Rac1 (IC50=12 μM)

Z62954982 (5-100 μM; 4-hour duration) exhibits a concentration-dependent reduction in intracellular Rac1-GTP levels, demonstrating the most inhibitory effect on Human SMCs with an IC50 of 12.2 μM[1]. In cultured SMCs, Z62954982 (25 μM; 4 hours) has the most inhibitory effect (86.0%) and dramatically lowers the ratio Rac1-GTP/Rac1[1]. In both HDMEC and HUVEC monolayers, Z62954982 (10-100 μM; 72 hours) results in a concentration-dependent reduction in transendothelial electrical resistance (TER)[2].

Z62954982 (intraperitoneal injection; 10 mg/kg every other day or 20 mg/kg daily; 3 weeks) has been shown to reduce p38 phosphorylation and released IL-6 in PASMCs in response to hypoxia in both bcr-/- and abr-/-mice[3]. It also shows no overt toxicity.

In vitro Cell Proliferation Assay and Flow Cytometric Cell Cycle Analysis[3]
PASMCs (3rd passage) from male bcr−/−, abr−/− and wt FVB/J mice were seeded in 6-well plates (1×104 cells/well) and cultured in serum-free medium for 24 hrs. Effective cell synchronization was confirmed by flow cytometry analysis. Triplicate wells of cells were then exposed to normoxia or hypoxia (5% O2), with or without 25 µmol/L of the Rac inhibitor Z62954982 (ZINC08010136). Z62954982 was made as a concentrated stock solution in DMSO (dimethyl sulfoxide). The final concentration of DMSO in the culture medium was 0.1%; control wells were treated with 0.1% DMSO only. Z62954982 did not have cytotoxic effects, since viability of the drug-treated PASMCs was around 95%, comparable with that of PASMCs treated with 0.1% DMSO. Medium was refreshed every 2 days. After 5 days, the supernatants were collected for measurement of IL-6. Cells were trypsinized and counted. PASMCs were then fixed with 70% ethanol and incubated with staining solution (20 µg/ml propidium iodide, 0.2 mg/ml DNase-free RNase, 0.01% Triton-100 in PBS). Cell cycle was analyzed using a flow cytometer. The proliferation index of the PASMCs was calculated as: (S+G2/M)/(G0/G1+ S + G2/M) ×100%. G0/G1, S, G2/M represents the percentage of the cells in G0/G1, S, G2/M phase, respectively.

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Quantitative Real-time PCR[3]
For the isolation of RNA and protein, 3rd passage PASMCs from Bcr−/−, Abr−/− and WT mice were plated in 10 cm culture dishes (3×104/dish). Cells were treated either with DMSO or with Z62954982 (25 µmol/L, dissolved in DMSO) in an incubator with normoxia or hypoxia (5% O2) for 5 days. Medium was refreshed every other day. Fresh Z62954982 was added along with the change of medium for the Z62954982 treatment cells. On day 5, cells were harvested, washed 3 times with PBS, harvested by scraping and processed for RNA (RNA mini kit) or for protein isolation in MLB (see below). 2 µg of RNA was converted to cDNA.

Animal/Disease Models: Male bcr-/-, abr-/- and wt mice (8 to 10weeks old littermates) are exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 weeks[3]
Doses: 10 mg/kg or 20 mg/kg
Route of Administration: intraperitoneal (ip) injection; 10 mg/kg every other day or 20 mg/kg daily; 3 weeks
Experimental Results: Promoted phosphorylation of p38 MAPK and increased IL-6 in Hypoxia in mice.

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Male bcr−/−, abr−/− and wt mice (8 to 10-week-old littermates) were exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 weeks. For in vivo Rac1 inhibitor treatment experiments, Z62954982 was administered intraperitoneally (i.p.) at 10 mg/kg every other day or 20 mg/kg daily. Control mice were injected with the same volume of vehicle (DMSO : corn oil  = 1∶ 9). During and at the end of the treatment, we monitored normoxia-exposed vehicle and Z62954982-treated wild type mice for possible obvious signs of toxicity of the drug. However there was no evidence for toxicity. Numbers of myeloid cells in the bone marrows were comparable and kidney (not shown) and liver appeared normal in both groups[3].

[1]. Virtual Screening Approach for the Identification of New Rac1 Inhibitors. J Med Chem. 2009 Jul 23;52(14):4087-90.

[2]. Activation of RhoA, but Not Rac1, Mediates Early Stages of S1P-Induced Endothelial Barrier Enhancement. PLoS One. 2016 May 17;11(5):e0155490.

[3]. Lack of BCR and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice. PLoS One. 2012;7(11):e49756.

Rac1 protein is implicated in several events of atherosclerotic plaque development and represents a new potential pharmacological target for cardiovascular diseases. In this paper we describe a pharmacophore virtual screening followed by molecular docking calculations leading to the identification of five new Rac1 inhibitors. These compounds were shown to be more effective than the reference compound NSC23766 in reducing the intracellular levels of Rac1-GTP, thus supporting this approach for the development of new Rac1 inhibitors.[1]

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Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell-substrate impedance sensor (ECIS). The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process.[2]

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Background: Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined. Methodology/principal findings: Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr-/- and abr-/- macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia. Conclusions: Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.[3]

C20H21N3O5S

415.46

415.12

1090893-12-1

9141046

White to off-white solid powder

1.4±0.1 g/cm3

1.618

1.49

2

7

6

29

668

0

OZZQJOAJXMUXCO-UHFFFAOYSA-N

InChI=1S/C20H21N3O5S/c1-12-7-8-16(10-19(12)29(21,25)26)22-20(24)15-5-4-6-17(9-15)27-11-18-13(2)23-28-14(18)3/h4-10H,11H2,1-3H3,(H,22,24)(H2,21,25,26)

3-[(3,5-dimethyl-1,2-oxazol-4-yl)methoxy]-N-(4-methyl-3-sulfamoylphenyl)benzamide

Rac1 Inhibitor II; 1090893-12-1; Z62954982; 3-[(3,5-dimethyl-1,2-oxazol-4-yl)methoxy]-N-(4-methyl-3-sulfamoylphenyl)benzamide; SCHEMBL15239985;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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