ITX3

ITX3 is a specific and nontoxic inhibitor of the TrioN (N-terminal GEF domain of the multidomain Trio protein) with IC50 of 76 uM; inhibits TrioN-stimulated RhoG exchange in vitro. IC50 value: 76 uM [1] Target: TrioN inhibitor In transfected mammalian cells, ITX3 blocked TrioN-mediated dorsal membrane ruffling and Rac1 activation while having no effect on GEF337-, Tiam1-, or Vav2-mediated RhoA or Rac1 activation. ITX3 specifically inhibited endogenous TrioN activity, as evidenced by its ability to inhibit neurite outgrowth in nerve growth factor (NGF)–stimulated PC12 cells or C2C12 differentiation into myotubes [1]. ITX3 repressed the Rac1 activity and dose dependently up-regulated the E-cadherin protein level in the Tara-KD cells [2].

References:
[1]. Bouquier N, et al. A cell active chemical GEF inhibitor selectively targets the Trio/RhoG/Rac1 signaling pathway. Chem Biol. 2009 Jun 26;16(6):657-66. [2]. Yano T, et al. Tara up-regulates E-cadherin transcription by binding to the Trio RhoGEF and inhibiting Rac signaling. J Cell Biol. 2011 Apr 18;193(2):319-32.

Supervillin binds the Rac/Rho-GEF Trio and increases Trio-mediated Rac1 activation.[Pubmed:25655724]

Cytoskeleton (Hoboken). 2015 Jan;72(1):47-64.

We investigated cross-talk between the membrane-associated, myosin II-regulatory protein supervillin and the actin-regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1-GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLa cells with normal levels of the Rac1-activating protein Trio. No reduction in Rac1-GTP loading was observed when supervillin levels were reduced in Trio-depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio-mediated Rac1 guanine nucleotide exchange activity with ITX3 partially blocked the SV4-mediated increase in Rac1-GTP. Both SV4 and SV1 co-localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4-7: SV1-171, which contains N-terminal residues found in both SV1 and SV4 and the SV4-specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4-E345; SV4 amino acids 276-669). In addition, SV4-E345 interacted with the homologous sequence in rat kalirin (repeats 4-7, amino acids 531-1101). Overexpressed SV1-174 and SV4-E345 affected Rac1-GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771-1057, which contain both supervillin-interaction sites, exerted a dominant-negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension.

Kalirin-9 and Kalirin-12 Play Essential Roles in Dendritic Outgrowth and Branching.[Pubmed:25146373]

Cereb Cortex. 2015 Oct;25(10):3487-501.

Proteins derived from the Kalrn gene, encoding 2 Rho guanine nucleotide exchange factor (GEF) domains, affect dendritic and axonal morphogenesis. The roles of endogenous Kalirin-9 (Kal9) and Kalirin-12 (Kal12), the Kalrn isoforms expressed before synaptogenesis, have not been studied in neurite growth and maturation during early development. The Caenorhabditis elegans and Drosophila melanogaster orthologues of Kalrn encode proteins equivalent to Kal9 but, lacking a kinase domain, neither organism expresses a protein equivalent to Kal12. Both in vivo and in vitro analyses of cortical neurons from total Kalrn knockout mice, lacking all major Kalirin isoforms, revealed a simplified dendritic arbor and reduced neurite length. Using isoform-specific shRNAs to reduce Kal9 or Kal12 expression in hippocampal cultures resulted in stunted dendritic outgrowth and branching in vitro, without affecting axonal polarity. Exposing hippocampal cultures to inhibitors of the first GEF domain of Kalirin (ITX3, Z62954982) blunted neurite outgrowth and branching, confirming its essential role, without altering the morphology of neurons not expressing Kalrn. In addition, exogenous expression of the active kinase domain unique to Kal12 increased neurite number and length, whereas that of the inactive kinase domain decreased neurite growth. Our results demonstrate that both endogenous Kal9 and endogenous Kal12 contribute to dendritic maturation in early development.

Tara up-regulates E-cadherin transcription by binding to the Trio RhoGEF and inhibiting Rac signaling.[Pubmed:21482718]

J Cell Biol. 2011 Apr 18;193(2):319-32.

The spatiotemporal regulation of E-cadherin expression is important during body plan development and carcinogenesis. We found that Tara (Trio-associated repeat on actin) is enriched in cadherin-based adherens junctions (AJs), and its knockdown in MDCK cells (Tara-KD cells) significantly decreases the expression of E-cadherin. Tara-KD activates Rac1 through the Trio RhoGEF, which binds to E-cadherin and subsequently increases the phosphorylation of p38 and Tbx3, a transcriptional E-cadherin repressor. Accordingly, the decrease in E-cadherin expression is abrogated by ITX3 and SB203580 (specific inhibitors of Trio RhoGEF and p38MAPK, respectively), and by dephosphomimetic Tbx3. Despite the decreased E-cadherin expression, the Tara-KD cells do not undergo an epithelial-mesenchymal transition and remain as an epithelial cell sheet, presumably due to the concomitant up-regulation of cadherin-6. Tara-KD reduces the actin-belt density in the circumferential ring, and the cells form flattened cysts, suggesting that Tara functions to modulate epithelial cell sheet formation and integrity by up-regulating E-cadherin transcription.

A cell active chemical GEF inhibitor selectively targets the Trio/RhoG/Rac1 signaling pathway.[Pubmed:19549603]

Chem Biol. 2009 Jun 26;16(6):657-66.

RhoGEFs (guanine nucleotide exchange factors of the Rho GTPase family) are upstream regulators of cell adhesion and migration pathways, thus representing attractive yet relatively unexplored targets for the development of anti-invasive drugs. We screened for chemical inhibitors of TrioN, the N-terminal GEF domain of the multidomain Trio protein, and identified ITX3 as a nontoxic inhibitor. In transfected mammalian cells, ITX3 blocked TrioN-mediated dorsal membrane ruffling and Rac1 activation while having no effect on GEF337-, Tiam1-, or Vav2-mediated RhoA or Rac1 activation. ITX3 specifically inhibited endogenous TrioN activity, as evidenced by its ability to inhibit neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells or C2C12 differentiation into myotubes. This study introduces a selective cell active inhibitor of the Trio/RhoG/Rac1 pathway and validates RhoGEFs as druggable targets.

ITX3 is a specific and nontoxic inhibitor of the TrioN (N-terminal GEF domain of the multidomain Trio protein) with IC50 of 76 uM; inhibits TrioN-stimulated RhoG exchange in vitro.

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