EHT 1864

EHT 1864 is a small-molecule inhibitor of Rac family small GTPases with Kd value of 40nM for Rac1 [1].

In NIH 3T3 mouse fibroblasts, EHT 1864 treatment shows an 80% reduction in PDGF-induced lamellipodia formation. EHT 1864 at concentration of 50μM can completely inhibit the complex formation between Rac1 and PAK-RBD and cause the release of nucleotide from Rac1. The reassociation of nucleotide with Rac1 induced by EDTA or the Tiam1 can also be blocked by EHT 1864. EHT 1864 is most potent to Rac1, both in the binding affinity and stimulation of nucleotide release. It binds to Rac1b, Rac2 and Rac3 with Kd values of 50nM, 60nM and 250nM, respectively. Moreover, EHT 1864 can suppress cellular transformation induced by Rac1 with an 80% inhibition of focus-forming activity [1].

References:
[1] Shutes A, Onesto C, Picard V, et al. Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases. Journal of Biological Chemistry, 2007, 282(49): 35666-35678.

EHT 1864, a small molecule inhibitor of Ras-related C3 botulinum toxin substrate 1 (Rac1), attenuates glucose-stimulated insulin secretion in pancreatic beta-cells.[Pubmed:25725286]

Cell Signal. 2015 Jun;27(6):1159-67.

Glucose-stimulated insulin secretion (GSIS) in the pancreatic beta-cells entails a variety of signaling mechanisms including activation of small GTP-binding proteins (G-proteins). Previous studies from our laboratory in human islets, rodent islets and clonal beta-cells have demonstrated that G-proteins (e.g., Arf6, Cdc42 and Rac1) play novel roles in cytoskeletal remodeling, which is a critical step in the trafficking of insulin-laden secretory granules for fusion with plasma membrane and release of insulin. To further understand regulatory roles of Rac1 in GSIS, we utilized, herein, EHT 1864, a small molecule inhibitor, which attenuates Rac1 activation by retaining the G-protein in an inert/inactive state, thereby preventing activation of its downstream effector proteins. We demonstrate that EHT 1864 markedly attenuated GSIS in INS-1 832/13 cells. In addition, EHT 1864 significantly reduced glucose-induced activation and membrane targeting of Rac1 in INS-1 832/13 cells. This Rac1 inhibitor also suppressed glucose-induced activation of ERK1/2 and p53, but not Akt. Lastly, unlike the inhibitors of protein prenylation (simvastatin), EHT 1864 did not exert any significant effects on cell morphology (cell rounding) under the conditions it attenuated Rac1-sensitive signaling steps leading to GSIS. Based on these findings, we conclude that EHT 1864 specifically inhibits glucose-induced Rac1 activation and membrane association and associated downstream signaling events culminating in inhibition of GSIS.

Characterization of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases.[Pubmed:18374160]

Methods Enzymol. 2008;439:111-29.

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that block Rho GTPase function indirectly, either by targeting enzymes involved in post-translational processing or downstream protein kinase effectors. We have reported the identification and characterization of the EHT 1864 small molecule as an inhibitor of Rac family small GTPases, placing Rac1 in an inert and inactive state and then impairing Rac1-mediated functions in vivo. Our work suggests that EHT 1864 selectively inhibits Rac1 downstream signaling and cellular transformation by a novel mechanism involving guanine nucleotide displacement. This chapter provides the details for some of the biochemical and biological methods used to characterize the mode of action of EHT 1864 on Rac1 and its impact on Rac1-dependent cellular functions.

Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases.[Pubmed:17932039]

J Biol Chem. 2007 Dec 7;282(49):35666-78.

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo.[Pubmed:16150730]

J Biol Chem. 2005 Nov 11;280(45):37516-25.

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.

EHT 1864 is an inhibitor of Rac family small GTPases. EHT 1864 directly binds and impairs the ability of this small GTPase to engage critical downstream effectors required for growth transformation. The Kd values are 40, 50, 60, and 230 nM for Rac1, Rac1b, Rac2 and Rac3, respectively. EHT 1864 also potently inhibits other Rac-dependent transformation processes, Tiam1- and Ras-mediated growth transformation. EHT 1864 prevents Aβ 40 and Aβ 42 production in vivo.

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