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1
Rowinsky, Eric K., Jolene J. Windle, and Daniel D. Von Hoff. "Ras Protein Farnesyltransferase: A Strategic Target for Anticancer Therapeutic Development." Journal of Clinical Oncology 17, no. 11 (November 1999): 3631–52. http://dx.doi.org/10.1200/jco.1999.17.11.3631.
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ABSTRACT: Ras proteins are guanine nucleotide–binding proteins that play pivotal roles in the control of normal and transformed cell growth and are among the most intensively studied proteins of the past decade. After stimulation by various growth factors and cytokines, Ras activates several downstream effectors, including the Raf-1/mitogen-activated protein kinase pathway and the Rac/Rho pathway. In approximately 30% of human cancers, including a substantial proportion of pancreatic and colon adenocarcinomas, mutated ras genes produce mutated proteins that remain locked in an active state, thereby relaying uncontrolled proliferative signals. Ras undergoes several posttranslational modifications that facilitate its attachment to the inner surface of the plasma membrane. The first—and most critical—modification is the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (FTase). It follows that inhibiting FTase would prevent Ras from maturing into its biologically active form, and FTase is of considerable interest as a potential therapeutic target. Different classes of FTase inhibitors have been identified that block farnesylation of Ras, reverse Ras-mediated cell transformation in human cell lines, and inhibit the growth of human tumor cells in nude mice. In transgenic mice with established tumors, FTase inhibitors cause regression in some tumors, which appears to be mediated through both apoptosis and cell cycle regulation. FTase inhibitors have been well tolerated in animal studies and do not produce the generalized cytotoxic effects in normal tissues that are a major limitation of most conventional anticancer agents. There are ongoing clinical evaluations of FTase inhibitors to determine the feasibility of administering them on dose schedules like those that portend optimal therapeutic indices in preclinical studies. Because of the unique biologic aspects of FTase, designing disease-directed phase II and III evaluations of their effectiveness presents formidable challenges.
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Graham, Timothy E., Janet R. Pfeiffer, Rebecca J. Lee, Donna F. Kusewitt, A. Marina Martinez, Terry Foutz, Bridget S. Wilson, and Janet M. Oliver. "MEK and ERK Activation in Ras-Disabled RBL-2H3 Mast Cells and Novel Roles for Geranylgeranylated and Farnesylated Proteins in FcεRI-Mediated Signaling." Journal of Immunology 161, no. 12 (December 15, 1998): 6733–44. http://dx.doi.org/10.4049/jimmunol.161.12.6733.
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Abstract Cross-linking the high affinity IgE receptor FcεRI of basophils and mast cells activates receptor-associated protein-tyrosine kinases and stimulates a signaling cascade leading to secretion, ruffling, spreading, and cytokine production. Previous evidence that the pan-prenylation inhibitor lovastatin blocks Ag-stimulated Ca2+ influx, secretion, and membrane/cytoskeletal responses implicated isoprenylated proteins in the FcεRI-coupled signaling cascade but could not distinguish between contributions of C15 (farnesylated) and C20 (geranylgeranylated) species. Here we establish concentrations of lovastatin and the farnesyl-specific inhibitor BZA-5B that inhibit the farnesylation and Ag-induced activation of Ras species in RBL-2H3 cells (H-Ras, K-RasA, and K-RasB). These inhibitors have little effect on tyrosine kinase activation, which initiates FcεRI signaling. Although Ras is disabled, only lovastatin substantially blocks Raf-1 activation, and neither inhibitor affects mitogen-activated protein kinase kinase/extracellular signal regulated kinase kinase (MEK) or ERK1/ERK2 activation. Thus, the pathway to FcεRI-mediated MEK/ERK and ERK activation can apparently bypass Ras and Raf-1. Predictably, only lovastatin inhibits Ag-induced ruffling, spreading, and secretion, previously linked to geranylgeranylated Rho and Rab family members. Additionally, only lovastatin inhibits phospholipase Cγ-mediated inositol (1,4,5) trisphosphate production, sustained Ca2+ influx, and Ca2+-dependent IL-4 production, suggesting novel roles for geranylgeranylated (lovastatin-sensitive, BZA-5B-insensitive) proteins in FcεRI signal propagation. Remarkably, BZA-5B concentrations too low to inactivate Ras reduce the lag time to Ag-induced Ca2+ stores release and enhance secretion. These results link a non-Ras farnesylated protein(s) to the negative regulation of Ca2+ release from intracellular stores and secretion. We identified no clear role for Ras in FcεRI-coupled signaling but suggest its involvement in mast cell growth regulation based on the inhibition of cell proliferation by both BZA-5B and lovastatin.
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Sugita, Kenji, and Mitsuaki Ohtani. "Inhibitors of Ras-Transformation." Current Pharmaceutical Design 3, no. 3 (June 1997): 323–34. http://dx.doi.org/10.2174/138161280303221007125314.
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Abstract: Ras-oncogene is now thought to be one of the most important oncogenes which is evidenced in the correlation with human cancers. Point mutation in ras-gene correlates with transformation caused by ras and is found in human cancers with high frequency, such as 90% in pancreatic cancer, 50% in colon cancer and 30% in lung cancer. Ras (product of ms-oncogene) has GTPase activity and loses its activity with point mutation, leading to transformation. Active GTP-binding form of Ras is a key molecule of signal transduction in cell growth, differentiation and transformation. Inhibitors of ms-transformation will be good candidates of anti-cancer agents. Although many inhibitors of ras transformation have been reported up to now including macromolecules (genes, nucleotides, antibodies), we summarize the small-molecule compounds, which inhibit ms-transformation in direct or indirect manner in this review. Direct inhibitors of ras include inhibitors of farnesylation of Ras. Farnesyltransferase inhibitors (L- 744,832, etc) include peptidomimetics with the rational drug design for C-terminus of Ras and natural products. Inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase (Lovastatin, etc) which block cholesterol metabolism, inhibit farnesylation of Ras by decreasing the amount of the substrate of farnesyltransferase (Frase). Indirect inhibitors of ras with known mechanisms include the inhibitor of inositol monophosphate (IMP) dehydrogenase (oxanosine), the inhibitor of mitogen-activated protein kinase kinase (MAPK kinase)(PD98059), the inhibitor of MAPK (apigenin), the inhibitors of protein kinase C (PKC) (UCN- 01, etc), the inhibitors of phosphatidylinositol 3-kinase (Pl3K) (Wortmannin and L-294002), the inducer of the transcription factor JunD (oxamflatin) and the inhibitors of histone deacetylase [trichostatin A (TSA) and trapoxins]. Almost all compounds are now under development, and will be evaluated in clinical studies as anticancer agents.
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Mattox, Tyler E., Xi Chen, Yulia Y. Maxuitenko, Adam B. Keeton, and Gary A. Piazza. "Exploiting RAS Nucleotide Cycling as a Strategy for Drugging RAS-Driven Cancers." International Journal of Molecular Sciences 21, no. 1 (December 24, 2019): 141. http://dx.doi.org/10.3390/ijms21010141.
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Oncogenic mutations in RAS genes result in the elevation of cellular active RAS protein levels and increased signal propagation through downstream pathways that drive tumor cell proliferation and survival. These gain-of-function mutations drive over 30% of all human cancers, presenting promising therapeutic potential for RAS inhibitors. However, many have deemed RAS “undruggable” after nearly 40 years of failed drug discovery campaigns aimed at identifying a RAS inhibitor with clinical activity. Here we review RAS nucleotide cycling and the opportunities that RAS biochemistry presents for developing novel RAS inhibitory compounds. Additionally, compounds that have been identified to inhibit RAS by exploiting various aspects of RAS biology and biochemistry will be covered. Our current understanding of the biochemical properties of RAS, along with reports of direct-binding inhibitors, both provide insight on viable strategies for the discovery of novel clinical candidates with RAS inhibitory activity.
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Zeng, Jun, Thao Nheu, Anna Zorzet, Bruno Catimel, Ed Nice, Hiroshi Maruta, Antony W.Burgess, and Herbert R.Treutlein. "Design of inhibitors of Ras–Raf interaction using a computational combinatorial algorithm." Protein Engineering, Design and Selection 14, no. 1 (January 2001): 39–45. http://dx.doi.org/10.1093/protein/14.1.39.
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Cruz-Migoni, Abimael, Peter Canning, Camilo E. Quevedo, Carole J. R. Bataille, Nicolas Bery, Ami Miller, Angela J. Russell, Simon E. V. Phillips, Stephen B. Carr, and Terence H. Rabbitts. "Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds." Proceedings of the National Academy of Sciences 116, no. 7 (January 25, 2019): 2545–50. http://dx.doi.org/10.1073/pnas.1811360116.
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The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein–protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein–protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein–protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein–protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.
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ZHAN, JIN-HUI, XI ZHAO, XU-RI HUANG, and CHIA-CHUNG SUN. "MOLECULAR DYNAMICS AND FREE ENERGY ANALYSES OF ERK2–PYRAZOLYLPYRROLE INHIBITORS INTERACTIONS: INSIGHT INTO STRUCTURE-BASED LIGAND DESIGN." Journal of Theoretical and Computational Chemistry 08, no. 05 (October 2009): 887–908. http://dx.doi.org/10.1142/s0219633609005131.
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The extracellular signal-regulated protein kinase 2 (ERK2) is a pivotal member involving in Ras/Raf/MEK/ERK signal transduction pathway, acting as a central point where multiple signaling pathways coalesce to drive transcription. The pyrazolylpyrrole compounds as ATP competitive inhibitors of ERK2 can bind target with a special binding mode and have higher inhibitory potency than other ERK2-inhibitors. We investigated the interaction mode of ERK2-inhibitor using molecular dynamics simulation. The molecular mechanics Poisson–Boltzmann surface area approach is used to calculate the binding free energy of ERK2 with pyrazolylpyrrole inhibitors to analyze the factors of improving the affinity. The results indicated that the electrostatic interactions play the most important role in keeping the stabilization of ERK2-inhibitor. The structural analyses showed that the protein motions can be controlled by changing the structures of inhibitors; furthermore, the full use of available space in the binding site by improving the flexibilities of inhibitors and introducing hydrophobic groups can increase the inhibitory effect.
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Überall, Florian, Karina Hellbert, Sonja Kampfer, Karl Maly, Andreas Villunger, Martin Spitaler, James Mwanjewe, Gabriele Baier-Bitterlich, Gottfried Baier, and Hans H. Grunicke. "Evidence That Atypical Protein Kinase C-λ and Atypical Protein Kinase C-ζ Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton." Journal of Cell Biology 144, no. 3 (February 8, 1999): 413–25. http://dx.doi.org/10.1083/jcb.144.3.413.
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Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.
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Tisdale, E. J., J. R. Bourne, R. Khosravi-Far, C. J. Der, and W. E. Balch. "GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex." Journal of Cell Biology 119, no. 4 (November 15, 1992): 749–61. http://dx.doi.org/10.1083/jcb.119.4.749.
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We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.
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Cooley, Rachel, Neesha Kara, Ning Sze Hui, Jonathan Tart, Chloë Roustan, Roger George, David C. Hancock, et al. "Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets." Wellcome Open Research 5 (February 6, 2020): 20. http://dx.doi.org/10.12688/wellcomeopenres.15675.1.
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Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLucâ ® Binary Technology (NanoBiTâ ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.
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Cooley, Rachel, Neesha Kara, Ning Sze Hui, Jonathan Tart, Chloë Roustan, Roger George, David C. Hancock, et al. "Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets." Wellcome Open Research 5 (June 2, 2020): 20. http://dx.doi.org/10.12688/wellcomeopenres.15675.2.
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Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc ® Binary Technology (NanoBiT ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.
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Kupperman, E., W. Wen, and J. L. Meinkoth. "Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase." Molecular and Cellular Biology 13, no. 8 (August 1993): 4477–84. http://dx.doi.org/10.1128/mcb.13.8.4477-4484.1993.
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Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.
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Kupperman, E., W. Wen, and J. L. Meinkoth. "Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase." Molecular and Cellular Biology 13, no. 8 (August 1993): 4477–84. http://dx.doi.org/10.1128/mcb.13.8.4477.
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Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.
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Al-Baldawi, N. F., J. D. Stockand, O. K. Al-Khalili, G. Yue, and D. C. Eaton. "Aldosterone induces Ras methylation in A6 epithelia." American Journal of Physiology-Cell Physiology 279, no. 2 (August 1, 2000): C429—C439. http://dx.doi.org/10.1152/ajpcell.2000.279.2.c429.
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Aldosterone increases Na+ reabsorption by renal epithelial cells: the acute actions (<4 h) appear to be promoted by protein methylation. This paper describes the relationship between protein methylation and aldosterone's action and describes aldosterone-mediated targets for methylation in cultured renal cells (A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/μg protein. Aldosterone stimulates protein methylation by increasing methyltransferase activity from 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/μg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce the aldosterone-induced increase in methyltransferase activity. One of these inhibitors, the isoprenyl-cysteine methyltransferase-specific inhibitor, S- trans, trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone induces protein methylation in two molecular weight ranges: near 90 kDa and around 20 kDa. The lower molecular weight range is the weight of small G proteins, and aldosterone does increase both Ras protein 1.6-fold and Ras methylation almost 12-fold. Also, Ras antisense oligonucleotides reduce the activity of Na+ channels by about fivefold. We conclude that 1) protein methylation is essential for aldosterone-induced increases in Na+ transport; 2) one target for methylation is p21ras; and 3) inhibition of Ras expression or Ras methylation inhibits Na+ channel activity.
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Kim, R., J. Rine, and S. H. Kim. "Prenylation of mammalian Ras protein in Xenopus oocytes." Molecular and Cellular Biology 10, no. 11 (November 1990): 5945–49. http://dx.doi.org/10.1128/mcb.10.11.5945-5949.1990.
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Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.
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Kim, R., J. Rine, and S. H. Kim. "Prenylation of mammalian Ras protein in Xenopus oocytes." Molecular and Cellular Biology 10, no. 11 (November 1990): 5945–49. http://dx.doi.org/10.1128/mcb.10.11.5945.
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Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.
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Manoharan, Ganesh Babu, Sunday Okutachi, and Daniel Abankwa. "Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells." PLOS ONE 17, no. 5 (May 26, 2022): e0268635. http://dx.doi.org/10.1371/journal.pone.0268635.
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Phenothiazines (PTZ) were developed as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they have been used for decades as antipsychotic drugs. In addition, they possess significant anti-cancer properties and several attempts for their repurposing were made. However, their incompletely understood polypharmacology is challenging. Here we examined the potential of the PTZ fluphenazine (Flu) and its mustard derivative (Flu-M) to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras- and MAPK-pathway, cell viability and features of cancer cell stemness. Consistently, we show that the combination of a CaM inhibitor and the PP2A activator DT-061 synergistically inhibited the 3D-spheroid formation of MDA-MB-231 (K-Ras-G13D), NCI-H358 (K-Ras-G12C) and A375 (B-raf-V600E) cancer cells, and increased apoptosis in MDA-MB-231. We reasoned that these activities remain combined in PTZ, which were the starting point for PP2A activator development, while several PTZ are known CaM inhibitors. We show that both Flu and Flu-M retained CaM inhibitory activity in vitro and in cells, with a higher potency of the mustard derivative in cells. In line with the CaM dependence of Ras plasma membrane organization, the mustard derivative potently reduced the functional membrane organization of oncogenic Ras, while DT-061 had a negligible effect. Like DT-061, both PTZ potently decreased c-MYC levels, a hallmark of PP2A activation. Benchmarking against the KRAS-G12C specific inhibitor AMG-510 in MIA PaCa-2 cells revealed a higher potency of Flu-M than combinations of DT-061 and a CaM inhibitor on MAPK-output and a strong effect on cell proliferation. While our study is limited, our results suggest that improved PTZ derivatives that retain both, their CaM inhibitory and PP2A activating properties, but have lost their neurological side-effects, may be interesting to pursue further as anti-cancer agents.
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Upadhyaya, Punit, Walaa Bedewy, and Dehua Pei. "Direct Inhibitors of Ras-Effector Protein Interactions." Mini-Reviews in Medicinal Chemistry 16, no. 5 (February 1, 2016): 376–82. http://dx.doi.org/10.2174/1389557515666151001141713.
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Graham, Samuel L., S. Jane deSolms, Elizabeth A. Giuliani, Nancy E. Kohl, Scott D. Mosser, Allen I. Oliff, David L. Pompliano, Elaine Rands, and Michael J. Breslin. "Pseudopeptide Inhibitors of Ras Farnesyl-Protein Transferase." Journal of Medicinal Chemistry 37, no. 6 (March 1994): 725–32. http://dx.doi.org/10.1021/jm00032a004.
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Wiechmann, Svenja, Pierre Maisonneuve, Britta M. Grebbin, Meike Hoffmeister, Manuel Kaulich, Hans Clevers, Krishnaraj Rajalingam, et al. "Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids." Journal of Biological Chemistry 295, no. 14 (February 20, 2020): 4526–40. http://dx.doi.org/10.1074/jbc.ra119.011025.
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The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.
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Chen, Shijie, Fengyang Li, Dan Xu, Kai Hou, Weirong Fang, and Yunman Li. "The Function of RAS Mutation in Cancer and Advances in its Drug Research." Current Pharmaceutical Design 25, no. 10 (August 5, 2019): 1105–14. http://dx.doi.org/10.2174/1381612825666190506122228.
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RAS (H-ras, K-ras, and N-ras), as the second largest mutated gene driver in various human cancers, has long been a vital research target for cancer. Its function is to transform the extracellular environment into a cascade of intracellular signal transduction. RAS mutant protein regulates tumor cell proliferation, apoptosis, metabolism and angiogenesis through downstream MAPK, PI3K and other signaling pathways. In KRAS or other RAS-driven cancers, current treatments include direct inhibitors and upstream/downstream signaling pathway inhibitors. However, the research on these inhibitors has been largely restricted due to their escape inhibition and off-target toxicity. In this paper, we started with the role of normal and mutant RAS genes in cancer, elucidated the relevant RAS regulating pathways, and highlighted the important research advancements in RAS inhibitor research. We concluded that for the crosstalk between RAS pathways, the effect of single regulation may be limited, and the multi-target drug combined compensation mechanism is becoming a research hotspot.
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Dillon, Martha, Antonio Lopez, Edward Lin, Dominic Sales, Ron Perets, and Pooja Jain. "Progress on Ras/MAPK Signaling Research and Targeting in Blood and Solid Cancers." Cancers 13, no. 20 (October 10, 2021): 5059. http://dx.doi.org/10.3390/cancers13205059.
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The mitogen-activated protein kinase (MAPK) pathway, consisting of the Ras-Raf-MEK-ERK signaling cascade, regulates genes that control cellular development, differentiation, proliferation, and apoptosis. Within the cascade, multiple isoforms of Ras and Raf each display differences in functionality, efficiency, and, critically, oncogenic potential. According to the NCI, over 30% of all human cancers are driven by Ras genes. This dysfunctional signaling is implicated in a wide variety of leukemias and solid tumors, both with and without viral etiology. Due to the strong evidence of Ras-Raf involvement in tumorigenesis, many have attempted to target the cascade to treat these malignancies. Decades of unsuccessful experimentation had deemed Ras undruggable, but recently, the approval of Sotorasib as the first ever KRas inhibitor represents a monumental breakthrough. This advancement is not without novel challenges. As a G12C mutant-specific drug, it also represents the issue of drug target specificity within Ras pathway; not only do many drugs only affect single mutational profiles, with few pan-inhibitor exceptions, tumor genetic heterogeneity may give rise to drug-resistant profiles. Furthermore, significant challenges in targeting downstream Raf, especially the BRaf isoform, lie in the paradoxical activation of wild-type BRaf by BRaf mutant inhibitors. This literature review will delineate the mechanisms of Ras signaling in the MAPK pathway and its possible oncogenic mutations, illustrate how specific mutations affect the pathogenesis of specific cancers, and compare available and in-development treatments targeting the Ras pathway.
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Onono, Fredrick O., Michael A. Morgan, H. Peter Spielmann, Douglas A. Andres, Michaela Scherr, Letizia Venturini, Iris Dallman, Arnold Ganser, and Christoph WM Reuter. "A Novel Proteomic Approach to Define Leukemia Cell Resistance to Farnesyltransferase Inhibitors." Blood 114, no. 22 (November 20, 2009): 3761. http://dx.doi.org/10.1182/blood.v114.22.3761.3761.
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Abstract Abstract 3761 Poster Board III-697 Farnesylation is a post-translational modification critical for the proper function of multiple physiologically important proteins, including small G-proteins, such as RAS. Methods allowing rapid and selective detection of protein farnesylation are fundamental for the understanding of farnesylated protein function and for monitoring efficacy of farnesyltransferase inhibitors (FTI). While the natural substrates for prenyltransferases are farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), FTase has been shown to incorporate isoprenoid analogues into protein substrates. Materials and Methods FTase targets in three different myeloid leukemia cell lines (HL-60, K562 and NB-4) were labeled using the unnatural FPP analogue 8-anilinogeranyl diphosphate (AGPP) in a tagging-via-substrate approach. Antibodies specific for the anilinogeranyl moiety were used to detect AG-modified proteins. This highly effective labeling/detection method was coupled with two-dimensional electrophoresis (2-DE) and subsequent Western blotting. Identities of individual protein spots were determined by overlaying anti-AG immunoblots and immunoblots probed with antibodies specific for different known prenylation target proteins, such as RAS-family proteins. The clinically tested FTIs BMS-214,662 and L-778,123 as well as the nitrogen-containing bisphosphonate zoledronate were used to further validate the specificity of this labeling technique. As a proof of principle of the ability of this approach to identify target proteins important for FTI resistance, RNA interference (RNAi) was applied to investigate the role of one of the specific protein targets (K-RAS) which remained AG-labelled in the presence of FTI BMS-214,662. Following confirmation of K-RAS silencing by Western blotting, flow cytometric analysis of Annexin-V stained cells was used to quantify the effect of K-RAS silencing on BMS-214,662-induced apoptosis. Results Metabolic labeling of prenylated proteins with anilinogeranyl (AG) occurred in a time-dependent manner. This method provided increased resolution of the prenylated proteome as we were able to detect as many as ten distinct protein spots corresponding to a single band at approximately 20 kDa observed by 1D SDS-PAGE. Some of the proteins we have identified using this overlay method include H-RAS, K-RAS, N-RAS, Rap1, RhoB, RhoC, pre-Lamin A, Lamin B, and LKB1. AG-specific signals decreased upon FTI treatment, further substantiating the specificity of this method. Additional evidence of the specificity of this approach was the observation that the bisphosphonate inhibitor zoledronate did not inhibit protein AG-labeling. Interestingly, this method allowed direct evaluation of specific FTI targets as demonstrated by the different efficacies we observed between the two FTIs studied here – BMS-214,662 and L-778,123. This method even allowed identification of subtle differences among the leukemia cell lines tested. Closer evaluation of the farnesylated proteome demonstrated clear differences between BMS-214,662 and L-778,123, consistent with earlier reports that FTIs elicit numerous cellular effects, including induction of apoptosis and cytostatic effects. While BMS-214,662 effectively inhibited farnesylation of the majority of larger molecular weight proteins, L-778,123 also blocked prenylation of smaller molecular weight proteins such as N-RAS and K-RAS. However, RhoB and RhoC remained farnesylated regardless of FTI treatment. BMS-214,662-induced apoptosis was substantially potentiated by RNAi knock-down of K-RAS expression. Conclusions This snapshot approach allowed simple, rapid and dynamic analysis of the complex farnesylated proteome in leukemia cells. Our results demonstrate that this method can be used to identify and validate specific inhibitor targets. Importantly, this approach also successfully identified proteins (e.g. K-RAS) which may be important for resistance to some FTIs, and thus may be useful in directing development of more efficient therapeutic regimens. Disclosures: No relevant conflicts of interest to declare.
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Upadhyay, Daya, Eduardo Correa-Meyer, Jacob I. Sznajder, and David W. Kamp. "FGF-10 prevents mechanical stretch-induced alveolar epithelial cell DNA damage via MAPK activation." American Journal of Physiology-Lung Cellular and Molecular Physiology 284, no. 2 (February 1, 2003): L350—L359. http://dx.doi.org/10.1152/ajplung.00161.2002.
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Cyclic stretch of alveolar epithelial cells (AEC) can alter normal lung barrier function. Fibroblast growth factor-10 (FGF-10), an alveolar type II cell mitogen that is critical for lung development, may have a role in promoting AEC repair. We studied whether cyclic stretch induces AEC DNA damage and whether FGF-10 would be protective. Cyclic stretch (30 min of 30% strain amplitude and 30 cycles/min) caused AEC DNA strand break formation, as assessed by alkaline unwinding technique and DNA nucleosomal fragmentation. Pretreatment of AEC with FGF-10 (10 ng/ml) blocked stretch-induced DNA strand break formation and DNA fragmentation. FGF-10 activated AEC mitogen-activated protein kinase (MAPK), and MAPK inhibitors prevented FGF-10-induced AEC MAPK activation and abolished the protective effects of FGF-10 against stretch-induced DNA damage. In addition, a Grb2-SOS inhibitor (SH3b-p peptide), a RAS inhibitor (farnesyl transferase inhibitor 277), and a RAF-1 inhibitor (forskolin) each prevented FGF-10-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in AEC. Moreover, N17-A549 cells that express a RAS dominant/negative protein prevented the FGF-10-induced ERK1/2 phosphorylation and RAS activation in AEC. We conclude that cyclic stretch causes AEC DNA damage and that FGF-10 attenuates these effects by mechanisms involving MAPK activation via the Grb2-SOS/Ras/RAF-1/ERK1/2 pathway.
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Feig, L. A., and G. M. Cooper. "Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP." Molecular and Cellular Biology 8, no. 8 (August 1988): 3235–43. http://dx.doi.org/10.1128/mcb.8.8.3235-3243.1988.
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Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.
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Feig, L. A., and G. M. Cooper. "Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP." Molecular and Cellular Biology 8, no. 8 (August 1988): 3235–43. http://dx.doi.org/10.1128/mcb.8.8.3235.
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Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.
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Zeng, Jun, and Herbert R. Treutlein. "A method for computational combinatorial peptide design of inhibitors of Ras protein." Protein Engineering, Design and Selection 12, no. 6 (June 1999): 457–68. http://dx.doi.org/10.1093/protein/12.6.457.
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Wennström, Stefan, and Julian Downward. "Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor." Molecular and Cellular Biology 19, no. 6 (June 1, 1999): 4279–88. http://dx.doi.org/10.1128/mcb.19.6.4279.
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ABSTRACT The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110α is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.
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Hauptschein, Robert, Simon Woodcock, Andrew Belfield, Helen Mason, Dorottya Keppel, Svetlana Markova, Kyuri Kim, et al. "Abstract 2677: JZP815, a potent and selective pan-RAF inhibitor, demonstrates efficacy in RAF and RAS mutant tumor pre-clinical models." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2677. http://dx.doi.org/10.1158/1538-7445.am2022-2677.
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Abstract Activation of the mitogen-activated protein kinase (MAPK) pathway by oncogenic mutations in RAS and RAF is a frequent driver of human cancer. Targeting specific components of the pathway is a precise and rational route to deliver benefits to cancer patients with high unmet therapeutic needs. The RAF kinase proteins (ARAF, BRAF, CRAF) are core pathway components which mediate MAPK signaling. Mutations in these critical signaling proteins leads to constitutive activation of the MAPK pathway and tumor growth. Using state of the art screening methodologies and medicinal chemistry we discovered and developed a next generation pan-RAF kinase inhibitor, JZP815, which potently inhibits wild-type and mutant RAF kinases in cell-free and cell-based assays. JZP815 is a selective RAF kinase inhibitor that suppresses all 3 RAF kinase family members at low-to-sub nanomolar potencies in cell-free assays. Moreover, in tumor cells JZP815 does not induce significant paradoxical pathway activation, observed with approved 1st generation BRAF-selective inhibitors, while demonstrating equivalent potencies for MAPK pathway inhibition driven by either mutant RAF monomers or RAS-induced RAF dimers in tumor cells. JZP815 significantly inhibited tumor growth as a single agent in multiple mouse xenograft solid tumor models harboring RAS and/or BRAF mutations. Furthermore, JZP815’s favorable pharmacokinetic profile sustained on-target pathway pharmacodynamic responses in a predictable dose and time dependent manner. JZP815 demonstrated enhanced activity when combined with inhibitors of other MAPK pathway components in both class 2 and class 3 mutant BRAF patient-derived tumor cells ex vivo, and KRAS mutant NSCLC and CRC xenografts in vivo. In summary, JZP815 is a potent and selective pan-RAF inhibitor that is orally bioavailable in multiple preclinical species with a well-behaved safety profile and pharmacokinetic properties predicted to provide drug exposure required for target engagement in humans, and therefore suitable for advancing into first-in-human trials. Citation Format: Robert Hauptschein, Simon Woodcock, Andrew Belfield, Helen Mason, Dorottya Keppel, Svetlana Markova, Kyuri Kim, Shane Roller, Caroline Phillips, Clifford D. Jones, Robin C. Humphreys. JZP815, a potent and selective pan-RAF inhibitor, demonstrates efficacy in RAF and RAS mutant tumor pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2677.
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Wu, Jianghong, Shawn McGinley, Yuan Wang, Peter Gallagher, and Haiching Ma. "Abstract 379: Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 379. http://dx.doi.org/10.1158/1538-7445.am2022-379.
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Abstract NanoBRET࣪ target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allow the measurement of compound binding to a selected cellular target protein. RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. Mutated RAS is locked in the activated GTP bound state and facilitates enhanced RAS signaling in cancer cells. While being a desirable target, the absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Here, we performed NanoBRET࣪ TE cellular assay with RAS inhibitors against transfected RAS and its mutants. Our data demonstrate that NanoBRET࣪ TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET࣪ RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), or HRAS fusion constructs co-transfected in live HEK293 cells. Z factor analysis indicates the assay is High Throughput Screening (HTS) compatible and reproducible. In addition, we are able to confirm the signaling inhibition of downstream phospho-ERK1/2 by KRAS (G12C)-specific inhibitors AMG 510 (Sotorasib), ARS-1620, MRTX849, and MRTX1257 in MIA PaCa-2 pancreas and SW837 colon cancer cells bearing KRAS (G12C) mutation by Western blot assay. Our results suggest NanoBRET࣪ TE cellular assay can serve as a great tool to facilitate RAS pathway drug discovery against human cancers. Citation Format: Jianghong Wu, Shawn McGinley, Yuan Wang, Peter Gallagher, Haiching Ma. Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 379.
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Hyun, Soonsil, and Dongyun Shin. "Small-Molecule Inhibitors and Degraders Targeting KRAS-Driven Cancers." International Journal of Molecular Sciences 22, no. 22 (November 9, 2021): 12142. http://dx.doi.org/10.3390/ijms222212142.
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Drug resistance continues to be a major problem associated with cancer treatment. One of the primary causes of anticancer drug resistance is the frequently mutated RAS gene. In particular, considerable efforts have been made to treat KRAS-induced cancers by directly and indirectly controlling the activity of KRAS. However, the RAS protein is still one of the most prominent targets for drugs in cancer treatment. Recently, novel targeted protein degradation (TPD) strategies, such as proteolysis-targeting chimeras, have been developed to render “undruggable” targets druggable and overcome drug resistance and mutation problems. In this study, we discuss small-molecule inhibitors, TPD-based small-molecule chemicals for targeting RAS pathway proteins, and their potential applications for treating KRAS-mutant cancers. Novel TPD strategies are expected to serve as promising therapeutic methods for treating tumor patients with KRAS mutations.
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Nonami, Atsushi, Chen Zhao, Liu Qingsong, Kristen Cowens, Amanda L. Christie, Yongfei Chen, Martin Sattler, et al. "Identification of Wee1 and IGF-1R As Novel Therapeutic Targets for Mutant RAS-Driven Acute Leukemia By Combinatory Chemical Screens." Blood 124, no. 21 (December 6, 2014): 3502. http://dx.doi.org/10.1182/blood.v124.21.3502.3502.
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Abstract RAS mutations, especially in NRAS, are common in AML, identified in over 10% of the patients. The mutations lead to constitutive RAS activation and activation of multiple signaling pathways, including RAF/MAPK, PI3K-AKT/mTOR, and others. Many efforts to directly target RAS itself with small molecules have been unsuccessful. Thus, efforts have been directed at targeting canonical downstream effectors of RAS, such as RAF, MEK, and others. The complexity of RAS signaling, including redundancy and activation of compensatory pathways, makes it difficult to predict clinical effects. For example, RAF inhibitors increase proliferation of RAS-transformed cells by paradoxically increasing activation of MEK. In an effort to identify new therapeutic targets related to RAS, we designed a novel chemical screen to identify agents capable of potentiating the activity of the MEK inhibitor, AZD6244 (A), or the mTOR inhibitor, Torin1 (B). As a cell line to be used in the screen, BaF/3 cells were transformed with G12D mutants of NRAS or KRAS (Ba/F3-NRAS or Ba/F3-KRAS cells, respectively) and were shown to be exquisitely dependent on each oncogene for viability. Screen A: We identified the IGF-1R inhibitor, GSK1904529A, as able to selectively potentiate the effects of AZD6244 against mutant RAS-positive leukemia. GSK1904529A and AZD6244 synergized against mutant NRAS-expressing cell lines, OCI-AML3 and HL60, as well as active KRAS-expressing and -dependent cell lines, NOMO-1, NB4, and SKM-1, but not against wild-type (wt) RAS-expressing HEL or MOLM14 cells, or normal mononuclear cells. This result was confirmed with an additional IGF-1R inhibitor, NVP-AEW541, which exhibits 100-fold more selectivity toward IGF-1R than the insulin receptor (IR), and the specificity of IGF-1R as the target of these inhibitors was validated by knockdown (KD) of IGF-1R by shRNA. Mechanistically, IGF-1R protein expression/activity was substantially increased in mutant RAS-expressing cells, and suppression of RAS led to a decrease in IGF-1R. It is hypothesized that the increased IGF-1R levels observed in mutant RAS-expressing cells may contribute significantly to RAS transformation of hematopoietic cells. The synergy between MEK and IGF-1R inhibitors correlated with induction of apoptosis, inhibition of cell cycle progression, and decreased phospho-S6 and phospho-4E-BP1. In vivo, NSG mice tail vein-injected with OCI-AML3-luc+ cells showed significantly lower tumor burden following one week of daily oral administration of 50 mg/kg NVP-AEW541 combined with 25 mg/kg AZD6244, as compared to mice treated with either agent alone, and the combination was more toxic to mutant NRAS-expressing primary AML patient cells, compared to either agent alone. Screen B: We identified the Wee1 kinase inhibitor, MK-1775, which was unexpectedly found to potentiate mTOR inhibition of mutant RAS- leukemia. Wee1 is a protein kinase and inhibitory regulator of the G2/M checkpoint that prevents cells from going through mitosis by inhibiting the activity of CDK1. In response to DNA damage, Wee1 inactivates CDK1, thus leading to G2 arrest; this allows transformed cells the time needed for repair of damaged DNA and thus confers a survival advantage. The synergy was observed in both mutant NRAS- and mutant KRAS-positive AML cell lines and primary patient samples. The observed synergy enhanced dephosphorylation of AKT, 4E-BP1 and S6K, and correlated with increased apoptosis. The specificity of Wee1 as the target of MK-1775 was validated by Wee1 KD, as well as partial reversal of drug combination-induced apoptosis by inhibition of CDK1 by the CDK1 inhibitor, RO-3306. In a mouse in vivo xenotransplantation model, the combination treatment (10 mg/kg MK-1775 combined with 10 mg/kg Torin2) was more effective than single agents at suppressing the growth of NB4-luc cells that were tail vein-injected to NSG mice. The present studies suggest that combinations of drugs that simultaneously inhibit IGF-1R and MEK, or that inhibit Wee1 and mTOR, represent novel targeted therapeutic strategies for mutant RAS-positive leukemias. Disclosures No relevant conflicts of interest to declare.
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Yaari-Stark, Shira, Yael Nevo-Caspi, Jasmine Jacob-Hircsh, Gideon Rechavi, Arnon Nagler, and Yoel Kloog. "Combining the Ras Inhibitor Salirasib and Proteasome Inhibitors: A Potential Treatment for Multiple Myeloma." Blood 116, no. 21 (November 19, 2010): 1810. http://dx.doi.org/10.1182/blood.v116.21.1810.1810.
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Abstract Abstract 1810 Multiple Myeloma (MM) is characterized by clonal proliferation of malignant plasma cells that eventually develop resistance to chemotherapy. Novel agents such as Thalidomide, Bortezomib (Velcade) and Lenalidomide improve response rates and prolong progression free and overall survival. Drug resistance, differentiation block and increased survival of the MM tumor cells result from genomic alterations, including high cyclin D and fibroblast growth factor receptor 3 (FGFR3) over-expression as well as mutations in NRas. Interactions between myeloma cells and stromal cells in the tumor microenvironment play a major role in MM resistance. Particularly, activation of NF-κB-mediated upregulation of IL-6 secretion by stromal cells is in connection with signal transduction of the Ras oncogene pathway. Activating mutations of Ras have been reported in 30–50% of MM patients. KRas and NRas are the most frequent mutated, suggesting that active Ras is an appropriate target in MM. Development of oncogenic Ras isoforms can be inhibited by Ras inhibitor, farnesylthiosalicylic acid (FTS, salirasib) which also inhibits fibroblast growth factor (FGF)-stimulated Ras activation. We, therefore, compared the effects of FTS on proliferation of NCIH929 (harboring oncogenic NRas) and of two other MM cell lines, MM1.S and U266, which do not harbor oncogenic NRas. Inhibition of cell proliferation was evident by the reduction in BrdU incorporation into the DNA of cells treated for 24 h with FTS (50, 75, or 100 μM) and by counting cells stained with alamarBlue. NCIH929 responded better than the others cell lines to FTS-induced growth inhibition (P<0.05). The IC50 values were 64μM, 82μM and 82–100μM for NCIH929, MM.1S and U266 MM tumor cell lines, respectively (n=3). Treatment with FTS also significantly reduced total Ras and NRas-GTP in NCIH929 but not in the two other cell types, which was accompanied by a significant decrease in the amount of c-Myc (62±2%), p-ERK (38±5%) and p-Akt (52±1.6%) (n=3). All of these proteins are essential for the proliferation, growth, and survival of myelomas. Gene-expression patterns of control and of FTS-treated NCIH929 cells demonstrated down-regulation of FGFR3 (by 2.44 fold) and FGFR3 protein expression declined significantly (36±5%) in these cells after FTS treatment. FTS also inhibited FGF-stimulated GTP loading of wild-type NRas, and hence ERK activation, in MM-NCIH929. These findings suggested that FGFR3 acts together with NRas to activate the MAPK pathway, and also pointing to the possibility that treatment with FTS affected both early Ras-dependent signaling and long-term Ras-dependent control of gene expression and protein translation. Proteasome inhibitors have emerged as powerful tools for inhibiting NFκB activity in myelomas. We therefore examined the combined effect of the proteasome inhibitor MG132 (0.5-2.5 μM) or bortezomib (2.5μM) and the Ras inhibitor FTS (50 or 75 μM) on the growth of NCIH929 cells. Combination of FTS with the proteasome inhibitor MG132 or bortezomib yielded synergistic inhibitory effect (up to 86±6.4%) of NCIH929 MM cell growth (P<0.001; P<0.05, respectively) (n=3). Lastly, we tested the potential inhibitory capabilities of new FTS derivatives including FTS-esters and amides. The FTS-amides exhibited substantially higher activity (50% higher) than FTS itself, while the FTS-esters were completely inactive. In conclusion, the dependence of MM on FGF3R and Ras pathways make them sensitive to Ras inhibitors such as FTS. The synergistic effects of bortezomib and FTS in NCIH929 cells and presumably in MM might be explained by the two distinct pathways that they affect. Based on these results, we suggest that salirasib (FTS) may be considered, both alone and moreover in combination with proteasome inhibitors, as a potential treatment for MM. Disclosures: No relevant conflicts of interest to declare.
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Jin, Ning, Tianyun Jiang, D. Marc Rosen, Barry D. Nelkin, and Douglas W. Ball. "Dual Inhibition of Mitogen-Activated Protein Kinase Kinase and Mammalian Target of Rapamycin in Differentiated and Anaplastic Thyroid Cancer." Molecular Endocrinology 23, no. 11 (November 1, 2009): 1937. http://dx.doi.org/10.1210/mend.23.11.9993.
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ABSTRACT Context Differentiated thyroid cancer and anaplastic thyroid cancer tumors frequently have activation of the ras/raf /MAPK kinase (MEK)/ERK and phosphatidylinositol 3-kinase (PI-3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathways. Objective The objective of the study was to investigate the efficacy of MEK and mTOR inhibitors in preclinical thyroid cancer treatment models with defined mutation status. Experimental Design The MEK inhibitor AZD6244 (ARRY-142886) and mTOR inhibitor rapamycin were tested separately and in combination in 10 differentiated thyroid cancer and anaplastic thyroid cancer cell lines and in a xenograft model for evidence of pathway inhibition, growth inhibition, apoptosis, and long-range adaptation and resistance. Results Seven of 10 tested lines had evidence of significant basal activity of the PI-3K/AKT/mTOR pathway, with elevated phosphorylated AKT and phosphorylated p70 S6 kinase. Activation of ras/RAF/MEK/ERK was equally common in this panel. All 10 lines exhibited better than 60% growth inhibition with combined MEK and mTOR inhibition, including lines with BRAF, Ret-PTC, ras, and PTEN mutations. Rapamycin or AZD6244 alone achieved this threshold in six and two lines, respectively. Dual-pathway inhibition in the Ret-PTC mutant cell line TPC1 caused an intense G1 arrest in cell culture and reversible cytostatic inhibition in a xenograft model. We did not observe significant feedback up-regulation of AKT activation in either acute or prolonged exposures. Conclusion These preclinical results support the inclusion of thyroid cancer patients in early-phase clinical trials combining ras/RAF/MEK/ERK and PI-3K/AKT/mTOR pathway inhibition.
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Lim, Jormay, Permeen Yusoff, Esther Sook Miin Wong, Sumana Chandramouli, Dieu-Hung Lao, Chee Wai Fong, and Graeme R. Guy. "The Cysteine-Rich Sprouty Translocation Domain Targets Mitogen-Activated Protein Kinase Inhibitory Proteins to Phosphatidylinositol 4,5-Bisphosphate in Plasma Membranes." Molecular and Cellular Biology 22, no. 22 (November 15, 2002): 7953–66. http://dx.doi.org/10.1128/mcb.22.22.7953-7966.2002.
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ABSTRACT Sprouty (Spry) proteins have been revealed as inhibitors of the Ras/mitogen-activated protein kinase (MAPK) cascade, a pathway crucial for developmental processes initiated by activation of various receptor tyrosine kinases. In COS-1 and Swiss 3T3 cells, all Spry isoforms translocate to the plasma membrane, notably ruffles, following activation. Here we show that microinjection of active Rac induced the translocation of Spry isoforms, indicating that the target of the Spry translocation domain (SpryTD) is downstream of active Rac. Targeted disruption of actin polymerization revealed that the SpryTD target appeared upstream of cytoskeletal rearrangements. Accumulated evidence indicated that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is the likely SpryTD target. Human Spry2TD (hSpry2TD) binds to PtdIns(4,5)P2 in vesicle-binding assays. hSpry2TD colocalizes with the pleckstrin homology domain of phospholipase Cδ, which binds PtdIns(4,5)P2. The plasma membrane localization of hSpry2TD was abolished in ionomycin-treated MDCK cells or when PtdIns(4,5)P2 was specifically dephosphorylated by overexpression of an engineered, green fluorescent protein-tagged inositol 5-phosphatase. Similarly, Spred, a novel Ras/MAPK inhibitor recently found to contain the conserved cysteine-rich SpryTD, also translocated to peripheral membranes and bound to PtdIns(4,5)P2. Alignment of the Spry and Spred proteins led us to identify a translocation-defective point mutant, hSpry2 D252. Targeting of hSpry2 to PtdIns(4,5)P2 was shown to be essential for the down-regulation of Ras/MAPK signaling.
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Jin, Ning, Tianyun Jiang, D. Marc Rosen, Barry D. Nelkin, and Douglas W. Ball. "Dual Inhibition of Mitogen-Activated Protein Kinase Kinase and Mammalian Target of Rapamycin in Differentiated and Anaplastic Thyroid Cancer." Journal of Clinical Endocrinology & Metabolism 94, no. 10 (October 1, 2009): 4107–12. http://dx.doi.org/10.1210/jc.2009-0662.
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Context: Differentiated thyroid cancer and anaplastic thyroid cancer tumors frequently have activation of the ras/raf /MAPK kinase (MEK)/ERK and phosphatidylinositol 3-kinase (PI-3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathways. Objective: The objective of the study was to investigate the efficacy of MEK and mTOR inhibitors in preclinical thyroid cancer treatment models with defined mutation status. Experimental Design: The MEK inhibitor AZD6244 (ARRY-142886) and mTOR inhibitor rapamycin were tested separately and in combination in 10 differentiated thyroid cancer and anaplastic thyroid cancer cell lines and in a xenograft model for evidence of pathway inhibition, growth inhibition, apoptosis, and long-range adaptation and resistance. Results: Seven of 10 tested lines had evidence of significant basal activity of the PI-3K/AKT/mTOR pathway, with elevated phosphorylated AKT and phosphorylated p70 S6 kinase. Activation of ras/RAF/MEK/ERK was equally common in this panel. All 10 lines exhibited better than 60% growth inhibition with combined MEK and mTOR inhibition, including lines with BRAF, Ret-PTC, ras, and PTEN mutations. Rapamycin or AZD6244 alone achieved this threshold in six and two lines, respectively. Dual-pathway inhibition in the Ret-PTC mutant cell line TPC1 caused an intense G1 arrest in cell culture and reversible cytostatic inhibition in a xenograft model. We did not observe significant feedback up-regulation of AKT activation in either acute or prolonged exposures. Conclusion: These preclinical results support the inclusion of thyroid cancer patients in early-phase clinical trials combining ras/RAF/MEK/ERK and PI-3K/AKT/mTOR pathway inhibition. Combined treatment with a MEK inhibitor (AZD6244/ARRY-142886) plus an mTOR inhibitor (Rapamycin) inhibited growth of thyroid cancer cells in vitro, and in a xenograft model, more potently than either agent alone.
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Correa-Meyer, Eduardo, Liuska Pesce, Carmen Guerrero, and Jacob I. Sznajder. "Cyclic stretch activates ERK1/2 via G proteins and EGFR in alveolar epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 282, no. 5 (May 1, 2002): L883—L891. http://dx.doi.org/10.1152/ajplung.00203.2001.
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Mechanical stimuli are transduced into intracellular signals in lung alveolar epithelial cells (AEC). We studied whether mitogen-activated protein kinase (MAPK) pathways are activated during cyclic stretch of AEC. Cyclic stretch induced a rapid (within 5 min) increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in AEC. The inhibition of Na+, L-type Ca2+ and stretch-activated ion channels with amiloride, nifedipine, and gadolinium did not prevent the stretch-induced ERK1/2 activation. The inhibition of Grb2-SOS interaction with an SH3 binding sequence peptide, Ras with a farnesyl transferase inhibitor, and Raf-1 with forskolin did not affect the stretch-induced ERK1/2 phosphorylation. Moreover, cyclic stretch did not increase Ras activity, suggesting that stretch-induced ERK1/2 activation is independent of the classical receptor tyrosine kinase-MAPK pathway. Pertussis toxin and two specific epidermal growth factor receptor (EGFR) inhibitors (AG-1478 and PD-153035) prevented the stretch-induced ERK1/2 activation. Accordingly, in primary AEC, cyclic stretch activates ERK1/2 via G proteins and EGFR, in Na+ and Ca2+ influxes and Grb2-SOS-, Ras-, and Raf-1-independent pathways.
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Bellazzo, Arianna, and Licio Collavin. "Cutting the Brakes on Ras—Cytoplasmic GAPs as Targets of Inactivation in Cancer." Cancers 12, no. 10 (October 21, 2020): 3066. http://dx.doi.org/10.3390/cancers12103066.
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The Ras pathway is frequently deregulated in cancer, actively contributing to tumor development and progression. Oncogenic activation of the Ras pathway is commonly due to point mutation of one of the three Ras genes, which occurs in almost one third of human cancers. In the absence of Ras mutation, the pathway is frequently activated by alternative means, including the loss of function of Ras inhibitors. Among Ras inhibitors, the GTPase-Activating Proteins (RasGAPs) are major players, given their ability to modulate multiple cancer-related pathways. In fact, most RasGAPs also have a multi-domain structure that allows them to act as scaffold or adaptor proteins, affecting additional oncogenic cascades. In cancer cells, various mechanisms can cause the loss of function of Ras inhibitors; here, we review the available evidence of RasGAP inactivation in cancer, with a specific focus on the mechanisms. We also consider extracellular inputs that can affect RasGAP levels and functions, implicating that specific conditions in the tumor microenvironment can foster or counteract Ras signaling through negative or positive modulation of RasGAPs. A better understanding of these conditions might have relevant clinical repercussions, since treatments to restore or enhance the function of RasGAPs in cancer would help circumvent the intrinsic difficulty of directly targeting the Ras protein.
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Suzuki, Jotaro, Yuji Yamazaki, Li Guang, Yoshito Kaziro, and Hiroshi Koide. "Involvement of Ras and Ral in Chemotactic Migration of Skeletal Myoblasts." Molecular and Cellular Biology 20, no. 13 (July 1, 2000): 4658–65. http://dx.doi.org/10.1128/mcb.20.13.4658-4665.2000.
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ABSTRACT In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca2+-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.
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Paruchuri, Sailaja, Bengt Hallberg, Maria Juhas, Christer Larsson, and Anita Sjölander. "Leukotriene D4 activates MAPK through a Ras-independent but PKCϵ-dependent pathway in intestinal epithelial cells." Journal of Cell Science 115, no. 9 (May 1, 2002): 1883–93. http://dx.doi.org/10.1242/jcs.115.9.1883.
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We have recently shown that leukotriene D4 (LTD4)increases cell survival in intestinal epithelial cells. Here we report and explore the complementary finding that LTD4 also enhances proliferation in these cells. This proliferative response was approximately half of that induced by epidermal growth factor (EGF) and its required activation of protein kinase C (PKC), Ras and the mitogen-activated protein kinase (MAPK) Erk-1/2. EGF also activated Erk-1/2 in these cells; however the EGF-receptor inhibitor PD153035 did not affect the LTD4-induced activation of Erk-1/2. In addition, LTD4 did not induce phosphorylation of the EGF receptor, nor did pertussis toxin (PTX) block EGF-induced activation of Erk-1/2, thus refuting a possible crosstalk between the receptors. Furthermore, LTD4-induced, but not EGF-induced,activation of Erk-1/2 was sensitive to PTX, PKC inhibitors and downregulation of PKCϵ. A definite role for PKCϵ in LTD4-induced stimulation of Erk-1/2 was documented by the inability of LTD4 to activate Erk-1/2 in cells transfected with either the regulatory domain of PKCϵ (an isoform specific dominant-negative inhibitor) or a kinase-dead PKCϵ. Although Ras and Raf-1 were both transiently activated by LTD4, only Raf-1 activation was abolished by abrogation of the PKC signal. Furthermore, the LTD4-induced activation of Erk-1/2 was unaffected by transfection with dominant-negative N17 Ras but blocked by transfection with kinase-dead Raf-1. Consequently, LTD4 regulates the proliferative response by a distinct Ras-independent, PKCϵ-dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway.
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Chen, Zhihong, Lora W. Forman, Kenneth A. Miller, Brandon English, Asami Takashima, Regine A. Bohacek, Robert M. Williams, and Douglas V. Faller. "Protein kinase Cδ inactivation inhibits cellular proliferation and decreases survival in human neuroendocrine tumors." Endocrine-Related Cancer 18, no. 6 (October 11, 2011): 759–71. http://dx.doi.org/10.1530/erc-10-0224.
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The concept of targeting cancer therapeutics toward specific mutations or abnormalities in tumor cells, which are not found in normal tissues, has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. Many human malignancies display activating mutations in the Ras family of signal-transducing genes or over-activity of p21Ras-signaling pathways. Carcinoid and other neuroendocrine tumors have been similarly demonstrated to have activation of Ras signaling directly by mutations in Ras, indirectly by loss of Ras-regulatory proteins, or via constitutive activation of upstream or downstream effector pathways of Ras, such as growth factor receptors or PI3-kinase and Raf/mitogen-activated protein kinases. We previously reported that aberrant activation of Ras signaling sensitizes cells to apoptosis when the activity of the PKCδ isozyme is suppressed and that PKCδ suppression is not toxic to cells with normal levels of p21Rassignaling. We demonstrate here that inhibition of PKCδ by a number of independent means, including genetic mechanisms (shRNA) or small-molecule inhibitors, is able to efficiently and selectively repress the growth of human neuroendocrine cell lines derived from bronchopulmonary, foregut, or hindgut tumors. PKCδ inhibition in these tumors also efficiently induced apoptosis. Exposure to small-molecule inhibitors of PKCδ over a period of 24 h is sufficient to significantly suppress cell growth and clonogenic capacity of these tumor cell lines. Neuroendocrine tumors are typically refractory to conventional therapeutic approaches. This Ras-targeted therapeutic approach, mediated through PKCδ suppression, which selectively takes advantage of the very oncogenic mutations that contribute to the malignancy of the tumor, may hold potential as a novel therapeutic modality.
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Lin, David Tse Shen, Nicholas G. Davis, and Elizabeth Conibear. "Targeting the Ras palmitoylation/depalmitoylation cycle in cancer." Biochemical Society Transactions 45, no. 4 (June 16, 2017): 913–21. http://dx.doi.org/10.1042/bst20160303.
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The Ras proteins are well-known drivers of many cancers and thus represent attractive targets for the development of anticancer therapeutics. Inhibitors that disrupt the association of the Ras proteins with membranes by blocking the addition of the farnesyl lipid moiety to the Ras C-terminus failed in clinical trials. Here, we explore the possibility of targeting a second lipid modification, S-acylation, commonly referred to as palmitoylation, as a strategy to disrupt the membrane interaction of specific Ras isoforms. We review the enzymes involved in adding and removing palmitate from Ras and discuss their potential roles in regulating Ras tumorigenesis. In addition, we examine other proteins that affect Ras protein localization and may serve as future drug targets.
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Bhagwat, Shripad V., Nenad Petrovic, Yasuhiro Okamoto, and Linda H. Shapiro. "The angiogenic regulator CD13/APN is a transcriptional target of Ras signaling pathways in endothelial morphogenesis." Blood 101, no. 5 (March 1, 2003): 1818–26. http://dx.doi.org/10.1182/blood-2002-05-1422.
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Angiogenesis, the formation of new blood vessels, is a critical step for tumor growth and metastasis and an integral component of the pathologic inflammatory response in arthritis and the proliferative retinopathies. The CD13/aminopeptidase N (CD13/APN) metalloprotease is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. Here, we show that cytokine induction of CD13/APN in endothelial cells is regulated by distinct Ras effector pathways involving Ras/mitogen-activated protein kinase (MAPK) or PI-3K. Signals transduced by activated Ras, Raf, and mitogen-induced extracellular kinase (MEK) stimulate transcription from theCD13/APN proximal promoter. Inhibition of these pathways and extracellular signal–regulated serine/threonine kinase (ERK-2) and PI-3K by expression of dominant-negative proteins or chemical inhibitors prevented induction of CD13/APNtranscription in response to basic fibroblast growth factor (bFGF). We show that Ras-induced signal transduction is required for growth factor–induced angiogenesis, because inhibition of downstream mediators of Ras signaling (MEK or PI-3K) abrogated endothelial cell migration, invasion, and morphogenesis in vitro. Reintroduction of CD13/APN, a shared downstream target of these pathways, overrode the suppressive effect of these inhibitors and restored the function of endothelial cells in migration/invasion and capillary morphogenesis assays. Similarly, inhibition of MEK abrogated cell invasion and the formation of endothelial-lined capillaries in vivo, which was effectively rescued by addition of exogenous CD13/APN protein. These studies provide strong evidence that CD13/APN is an important target of Ras signaling in angiogenesis and is a limiting factor in angiogenic progression.
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Saber, Sameh, Amr A. A. Mahmoud, Noha S. Helal, Eman El-Ahwany, and Rasha H. Abdelghany. "Renin–angiotensin system inhibition ameliorates CCl4-induced liver fibrosis in mice through the inactivation of nuclear transcription factor kappa B." Canadian Journal of Physiology and Pharmacology 96, no. 6 (June 2018): 569–76. http://dx.doi.org/10.1139/cjpp-2017-0728.
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Therapeutic interventions for liver fibrosis are still limited due to the complicated molecular pathogenesis. Renin–angiotensin system (RAS) seems to contribute to the development of hepatic fibrosis. Therefore, we aimed to examine the effect of RAS inhibition on CCl4-induced liver fibrosis. Mice were treated with silymarin (30 mg·kg−1), perindopril (1 mg·kg−1), fosinopril (2 mg·kg−1), or losartan (10 mg·kg−1). The administration of RAS inhibitors improved liver histology and decreased protein expression of alpha smooth muscle actin (α-SMA) and hepatic content of hydroxyproline. These effects found to be mediated via inactivation of nuclear transcription factor kappa B (NFκB) pathway by the inhibition of NFκB p65 phosphorylation at the Ser536 residue and phosphorylation-induced degradation of nuclear factor kappa-B inhibitor alpha (NFκBia) subsequently inhibited NFκB-induced TNF-α and TGF-β1, leading to lower levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF). We concluded that the tissue affinity of the angiotensin converting enzyme inhibitors (ACEIs) has no impact on its antifibrotic activity and that interfering the RAS either through the inhibition of ACE or the blockade of AT1R has the same therapeutic benefit. These results suggest RAS inhibitors as promising candidates for further clinical trials in the management of hepatic fibrosis.
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Patel, Dinesh V., Robert J. Schmidt, Scott A. Biller, Eric M. Gordon, Simon S. Robinson, and Veeraswamy Manne. "Farnesyl Diphosphate-Based Inhibitors of Ras Farnesyl Protein Transferase." Journal of Medicinal Chemistry 38, no. 15 (July 1995): 2906–21. http://dx.doi.org/10.1021/jm00015a013.
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Patel, Dinesh V., Manorama M. Patel, Simon S. Robinson, and Eric M. Gordon. "Phenol based tripeptide inhibitors of ras farnesyl protein transferase." Bioorganic & Medicinal Chemistry Letters 4, no. 15 (August 1994): 1883–88. http://dx.doi.org/10.1016/s0960-894x(01)80390-5.
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Marchwicka, Aleksandra, Daria Kamińska, Mohsen Monirialamdari, Katarzyna M. Błażewska, and Edyta Gendaszewska-Darmach. "Protein Prenyltransferases and Their Inhibitors: Structural and Functional Characterization." International Journal of Molecular Sciences 23, no. 10 (May 12, 2022): 5424. http://dx.doi.org/10.3390/ijms23105424.
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Protein prenylation is a post-translational modification controlling the localization, activity, and protein–protein interactions of small GTPases, including the Ras superfamily. This covalent attachment of either a farnesyl (15 carbon) or a geranylgeranyl (20 carbon) isoprenoid group is catalyzed by four prenyltransferases, namely farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase-I), Rab geranylgeranyltransferase (GGTase-II), and recently discovered geranylgeranyltransferase type III (GGTase-III). Blocking small GTPase activity, namely inhibiting prenyltransferases, has been proposed as a potential disease treatment method. Inhibitors of prenyltransferase have resulted in substantial therapeutic benefits in various diseases, such as cancer, neurological disorders, and viral and parasitic infections. In this review, we overview the structure of FTase, GGTase-I, GGTase-II, and GGTase-III and summarize the current status of research on their inhibitors.
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Lebowitz, P. F., J. P. Davide, and G. C. Prendergast. "Evidence that farnesyltransferase inhibitors suppress Ras transformation by interfering with Rho activity." Molecular and Cellular Biology 15, no. 12 (December 1995): 6613–22. http://dx.doi.org/10.1128/mcb.15.12.6613.
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Small-molecule inhibitors of the housekeeping enzyme farnesyltransferase (FT) suppress the malignant growth of Ras-transformed cells. Previous work suggested that the activity of these compounds reflected effects on actin stress fiber regulation rather than Ras inhibition. Rho proteins regulate stress fiber formation, and one member of this family, RhoB, is farnesylated in vivo. Therefore, we tested the hypothesis that interference with RhoB was the principal basis by which the peptidomimetic FT inhibitor L-739,749 suppressed Ras transformation. The half-life of RhoB was found to be approximately 2 h, supporting the possibility that it could be functionally depleted within the 18-h period required by L-739,749 to induce reversion. Cell treatment with L-739,749 disrupted the vesicular localization of RhoB but did not effect the localization of the closely related RhoA protein. Ras-transformed Rat1 cells ectopically expressing N-myristylated forms of RhoB (Myr-rhoB), whose vesicular localization was unaffected by L-739,749, were resistant to drug treatment. The protective effect of Myr-rhoB required the integrity of the RhoB effector domain and was not due to a gain-of-function effect of myristylation on cell growth. In contrast, Rat1 cells transformed by a myristylated Ras construct remained susceptible to growth inhibition by L-739,749. We concluded that Rho is necessary for Ras transformation and that FT inhibitors suppress the transformed phenotype at least in part by direct or indirect interference with Rho, possibly with RhoB itself.
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Mahgoub, Nidal, Brigit R. Taylor, Mary Gratiot, Nancy E. Kohl, Jackson B. Gibbs, Tyler Jacks, and Kevin M. Shannon. "In Vitro and In Vivo Effects of a Farnesyltransferase Inhibitor onNf1-Deficient Hematopoietic Cells." Blood 94, no. 7 (October 1, 1999): 2469–76. http://dx.doi.org/10.1182/blood.v94.7.2469.419a01_2469_2476.
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Oncogenic RAS alleles encode proteins that accumulate in the guanosine triphosphate (GTP)-bound state. Because post-translational processing of Ras by farnesyltransferase is essential for biologic function, inhibitors of this enzyme have been developed as rational cancer therapeutics. We have investigated farnesyltransferase inhibitor (FTI) L-744,832 in an in vivo murine model of myeloid leukemia that is associated with inactivation of the Nf1 tumor suppressor gene.Nf1 encodes a GTPase activating protein for Ras, andNf1-deficient (Nf1−/−) hematopoietic cells show hyperactive Ras signaling through the mitogen-activated protein (MAP) kinase pathway. L-744,832 inhibited H-Ras prenylation in cell lines and in primary hematopoietic cells and abrogated the in vitro growth of myeloid progenitor colonies in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). This FTI also partially blocked GM-CSF–induced MAP kinase activation, but did not reduce constitutively elevated levels of MAP kinase activity in primaryNf1−/− cells. Injection of a single dose of 40 or 80 mg/kg of L-744,832 increased the amount of unprocessed H-Ras in bone marrow cells, but had no detectable effect on N-Ras. Adoptive transfer ofNf1−/− hematopoietic cells into irradiated mice induces a myeloproliferative disorder that did not respond to L-744,832 treatment. We speculate that the lack of efficacy in this model is due to the resistance of N-Ras and K-Ras processing to inhibition by this FTI.
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Dobrowolski, S., M. Harter, and D. W. Stacey. "Cellular ras activity is required for passage through multiple points of the G0/G1 phase in BALB/c 3T3 cells." Molecular and Cellular Biology 14, no. 8 (August 1994): 5441–49. http://dx.doi.org/10.1128/mcb.14.8.5441-5449.1994.
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Microinjection experiments demonstrated a requirement for cellular ras activity late in G1. In this study, we used two separate methods to identify an additional requirement for cellular ras activity early in the G0/G1 phase of the cell cycle. Quiescent BALB/c cells were injected with anti-ras antibody prior to stimulation with serum. The cells would therefore be inhibited in progression through the cell cycle at the earliest point requiring ras function. Alternatively, cells were inhibited in late G1 as in previous studies by injecting anti-ras several hours after serum addition to quiescent cells. The injected cultures were then treated with chemical cell cycle inhibitors known to function in mid-G1. Cells injected with anti-ras prior to serum stimulation were retained at a point of ras requirement prior to the execution point of the chemical inhibitor, while cells injected 3 to 5 h after serum stimulation were retained at a point of ras requirement downstream of the execution point of the chemical inhibitor. To confirm these results, quiescent BALB/c cells were injected with anti-ras antibody prior to or several hours following serum addition. In this case, however, second injections of oncogenic ras or adenoviral E1A protein were performed to overcome the inhibitory effects of the anti-ras antibody. Cells injected prior to serum addition were clearly inhibited at an early point of Ras requirement since they required 5 or 6 h longer to enter S phase than cells injected with anti-ras antibody after serum addition.