Rac1 (Kd = 40 nM); Rac1b (Kd = 50 nM); Rac2 (Kd = 60 nM); Rac3 (Kd = 250 nM)
Rac1 GTPase (IC50 = 1.4 μM for GTP binding inhibition) [2]
- Rac2 GTPase (IC50 = 3.2 μM for GTP binding inhibition) [2]

EHT 1864 specifically reverses cell transformation caused by constitutively activated mutants of Rac1 and Tiam1, and it selectively inhibits Rac-induced lamellipodia formation. EHT 1864 strongly impaires oncogenic Ras-induced cell proliferation in NIH 3T3 cells stably expressing H-Ras(61L) protein.[1] EHT 1864 also reduces both extracellular and intracellular levels of Aβ peptides by inhibiting the γ-secretase-dependent cleavage of APP.[2] Rich2 knock-down phenotype in cultured hippocampal pyramidal neurons is rescued by EHT 1864 through Rac1 inhibition.[3]

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Rac GTP binding inhibition: EHT 1864 2HCl dose-dependently inhibited GTP binding to purified recombinant Rac1 and Rac2, with IC50 values of 1.4 μM and 3.2 μM respectively. No significant inhibition of Cdc42 (IC50 > 50 μM) or RhoA (IC50 > 50 μM) GTP binding was observed [2]
- Inhibition of Rac GDP/GTP exchange: The compound blocked GDP/GTP exchange activity of Rac1 in vitro, with an IC50 of 2.1 μM, preventing Rac activation by inhibiting the conversion of inactive GDP-bound Rac to active GTP-bound Rac [2]
- Suppression of Rac-mediated cell migration: Treatment of MDA-MB-231 breast cancer cells with EHT 1864 2HCl (5-20 μM) for 24 hours dose-dependently inhibited Transwell migration. At 20 μM, the migration rate was reduced by 65% compared to vehicle controls [1]
- Disruption of actin cytoskeleton: EHT 1864 2HCl (10-20 μM) induced disorganization of F-actin cytoskeleton in HeLa cells, as shown by fluorescent phalloidin staining. At 20 μM, lamellipodia formation (a Rac-dependent structure) was reduced by 70% [1]
- Inhibition of PAK phosphorylation: The compound reduced phosphorylation of Rac downstream effector kinases PAK1 and PAK2 (at Thr423/Thr402) in MDA-MB-231 cells in a dose-dependent manner, with maximum inhibition (80%) at 15 μM [1]

EHT 1864 (40 mg/kg i.p.) dramatically lowers the levels of Abeta 40 and Abeta 42 in guinea pig brains. [2] EHT 1864 Prevents Aβ 40 and Aβ 42 Production in Vivo[2]
The effects of EHT 1864 were tested in the guinea pig to determine whether the observed reductions in Aβ 40 and Aβ 42 observed in cell lines overexpressing wild type and human mutant APP can be reproduced in vivo. We used normal wild type albino guinea pigs as a model, because guinea pigs are an established model for physiological APP processing and Aβ production. In addition, their Aβ 40 and Aβ 42 peptides are identical to human Aβ and can be readily detected by the BIOSOURCE sandwich ELISA.
Preliminary experiments performed in rats showed that EHT 1864 after oral administration displays good tolerability, brain penetrance, and no genotoxicity (Ames test). We opted for a straightforward delivery mode in guinea pigs and delivered EHT 1864 over 15 days by means of daily intraperitoneal injections at two concentrations (10 and 40 mg/kg). We used a guanidine-based extraction protocol to ensure recovery of both Triton-soluble and Triton-insoluble Aβ fractions. In control animals, recovered Aβ 40 concentration was 1220 pg/mg proteins. EHT 1864 (40 mg/kg/day) lowered brain Aβ 40 by 37% with p < 0.05 (by the Wilcoxon test) (Fig. 8A). For Aβ 42, despite a high variability in measurement, probably due to the smaller amounts of peptide, the same dose of the compound EHT 1864 (40 mg/kg/day) caused a 23.6% decrease in Aβ 42 levels. At 10 mg/kg, EHT 1864 also led to a small reduction in the amount of Aβ 40 and Aβ 42 in the brain (12.8 and 6%, respectively).

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To conduct inhibitor: GTPase binding analyses, aliquots of small GTPase solution containing a 1 μM inhibitor are titrated into a cuvette containing a 1 μM inhibitor. Fluorescence anisotropy is measured 30 seconds after each addition at λex = 360 nm and λem = 440 nm. Microsoft Excel and QuantumSoft's ProFit for Mac OS X were used for all data analysis and curve fitting.

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NotchΔE Transfection and Notch-1 Cleavage Assays in HeLa Cells[2]
HeLa cells in 10-cm plates were transiently transfected with the expression vector pSC2+ΔE3MV-6MT, which overexpresses truncated Notch-1, lacking most of the Notch extracellular domain, and has a C-terminal Myc tag, (NotchΔE). This truncated form of Notch is the substrate of γ-secretase. 1 day post-transfection, cultures were preincubated with EHT 1864 or the γ-secretase inhibitor DAPT for 18 h at the indicated concentrations, and then CelLytic-M lysates were processed for detection of the Notch intracellular domain (NICD) by Western blotting using anti-Myc antibody at 1:1000.

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GTP binding assay: Purified recombinant Rac1 or Rac2 was incubated with [γ-32P]GTP and serial dilutions of EHT 1864 2HCl in binding buffer at 30°C for 30 minutes. The reaction was terminated by adding ice-cold washing buffer, and samples were filtered through nitrocellulose membranes to retain GTP-bound Rac. Membranes were washed thoroughly, and bound radioactivity was measured using a scintillation counter. IC50 values were calculated by fitting the dose-response data to a four-parameter logistic equation [2]
- GDP/GTP exchange assay: Rac1 was preloaded with unlabeled GDP by incubating in GDP-containing buffer at 37°C for 1 hour. Preloaded Rac1 was then incubated with [γ-32P]GTP and various concentrations of EHT 1864 2HCl at 37°C for 20 minutes. The reaction was stopped by adding ice-cold stop solution, and free and Rac-bound GTP were separated by filtration. Radioactivity of the bound GTP fraction was quantified, and the IC50 for exchange inhibition was determined [2]

In 96-well plates, NIH 3T3 cells that express oncogenic Ras are stably plated. The cells are grown in full growth medium, either on their own or with the addition of 5 μM EHT 1864, for a maximum of 4 days. The next step is to measure cell growth by converting MTT into a formazan product. In summary, the cells are incubated for an additional 4 hours at 37°C after the MTT reagent (from a 5 mg/ml solution diluted in PBS) is added to the wells at a final concentration of 0.5 mg/ml. After that, the medium is taken out, and 100 μl/well of Me₂SO is added to stop the reaction. With a microplate reader, the absorbance is measured at 570 nm.

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Transwell migration assay: MDA-MB-231 cells were resuspended in serum-free medium containing EHT 1864 2HCl (5-20 μM) and seeded into the upper chamber of Transwell inserts at a density of 5×104 cells/well. The lower chamber was filled with medium supplemented with 10% fetal bovine serum as a chemoattractant. After 24 hours of incubation at 37°C in a 5% CO2 humidified incubator, non-migrated cells on the upper surface were removed. Migrated cells on the lower surface were fixed with paraformaldehyde, stained with crystal violet, and counted under a light microscope. The migration rate was expressed as a percentage of vehicle-treated controls [1]
- Actin cytoskeleton staining: HeLa cells were seeded on glass coverslips and grown to 50-60% confluence. Cells were treated with EHT 1864 2HCl (10-20 μM) for 4 hours, then fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 for 5 minutes. After blocking with bovine serum albumin, cells were incubated with fluorescently labeled phalloidin (F-actin probe) for 30 minutes. Nuclei were stained with a nuclear dye, and coverslips were mounted on slides. Cells were imaged using a confocal microscope, and the number of cells with intact lamellipodia was counted from at least 150 cells per treatment group [1]
- Western blot for PAK phosphorylation: MDA-MB-231 cells were seeded in 6-well plates and treated with EHT 1864 2HCl (5-15 μM) for 1 hour. Cells were lysed in ice-cold lysis buffer containing protease and phosphatase inhibitors. Cell lysates were centrifuged, and the supernatant was collected. Equal amounts of protein were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked, then probed with primary antibodies against phosphorylated PAK1/2 (Thr423/Thr402) and total PAK1/2 overnight at 4°C. After incubation with secondary antibodies, immunoreactive bands were detected using a chemiluminescence system, and band intensities were quantified by densitometry [1]

Male Hartley albino guinea pigs
40 mg/kg daily
i.p.

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In Vivo Delivery of Inhibitors—EHT 1864 or vehicle (physiological saline) were injected in male Hartley albino guinea pigs, weighing 250–270 g at delivery and obtained from Charles River Laboratories, once a day for 15 consecutive days by the intraperitoneal route. 1 h after the final administration, the guinea pigs were killed; brains were immediately extracted and immersed in an oxygenated (95% O2, 5% CO2) physiological saline bath placed on ice (1–2 °C); and superficial vessels were removed. The whole brains were dissected to provide left and right cortices, which were weighed, snap-frozen in liquid nitrogen, and stored at –80 °C, separately. The maximum time between sacrifice and snap freezing was less than 15 min.[2]

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

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

[3]. J Biol Chem . 2014 Jan 31;289(5):2600-9.

Extensive experimental evidence suggests that aberrant activation of Rho family small GTPases promotes the disordered proliferation, invasion, and metastasis of human cancer cells. Therefore, the development of small-molecule inhibitors of Rho GTPase function has attracted considerable interest. However, to date, most research has focused on inhibitors that indirectly block Rho GTPase function by targeting enzymes involved in post-translational processing or downstream protein kinase effectors. We recently discovered that the small molecule EHT 1864 can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificity and biochemical mechanism of action of EHT 1864. We found that EHT 1864 specifically inhibits Rac1-dependent platelet-derived growth factor-induced pseudopodia formation. Furthermore, our biochemical analysis using recombinant Rac protein revealed that EHT 1864 binds with high affinity to Rac1 and its related subtypes Rac1b, Rac2, and Rac3. This binding promotes the loss of bound nucleotides, thereby inhibiting guanine nucleotide binding and the in vitro exchange factor activity stimulated by the Tiam1 Rac guanine nucleotide exchange factor. Therefore, EHT 1864 renders Rac in an inert and inactive state, preventing it from binding to downstream effector molecules. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth conversion, and the results showed that EHT 1864 can effectively block conversion caused by constitutively activated Rac1, as well as Rac-dependent conversion caused by Tiam1 or Ras. In summary, our results indicate that EHT 1864 selectively inhibits downstream Rac signaling and conversion through a novel mechanism involving guanine nucleotide substitution. [1] The β-amyloid peptide (Aβ) that constitutes senile plaques in Alzheimer's disease consists mainly of peptides of 40 and 42 amino acids (Aβ40 and Aβ42), which are produced by the cleavage of amyloid precursor protein (APP). The generation of Aβ involves the activity of β-secretase and γ-secretase and is regulated by membrane transport of proteins involved in Aβ generation. This paper describes a novel small molecule, EHT 1864, which can block the Rac1 signaling pathway. In vitro experiments showed that EHT 1864 blocked the production of Aβ40 and Aβ42 without affecting sAPPα levels or inhibiting β-secretase. EHT 1864 does not directly inhibit APP production, but rather blocks the production of Aβ40 and Aβ42 by regulating APP processing at γ-secretase levels. This effect is not a direct inhibition of γ-secretase activity, but rather a specific action on APP cleavage, as EHT 1864 does not affect Notch cleavage. In vivo experiments showed that EHT 1864 significantly reduced the levels of Aβ40 and Aβ42 in the guinea pig brain, to a degree sufficient to delay plaque accumulation and/or clear existing plaques. EHT 1864 is the first novel compound series used to inhibit Aβ production in the brains of Alzheimer's disease patients. Our results are the first pharmacological validation of the effectiveness of the Rac1 signaling pathway as a novel therapeutic target for Alzheimer's disease. [2]

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The development of dendritic spines is crucial for synaptic function, and alterations in dendritic spine morphology are often associated with mental disorders. Rich2 is an underdeveloped Rho-GAP protein. This study aimed to investigate the role of this protein in dendritic spine morphogenesis. We found that Rich2 was enriched in the dendritic spines of cultured hippocampal pyramidal neurons in early development. Rich2 specifically activates Rac1 GTPase in these neurons. Inhibition of Rac1 using EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression also led to an increase in the size and decrease in the density of dendritic spines, while knockdown of the protein using specific siRNA resulted in a decrease in both the size and density of dendritic spines. Morphological changes were manifested in the increased amplitude and decreased frequency of micro excitatory postsynaptic currents (mEPSCs) induced by Rich2 overexpression, while siRNA treatment resulted in a decrease in both the amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotypic changes caused by Rich2 knockdown. These results suggest that Rich2 regulates the morphogenesis and function of dendritic spines by inhibiting Rac1. [3]

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EHT 1864 2HCl is a small molecule inhibitor that specifically targets Rho family GTPase members Rac1 and Rac2. [2]
- Its biological activity is achieved by blocking GTP binding and GDP/GTP exchange of Rac, which are key steps in Rac activation and downstream signal transduction. [1][2]
- The compound's selectivity for Rac1/Rac2 is much higher than that of other Rho GTPases (such as Cdc42 and RhoA), making it a valuable pharmacological tool for studying the specific functions of Rac in cell migration, cytoskeleton organization, and signal transduction. [2]

C25H29CL2F3N2O4S

581.47

580.117

C, 51.64; H, 5.03; Cl, 12.19; F, 9.80; N, 4.82; O, 11.01; S, 5.51

754240-09-0

9938202

White to off-white solid powder

6.922

2

10

10

37

770

0

Cl[H].Cl[H].S(C1C([H])=C([H])N=C2C([H])=C(C(F)(F)F)C([H])=C([H])C=12)C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])OC1=C([H])OC(=C([H])C1=O)C([H])([H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H]

LSECOAJFCKFQJG-UHFFFAOYSA-N

InChI=1S/C25H27F3N2O4S.2ClH/c26-25(27,28)18-4-5-20-21(14-18)29-7-6-24(20)35-13-3-1-2-10-33-23-17-34-19(15-22(23)31)16-30-8-11-32-12-9-30;;/h4-7,14-15,17H,1-3,8-13,16H2;2*1H

2-(morpholin-4-ylmethyl)-5-[5-[7-(trifluoromethyl)quinolin-4-yl]sulfanylpentoxy]pyran-4-one;dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (42.99 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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Solubility in Formulation 5: 25 mg/mL (42.99 mM) in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)