Rac1 (Kd = 40 nM); Rac1b (Kd = 50 nM); Rac2 (Kd = 60 nM); Rac3 (Kd = 250 nM)

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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EHT 1864 (40 mg/kg i.p.) dramatically lowers the levels of Abeta 40 and Abeta 42 in guinea pig brains. [2]

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.

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.

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.

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.[1]

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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.[2]

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Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.[3]

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.)