Small molecule inhibitors of GEF-mediated GTPase signaling

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.

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 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.


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.

Table 1. Drugs directly targeting GEFs
GEFDrugBinding Affinity (Kd)Ref #
TrioCTripαNR*; IC50 of 4 µMz in TrioC-mediated RhoA exchange activity8
TgatTripE32GNR*; apparent inhibition constant (Kiapp) of 2.4-12.4 µM in Tgat-mediated RhoA exchange assay9
LARGY1680 nM10
SOS1NSC-6584977 µM13
SOS1UC-7735873.4 µM14
SOS1UC-85799314.7 µM14
TrioNITX3NR*; IC50 of 76 µM in TrioN-mediated RhoG exhange assay15
  *NR, not reported

Related Products & Resources

Custom GEF Protein Production

SOS1 exchange domain: His tagged, human recombinant (564-1049) (Cat. # CS-SOS1)

Tiam1 MBP-DHPH domain: human recombinant (1040– 1406) (Cat. # CS-TIAM1)

K-Ras 4B His tagged wild-type: human recombinant (Cat. # CS-RS03)

K-Ras protein His tagged G12V mutant: human recombinant (Cat. # CS-RS04)

G-protein Effector Proteins and Kits

RhoGEF Exchange Assay Biochem Kit™ (Cat. # BK100)

Dbs protein GEF domain: His tagged: human recombinant (Cat. # GE01)

RhoA protein: His tagged: human wild type (Cat. # RH01)

p50RhoGAP GST Protein, GAP domain: GAP for Cdc42, Rac, and Rho, >90% Pure (Cat. # RC01)

RhoGDI GST Protein: Inhibitor of Cdc42, Rac, and Rho, >90% Pure (Cat. # CD01)

G-Switch Activators and Inhibitors

Rho Activator I: Cell Permeable: Indirect (Cat. # CN01)

Rac/Cdc42 Activator II: Receptor Mediated: Indirect (Cat. # CN02)

Rho Activator II: Cell Permeable: Direct (Cat. # CN03)

Rho/Rac/Cdc42 Activator I: Cell Permeable: Direct (Cat. # CN04)

C3 Transferase Protein: Pinocytosis: Direct (Cat. # CT03)

Rho Inhibitor I: Cell Permeable: Direct (Cat. # CT04)


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