Rho family proteins (20 human members) comprise a major branch of the Ras superfamily of small GTPases. Like Ras, Rho GTPases function as GDP/GTP-regulated binary on-off switches. Rho GTPases are regulators of a diverse variety of cellular processes that include regulation of cell proliferation, actin cytoskeleton reorganization, and gene expression. 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. EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms with KD of 40 nM, 50 nM, 60 nM and 250 nM, respectively. EHT 1864 can inhibit a variety of downstream signaling activities activated by ectopic expression of constitutively-activated Rac1(G12V) and decreased Rac1 association with the isolated Rho GTPase binding domain (RBD) of the p21-activated serine/threonine kinase effector PAK1in vivo. NIH 3T3 cells were incubated for 16 h in serum-free growth medium, either alone, or supplemented with 5 μM EHT 1864 for the last 4 h. It was found that EHT 1864-treated cells showed an ∼80% reduction in PDGF stimulation of lamellipodia which demonstrated that EHT 1864 is a potent and specific inhibitor of PDGF-induced lamellipodia formation[3]. In vitro study, after pre-incubation at varying concentrations of EHT 1864 (0–10 μM) for 1 h, INS-1 832/13 cells were further incubated in the presence of low (2.5 mM) or varying concentrations of glucose (2.5-20 mM) or KCl (40 mM) for 30 min at 37 °C, the result showed that EHT 1864 inhibited glucose-induced Rac1 activation in INS-1 832/13 cells, which resulted in inhibition of GSIS (Glucose-stimulated insulin secretion)[4]. C57BL/6 mice were injected EHT-1864 once a day for 7 days. It was determined that EHT-1864 efficiently inhibits the expression of Rac1, Rac2, and Rac3. And colon length was observed recovered after EHT-1864 treatment for 5 days. Furthermore, less inflammation-associated rectal bleeding and soft stool were observed in the EHT-1864 treatment group, which demonstrate the preventive role of EHT-1864 on acute colitis in mice[5].
作用机制
EHT 1864 binds to Rac1 with high affinity, and retains Rac1 in an inert and inactive state by displacement of pre-bound guanine nucleotide (GDP/GTP).
EHT 1864 细胞实验
Cell Line
Concentration
Treated Time
Description
References
Platelets
100 µM
10 minutes
Inhibition of Rac1 activation, reduced α-granule and dense granule release
To investigate the effect of Rac1 GTPase inhibition on Bmp4 expression. Results showed that Rac1 inhibition significantly increased Bmp4 expression.
Cell Rep. 2021 Oct 5;37(1):109789.
Dendritic cells (DCs)
50 µM
20 hours
To investigate the effect of Rac1 GTPase inhibition on Il12p40 expression. Results showed that Rac1 inhibition significantly increased Il12p40 expression.
Cell Rep. 2021 Oct 5;37(1):109789.
INS-1 832/13 cells
10 µM
24 hours
Inhibition of HG-induced p53 activation, suggesting that Rac1 activation is requisite for HG-induced activation of p53 in INS-1 832/13 cells.
Apoptosis. 2017 May;22(5):597-607.
Rat islets
10 µM
24 hours
Inhibition of HG-induced p53 phosphorylation, indicating the regulatory role of Rac1 in p53 activation.
Apoptosis. 2017 May;22(5):597-607.
WPMY-1 cells
25 µM
24 hours
To assess the effect of EHT 1864 on the survival of WPMY-1 cells, results showed 81% cell survival after 24 hours.
Br J Pharmacol. 2015 Jun;172(11):2905-17.
WPMY-1 cells
100 µM
24 hours
To assess the effect of EHT 1864 on the survival of WPMY-1 cells, results showed 64% cell survival after 24 hours.
Br J Pharmacol. 2015 Jun;172(11):2905-17.
INS-1 832/13 cells
10 µM
24 hours
Inhibits HG-induced p38MAPK activation, suggesting a regulatory role for Rac1 in the cascade of events leading to HG-induced p38MAPK activation.
Biochem Pharmacol. 2015 Jun 15;95(4):301-10.
Human microvascular endothelial cells
1 µM
30 minutes
EHT1864 did not interfere with Ang II and ET-1-induced NADPH oxidase activation, but decreased Ang II and ET-1-stimulated Rac-1 translocation, as evidenced by decreased membrane expression of Rac-1.
Circ Res. 2010 Apr 30;106(8):1363-73.
MCF-7 cells
2 µM, 5 µM, 10 µM
5 days
EHT 1864 was more effective than 4-hydroxytamoxifen (OHT) in blocking E2-stimulated MCF-7 cell growth; 5 or 10 μM EHT 1864 prevented all E2-stimulated cell growth
Inhibition of Rac activity to rescue the enhanced migration ability in DP knockdown cells
J Invest Dermatol. 2019 Jun;139(6):1227-1236.
EHT 1864 动物实验
Species
Animal Model
Administration
Dosage
Frequency
Description
References
Mice
C57BL/6 WT or Nod2−/− mice
Intraperitoneal injection
40 mg/kg
Injected on days 3, 5, and 7, continued until day 14
To investigate the effect of Rac1 GTPase inhibition on thymic regeneration and peripheral T cell recovery. Results showed that EHT1864 significantly increased thymic cellularity and promoted the recovery of peripheral CD4+ naive T cells.
Cell Rep. 2021 Oct 5;37(1):109789.
BALB/c mice
Subchronic mouse lung tissue damage model
Intraperitoneal injection
5 mg/kg
Three times per week for six weeks
EHT1864 attenuated the IR-induced increase in breathing frequency and reduced the percentage of γH2AX and 53BP1-positive cells. This indicates that inhibition of Rac1 signaling lowers IR-induced residual DNA damage by promoting DNA repair. Moreover, EHT1864 protected lung tissue from IR-triggered apoptosis and mitigated the IR-stimulated increase in regenerative proliferation.
Cell Death Dis. 2017 Aug 10;8(8):e2978
Mice
S100β-v-erbB/p53−/− tumor allograft model
Intraperitoneal injection
80 mg/kg
Once a day for 10 days
EHT-1864 significantly reduced VEGF secretion, slowed tumor growth, and increased the survival of mice allografted with S100β-v-erbB/p53?/? glioma stem-like cells.
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