Post-translational Modifications - Essential for Protein Regulation · 2018-10-30 · 12....
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Ras and Rho-family GTPases regulate mulple cellular processes, including development, growth, molity, intracellular trafficking, gene expression, and the cell cycle 1,2 . Moreover, dysfuncon of these GTPase are correlated with several human diseases (e.g., cancer, neurodegeneraon, bacterial pathogenesis) 3-5 . Like all GTPases, Ras and Rho GTPases cycle between acve (GTP-bound) and inacve (GDP-bound) states. Guanine nucleode exchange factors (GEFs) regulate GTPase acvaon, driving the exchange of GDP for GTP in response to a variety of physiological and pathological extracellular signals 1,2 . Thus, GEFs are therapeuc targets; however, small molecule inhibitors require hydrophobic pockets for binding which are not typically found on GEFs (or GTPases). Only recently have novel binding pockets on GEFs and GTPases been discovered 6,7 . In this light, the current newsleer explores small molecule inhibitors of Ras (N-, H-, K-Ras) and Rho (RhoA, Rac1, Cdc42) GEFs that inhibit through direct binding. Rho GEFs Trio is a mul-domain Rho GEF that acvates RhoA, RhoG, and Rac. Trio’s C-terminal domain (TrioC) acvates RhoA while the N-terminal domain (TrioN) acvates RhoG and Rac 8 . Using pepde aptamer screening, the first Rho GEF inhibitor, TRIPa (Trio inhibitory pepde a), was discovered. This inhibitor binds TrioC’s Rho GEF domain (TrioGEFD2) 8 and inhibits the catalyc acvity of TrioGEFD2 and Rho acvaon in COS cells co-expressing TRIPα and TrioGEFD2 8 (Table 1). Pepde aptamer screening also discovered TRIP E32G , the first inhibitor to the oncogenic Rho GEF Tgat (Trio-related transforming gene in ATL tumor cells) 9 . TRIP E32G inhibited Tgat’s GEF acvity and transforming acvity in Swiss 3T3 fibroblasts (Table 1). Subcutaneous injecon of fibroblasts co-expressing Tgat and TRIPE32G decreased the presence of tumors and delayed tumor development and weight in nude mice 9 . The Rho GEF LARG (leukemia-associated Rho GEF), a G-protein- regulated, RGS (regulators of G-protein signaling)-domain containing GEF 10 is inhibited by the Y16 compound. Y16 suppressed the interacon of LARG with RhoA by binding to the DH-PH juncon of LARG (Table 1). Y16 also inhibits RhoA's interacon with two other RGS domain-containing GEFs, p115 RhoGEF and PDZ RhoGEF. In MCF-7 breast cancer cells, Y16 inhibits growth, migraon, and invasion 10 . Ras GEFs Acvang Ras mutaons are associated with ~30% of all human cancers. Although the need for GEF-mediated acvaon of constuvely-acve Ras mutants remains unclear, Ras GEF inhibitors remain an acve research interest 7 . The most studied Ras GEF is SOS1, a mul-domain protein with a catalyc domain consisng of a Ras exchanger mof domain and cell division cycle 25 homology domain, both necessary for Ras acvaon. These domains contain a catalyc and an allosteric site for binding of Ras. The catalyc site binds GDP-Ras and smulates exchange of GDP for GTP 7,11,12 . A recent virtual screen for small molecule inhibitors directly targeng the catalyc site offered several promising candidates 13 . Table 1. Drugs directly targeng GEFs GEF Drug Binding Affinity (K d ) Ref # TrioC Tripa NR*; IC 50 of 4 mM in TrioC- mediated RhoA exchange acvity 8 Tgat Trip E32G NR*; apparent inhibion constant (Ki app )of 2.4-12.4 mM in Tgat-mediated RhoA exchange assay 9 LARG Y16 80 nM 10 SOS1 NSC-658497 7 mM 13 SOS1 UC-773587 3.4 mM 14 SOS1 UC-857993 14.7 mM 14 TrioN ITX3 NR*; IC 50 of 76 mM in TrioN- mediated RhoG exchange assay 15 *NR, not reported NSC-658497 is a small molecule SOS1 inhibitor that specifically and directly binds at its catalyc site (Table 1) and compevely inhibits Ras binding to SOS1 and decreases SOS1-mediated Ras acvaon, all in a concentraon-dependent manner 13 . Addionally, NSC-658497 inhibited EGF-smulated Ras acvaon and downstream signaling and proliferaon of 3T3 mouse fibroblasts as well as prostate, lung, and ovarian cancer cells that specifically express wild-type K-Ras rather Small molecule inhibitors of GEF-mediated GTPase signaling v News Publications Research Tools NOV/DEC 2016 www.cytoskeleton.com CYTOSKELETON NEWS NEWS FROM CYTOSKELETON INC. Meetings Society for Neuroscience November, 12-16 Booth # 2417 San Diego, CA American Society for Cell Biology December 3-7 Booth # 928 San Francisco, CA Cytoskeleton Products Actin Proteins Activation Assays Antibodies ECM Proteins ELISA Kits G-LISA® Kits Pull-down Assays Motor Proteins Small G-Proteins Tubulin & FtsZ Proteins Contact Us P: 1 (303) 322.2254 F: 1 (303) 322.2257 E: [email protected] W: cytoskeleton.com Small molecule inhibitors of GEF-mediated GTPase signaling Related Publications Research Tools
Transcript of Post-translational Modifications - Essential for Protein Regulation · 2018-10-30 · 12....
Ras and Rho-family GTPases regulate multiple cellular processes, including development, growth, motility, intracellular trafficking, gene expression, and the cell cycle1,2. Moreover, dysfunction of these GTPase are correlated with several human diseases (e.g., cancer, neurodegeneration, bacterial pathogenesis)3-5. Like all GTPases, Ras and Rho GTPases cycle between active (GTP-bound) and inactive (GDP-bound) states. Guanine nucleotide exchange factors (GEFs) regulate GTPase activation, driving the exchange of GDP for GTP in response to a variety of physiological and pathological extracellular signals1,2. Thus, GEFs are therapeutic targets; however, small molecule inhibitors require hydrophobic pockets for binding which are not typically found on GEFs (or GTPases). Only recently have novel binding pockets on GEFs and GTPases been discovered6,7. In this light, the current newsletter explores small molecule inhibitors of Ras (N-, H-, K-Ras) and Rho (RhoA, Rac1, Cdc42) GEFs that inhibit through direct binding.
Rho GEFs
Trio is a multi-domain Rho GEF that activates RhoA, RhoG, and Rac. Trio’s C-terminal domain (TrioC) activates RhoA while the N-terminal domain (TrioN) activates RhoG and Rac8. Using peptide aptamer screening, the first Rho GEF inhibitor, TRIPa (Trio inhibitory peptide a), was discovered. This inhibitor binds TrioC’s Rho GEF domain (TrioGEFD2)8 and inhibits the catalytic activity of TrioGEFD2 and Rho activation in COS cells co-expressing TRIPα and TrioGEFD28 (Table 1). Peptide aptamer screening also discovered TRIPE32G, the first inhibitor to the oncogenic Rho GEF Tgat (Trio-related transforming gene in ATL tumor cells)9. TRIPE32G inhibited Tgat’s GEF activity and transforming activity in Swiss 3T3 fibroblasts (Table 1). Subcutaneous injection of fibroblasts co-expressing Tgat and TRIPE32G decreased the presence of tumors and delayed tumor development and weight in nude mice9.
The Rho GEF LARG (leukemia-associated Rho GEF), a G-protein-regulated, RGS (regulators of G-protein signaling)-domain containing GEF10 is inhibited by the Y16 compound. Y16 suppressed the interaction of LARG with RhoA by binding to the DH-PH junction of LARG (Table 1). Y16 also inhibits RhoA's interaction with two other RGS domain-containing GEFs, p115 RhoGEF and PDZ RhoGEF. In MCF-7 breast cancer cells, Y16 inhibits growth, migration, and invasion10.
Ras GEFs
Activating Ras mutations are associated with ~30% of all human cancers. Although the need for GEF-mediated activation of constitutively-active Ras mutants remains unclear, Ras GEF inhibitors remain an active research interest7. The most studied Ras GEF is SOS1, a multi-domain protein with a catalytic domain consisting of a Ras exchanger motif domain and cell division cycle 25 homology domain, both necessary for Ras activation. These domains contain a catalytic and an allosteric site for binding of Ras. The catalytic site binds GDP-Ras and stimulates exchange of GDP for GTP7,11,12. A recent virtual screen for small molecule inhibitors directly targeting the catalytic site offered several promising candidates13.
Table 1. Drugs directly targeting GEFs
GEF Drug Binding Affinity (Kd) Ref #
TrioC Tripa NR*; IC50 of 4 mM in TrioC-mediated RhoA exchange
activity
8
Tgat TripE32G NR*; apparent inhibition constant (Kiapp)of 2.4-12.4 mM in Tgat-mediated RhoA
exchange assay
9
LARG Y16 80 nM 10
SOS1 NSC-658497 7 mM 13
SOS1 UC-773587 3.4 mM 14
SOS1 UC-857993 14.7 mM 14
TrioN ITX3 NR*; IC50 of 76 mM in TrioN-mediated RhoG exchange
assay
15
*NR, not reported
NSC-658497 is a small molecule SOS1 inhibitor that specifically and directly binds at its catalytic site (Table 1) and competitively inhibits Ras binding to SOS1 and decreases SOS1-mediated Ras activation, all in a concentration-dependent manner13. Additionally, NSC-658497 inhibited EGF-stimulated Ras activation and downstream signaling and proliferation of 3T3 mouse fibroblasts as well as prostate, lung, and ovarian cancer cells that specifically express wild-type K-Ras rather
Small molecule inhibitors of GEF-mediated GTPase signaling v
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than oncogenic K-Ras mutants13. This same research group14 identified two more compounds that specifically inhibit SOS1, designated UC-773587 and UC-857993. Further biochemical and structural analyses and pharmacological studies demonstrated that each compound inhibited EGF-stimulated Ras activity in 3T3 mouse fibroblasts and various human cancer cell lines, but through binding at different sites within SOS1’s domain, and with micromolar affinity in a concentration-dependent manner (Table 1). Furthermore, both compounds inhibited Ras activation and downstream signaling in cell lines14.
Rac GEFs
Like other Rho-family GTPases, Rac is activated by multiple GEFs, including Trio and Vav1. Screening efforts identified the small molecule ITX3 as a directly-binding TrioN inhibitor, blocking TrioN-induced nucleotide exchange on both RhoG and Rac1 and antagonizing TrioN-mediated changes in cell morphology (e.g., NGF-induced neurite outgrowth). ITX3 inhibited TrioN-mediated Rac1 activation (Table 1)15.
Vav1, a GEF for Rac (and Cdc42) has been implicated in melanoma and pancreatic cancers16. Azathioprine, a purine analogue, is well-characterized as an anti-inflammatory molecule, but is now also recognized as a Vav1 inhibitor in immune cells. Extending this work to pancreatic cancer cells, the authors found that azathioprine inhibited Vav1-mediated cancer cell invasion and migration and degradation of the extracellular matrix, both in vitro and in vivo16.
Summary
GTPase activation can be inhibited in a number of ways, one of which is direct binding of GEFs with a small molecule that prevents binding of the GEF to the GTPase and its subsequent activation (GDP exchange for GTP). With the discovery of novel binding pockets on GEFs (and GTPases), drug discovery efforts aimed at inhibiting GTPases received a much needed shot in the arm. To assist in these efforts, Cytoskeleton, Inc. offers a variety of GTPase activators and inhibitors, purified GTPases and GEFs, and activation assay kits to stimulate and measure activation of Ras and Rho GTPases in cell and tissue lysates.
ReferencesContinued from Page 1
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GTPase and GEF PRODUCTS
1. Jaffe A.B. and Hall A. 2005. Rho GTPases: biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21, 247-269.
2. Heasman S.J. and Ridley A.J. 2008. Mammalian Rho GTPases: new insighs into their func-tions from in vivo studies. Nat. Rev. Mol. Cell Biol. 9, 690-701.
3. Kasri Nadif N. and Van Aelst L.. 2008. Rho-linked genes and neurological disorders. Pflugers Arch. 455, 787-797.
4. Vega F.M. and Ridley A.J. 2008. Rho GTPases in cancer cell biology. FEBS Lett. 582, 2093-2101.
5. Aktories K. 2011. Bacterial protein toxins that modify host regulatory GTPases. Nat. Rev. Microbiol. 9, 487-498.
6. Quah S.Y. et al. 2016. Pharmacological modulation of oncogenic Ras by natural products and their derivatives: Renewed hope in the discovery of novel anti-Ras drugs. Pharmacol. Ther. 162, 35-57.
7. Shima F. et al. 2015. Current status of the development of Ras inhibitors. J. Biochem. 158, 91-99.
8. Schmidt S. et al. 2002. Identification of the first Rho-GEF inhibitor, TRIPa, which targets the RhoA-specific GEF domain of Trio. FEBS Lett. 523, 35-42.
9. Bouquier N. et al. 2009. Aptamer-derived peptides as potent inhibitors of the oncogenic RhoGEF Tgat. Chem. Biol. 16, 391-400.
10. Shang X. et al. 2013. Small-molecule inhibitors targeting G-protein-coupled Rho guanine nucleotide exchange factors. Proc. Natl. Acad. Sci. U.S.A. 110, 3155-3160.
11. Margarit S.M. et al. 2003. Structural evidence for feedback activation by Ras·GTP of the Ras-specific nucleotide exchange factor SOS. Cell. 112, 685-695.
12. Sondermann H. et al. 2004. Structural analysis of autoinhibition in the Ras activator son of sevenless. Cell. 119, 393–405.
13. Evelyn C.R. et al. 2014. Rational design of small molecule inhibitors targeting the Ras GEF, SOS1. Chem. Biol. 21, 1618-1628.
14. Evelyn C.R. et al. 2015. Combined rational design and a high throughput screening platform for identifying chemical inhibitors of a Ras-activating enzyme. J. Biol. Chem. 290, 12879-12898.
15. Bouquier N. et al. 2009. A cell active chemical GEF inhibitor selectively targets the Trio/RhoG/Rac1 signaling pathway. Chem. Biol. 16, 657-666.
16. Razidlo G.L. et al. 2015. Targeting pancreatic cancer metastasis by inhibition of Vav1, a driver of tumor cell invasion. Cancer Res. 75, 2907-2915.
Protein Purification Amount Cat. #
SOS1 exchange domainHis tagged, human recombinant (564-1049)
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Tiam1 MBP-DHPH domainhuman recombinant (1040– 1406) 1 x 100 µg CS-TIAM1
K-Ras 4B His tagged wild-typehuman recombinant 1 x 300 µg CS-RS03
K-Ras protein His tagged G12V mutanthuman recombinant 1 x 100 µg CS-RS04
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p50RhoGAP GST Protein, GAP domain GAP for Cdc42, Rac, and Rho, >90% Pure
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