scholar 

  • Quevedo CE et al (2018) Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment. Nat Commun 9:3169

    ADS  Google Scholar 

  • Raponi M, Winkler H, Dracopoli NC (2008) KRAS mutations predict response to EGFR inhibitors. Curr Opin Pharmacol 8:413–418

    Google Scholar 

  • Renaud JP et al (2018) Cryo-EM in drug discovery: achievements, limitations and prospects. Nat Rev Drug Discov 17:471–492

    Google Scholar 

  • Riely GJ et al (2011) A phase II trial of Salirasib in patients with lung adenocarcinomas with KRAS mutations. J Thorac Oncol 6:1435–1437

    Google Scholar 

  • Rocks O et al (2005) An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307:1746–1752

    ADS  Google Scholar 

  • Rocks O, Peyker A, Bastiaens PI (2006) Spatio-temporal segregation of Ras signals: one ship, three anchors, many harbors. Curr Opin Cell Biol 18:351–357

    Google Scholar 

  • Rocks O et al (2010) The palmitoylation machinery is a spatially organizing system for peripheral membrane proteins. Cell 141:458–471

    Google Scholar 

  • Rosnizeck IC et al (2010) Stabilizing a weak binding state for effectors in the human ras protein by cyclen complexes. Angew Chem Int Ed Engl 49:3830–3833

    Google Scholar 

  • Rosnizeck IC et al (2012) Metal-bis(2-picolyl)amine complexes as state 1(T) inhibitors of activated Ras protein. Angew Chem Int Ed Engl 51:10647–10651

    Google Scholar 

  • Ruess DA et al (2018) Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase. Nat Med 24:954–960

    Google Scholar 

  • Ryan MB, Der CJ, Wang-Gillam A, Cox AD (2015) Targeting RAS-mutant cancers: is ERK the key? Trends Cancer 1:183–198

    Google Scholar 

  • Saito N, Mine N, Kufe DW, Von Hoff DD, Kawabe T (2017) CBP501 inhibits EGF-dependent cell migration, invasion and epithelial-to-mesenchymal transition of non-small cell lung cancer cells by blocking KRas to calmodulin binding. Oncotarget 8:74006–74018

    Google Scholar 

  • Saur M et al (2019) Fragment-based drug discovery using cryo-EM. Drug Discov Today 61(3):543–560

    Google Scholar 

  • Saxton RA, Sabatini DM (2017) mTOR signaling in growth, metabolism, and disease. Cell 168:960–976

    Google Scholar 

  • Schapira M, Calabrese MF, Bullock AN, Crews CM (2019) Targeted protein degradation: expanding the toolbox. Nat Rev Drug Discov 18:949–963

    Google Scholar 

  • Schmick M et al (2014) KRas localizes to the plasma membrane by spatial cycles of solubilization, trapping and vesicular transport. Cell 157:459–471

    Google Scholar 

  • Shima F et al (2010) Structural basis for conformational dynamics of GTP-bound Ras protein. J Biol Chem 285:22696–22705

    Google Scholar 

  • Shima F et al (2013) In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction. Proc Natl Acad Sci USA 110:8182–8187

    ADS  Google Scholar 

  • Shima F et al (2015) Current status of the development of Ras inhibitors. J Biochem 158:91–99

    Google Scholar 

  • Shuker SB, Hajduk PJ, Meadows RP, Fesik SW (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274:1531–1534

    ADS  Google Scholar 

  • Siddiqui FA et al (2020) PDE6D inhibitors with a new design principle selectively block K-Ras activity. ACS Omega 5:832–842

    Google Scholar 

  • Silvius JR, l'Heureux F (1994) Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers. Biochemistry 33:3014–3022

    Google Scholar 

  • Silvius JR, Bhagatji P, Leventis R, Terrone D (2006) K-ras4B and prenylated proteins lacking "second signals" associate dynamically with cellular membranes. Mol Biol Cell 17:192–202

    Google Scholar 

  • Simanshu DK, Nissley DV, McCormick F (2017) RAS Proteins and Their Regulators in Human Disease. Cell 170:17–33

    Google Scholar 

  • Singh J, Petter RC, Baillie TA, Whitty A (2011) The resurgence of covalent drugs. Nat Rev Drug Discov 10:307–317

    Google Scholar 

  • Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317

    ADS  Google Scholar 

  • Smith MJ, Ikura M (2014) Integrated RAS signaling defined by parallel NMR detection of effectors and regulators. Nat Chem Biol 10:223–230

    Google Scholar 

  • Smith MJ, Neel BG, Ikura M (2013) NMR-based functional profiling of RASopathies and oncogenic RAS mutations. Proc Natl Acad Sci USA 110:4574–4579

    ADS  Google Scholar 

  • Spencer-Smith R et al (2017) Inhibition of RAS function through targeting an allosteric regulatory site. Nat Chem Biol 13:62–68

    Google Scholar 

  • Sperlich B, Kapoor S, Waldmann H, Winter R, Weise K (2016) Regulation of K-Ras4B Membrane Binding by Calmodulin. Biophys J 111:113–122

    Google Scholar 

  • Spiegel J, Cromm PM, Zimmermann G, Grossmann TN, Waldmann H (2014) Small-molecule modulation of Ras signaling. Nat Chem Biol 10:613–622

    Google Scholar 

  • Spoerner M, Herrmann C, Vetter IR, Kalbitzer HR, Wittinghofer A (2001) Dynamic properties of the Ras switch I region and its importance for binding to effectors. Proc Natl Acad Sci USA 98:4944–4949

    ADS  Google Scholar 

  • Spoerner M, Wittinghofer A, Kalbitzer HR (2004) Perturbation of the conformational equilibria in Ras by selective mutations as studied by 31P NMR spectroscopy. FEBS Lett 578:305–310

    Google Scholar 

  • Spoerner M, Graf T, Konig B, Kalbitzer HR (2005) A novel mechanism for the modulation of the Ras-effector interaction by small molecules. Biochem Biophys Res Commun 334:709–713

    Google Scholar 

  • Stephen AG, Esposito D, Bagni RK, McCormick F (2014) Dragging ras back in the ring. Cancer Cell 25:272–281

    Google Scholar 

  • Sun Q et al (2012) Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angew Chem Int Ed Engl 51:6140–6143

    Google Scholar 

  • Sun Q et al (2014) A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand. J Biomol NMR 60:11–14

    Google Scholar 

  • Sung PJ et al (2013) Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors. Proc Natl Acad Sci USA 110:20593–20598

    ADS  Google Scholar 

  • Tejpar S et al (2012) Association of KRAS G13D tumor mutations with outcome in patients with metastatic colorectal cancer treated with first-line chemotherapy with or without cetuximab. J Clin Oncol 30:3570–3577

    Google Scholar 

  • Thorpe LM, Yuzugullu H, Zhao JJ (2015) PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting. Nat Rev Cancer 15:7–24

    Google Scholar 

  • Tran TH et al (2020) The small molecule BI-2852 induces a nonfunctional dimer of KRAS. Proc Natl Acad Sci USA 117:3363–3364

    Google Scholar 

  • Traut TW (1994) Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140:1–22

    Google Scholar 

  • Travers T et al (2018) Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain. Sci Rep 8:8461

    ADS  Google Scholar 

  • Tugarinov V, Kanelis V, Kay LE (2006) Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protoc 1:749–754

    Google Scholar 

  • Vidimar V et al (2020) An engineered chimeric toxin that cleaves activated mutant and wild-type RAS inhibits tumor growth. Proc Natl Acad Sci USA

  • Vigil D, Cherfils J, Rossman KL, Der CJ (2010) Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nat Rev Cancer 10:842–857

    Google Scholar 

  • Villalonga P et al (2001) Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling. Mol Cell Biol 21:7345–7354

    Google Scholar 

  • Wahlstrom AM et al (2008) Inactivating Icmt ameliorates K-RAS-induced myeloproliferative disease. Blood 112:1357–1365

    Google Scholar 

  • Wang MT et al (2015) K-Ras promotes tumorigenicity through suppression of non-canonical Wnt signaling. Cell 163:1237–1251

    Google Scholar 

  • Welsch ME et al (2017) Multivalent small-molecule Pan-RAS inhibitors. Cell 168:878–889

    Google Scholar 

  • Whyte DB et al (1997) K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem 272:14459–14464

    Google Scholar 

  • Willumsen BM, Christensen A, Hubbert NL, Papageorge AG, Lowy DR (1984) The p21 ras C-terminus is required for transformation and membrane association. Nature 310:583–586

    ADS  Google Scholar 

  • Winter-Vann AM, Casey PJ (2005) Post-prenylation-processing enzymes as new targets in oncogenesis. Nat Rev Cancer 5:405–412

    Google Scholar 

  • Wolfson E, Schmukler E, Schokoroy ST, Kloog Y, Pinkas-Kramarski R (2015) Enhancing FTS (Salirasib) efficiency via combinatorial treatment. Biol Cell 107:130–143

    Google Scholar 

  • Wong GS et al (2018) Targeting wild-type KRAS-amplified gastroesophageal cancer through combined MEK and SHP2 inhibition. Nat Med 24:968–977

    Google Scholar 

  • Xie J, Wang X, Proud CG (2016) mTOR inhibitors in cancer therapy. F1000Res 5:F1000

    Google Scholar 

  • Xie C et al (2017) Identification of a New Potent Inhibitor Targeting KRAS in Non-small Cell Lung Cancer Cells. Front Pharmacol 8:823

    Google Scholar 

  • Xiong Y et al (2017) Covalent guanosine mimetic inhibitors of G12C KRAS. ACS Med Chem Lett 8:61–66

    Google Scholar 

  • Yamasaki K et al (1989) Conformation change of effector-region residues in antiparallel beta-sheet of human c-Ha-ras protein on GDP––GTP gamma S exchange: a two-dimensional NMR study. Biochem Biophys Res Commun 162:1054–1062

    Google Scholar 

  • Yang MH et al (2012) Regulation of RAS oncogenicity by acetylation. Proc Natl Acad Sci USA 109:10843–10848

    ADS  Google Scholar 

  • Yang JL et al (2016) A novel anti-p21Ras scFv antibody reacting specifically with human tumour cell lines and primary tumour tissues. BMC Cancer 16:131

    Google Scholar 

  • Ye M et al (2005) Crystal structure of M-Ras reveals a GTP-bound "off" state conformation of Ras family small GTPases. J Biol Chem 280:31267–31275

    Google Scholar 

  • Zhang Z, Shokat KM (2019) Bifunctional small-molecule ligands of K-Ras induce its association with immunophilin proteins. Angew Chem Int Ed Engl 58:16314–16319

    Google Scholar 

  • Zhang J et al (2010) Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors. Nature 463:501–506

    ADS  Google Scholar 

  • Zhang Y et al (2018) Design of small molecules that compete with nucleotide binding to an engineered oncogenic KRAS allele. Biochemistry 57:1380–1389

    Google Scholar 

  • Zhou Y, Hancock JF (2015) Ras nanoclusters: Versatile lipid-based signaling platforms. Biochim Biophys Acta 1853:841–849

    Google Scholar 

  • Zhou Y et al (2017) Lipid-sorting specificity encoded in K-Ras membrane anchor regulates signal output. Cell 168:239–251

    Google Scholar 

  • Zimmermann G et al (2013) Small molecule inhibition of the KRAS-PDEdelta interaction impairs oncogenic KRAS signalling. Nature 497:638–642

    ADS  Google Scholar 

  • Zimmermann G et al (2014) Structure guided design and kinetic analysis of highly potent benzimidazole inhibitors targeting the PDEdelta prenyl binding site. J Med Chem 57:5435–5448

    Google Scholar 

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Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research (410008598), the Canadian Cancer Society Research Institute (703209, 706696), Canada Foundation for Innovation (CFI), the Princess Margaret Cancer Foundation (PMCF) and Canada Research Chairs.

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Authors and Affiliations

  1. Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada

    Christopher B. Marshall, Fenneke KleinJan, Teklab Gebregiworgis, Ki-Young Lee, Zhenhao Fang, Ben J. Eves, Ningdi F. Liu, Geneviève M. C. Gasmi-Seabrook, Masahiro Enomoto & Mitsuhiko Ikura

  2. Department of Medical Biophysics, University of Toronto, Toronto, ON, M5G 1L7, Canada

    Zhenhao Fang, Ningdi F. Liu & Mitsuhiko Ikura

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  1. Christopher B. Marshall
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Marshall, C.B., KleinJan, F., Gebregiworgis, T. et al. NMR in integrated biophysical drug discovery for RAS: past, present, and future. J Biomol NMR 74, 531–554 (2020). https://doi.org/10.1007/s10858-020-00338-6

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