Rac1 (IC50=12 μM)
Rac1 (Ras-related C3 botulinum toxin substrate 1). Z62954982 is a selective Rac1 inhibitor. It functions by disrupting the interaction between Rac1 and its guanine nucleotide exchange factor (GEF) Tiam1, thereby preventing Rac1 activation (GTP loading).

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].

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In cultured smooth muscle cells (SMCs), 25 uM Z62954982 significantly reduces the ratio of Rac1-GTP to total Rac1, achieving 86% inhibitory activity. In endothelial cells (10-100 uM for 72 hours), it causes a concentration-dependent decrease in transendothelial electrical resistance, reflecting effects on cell barrier function.

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.

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In vivo, Z62954982 is administered intraperitoneally (i.p.) at 10 mg/kg every other day or 20 mg/kg daily in mouse models. It has been shown to neutralize cathode-directed growth in retinal explant cultures and is used to study the role of Rac1 in vascular barrier function and neuronal development.

A cell-free binding assay is not applicable for Rac1 inhibitors, as they function by disrupting protein-protein interactions rather than binding to an active site. Surface plasmon resonance (SPR) or fluorescence polarization assays can be used to measure the binding of Z62954982 to purified Rac1 or the Rac1/Tiam1 complex, but the functional assay is typically performed in cells.

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.

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For cellular Rac1 activation assays, cells are treated with Z62954982 and then stimulated with a Rac1 activator (e.g., PDGF-BB). After lysis, active Rac1 (GTP-bound) is pulled down using a GST-fusion protein of the Rac1 binding domain (e.g., PAK-PBD). The amount of precipitated Rac1 is then quantified by Western blot. Cell viability and barrier function are assessed by standard assays.

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].

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A standard in vivo protocol involves male mice (e.g., bcr-/-, abr-/-) exposed to hypoxia (10% O2) for 3 weeks. Z62954982 is administered intraperitoneally at 10 mg/kg every other day or 20 mg/kg daily. Endpoints include measurement of pulmonary vascular resistance, assessment of vascular leakage using Evans blue dye, and analysis of lung tissue for Rac1 activation and downstream signaling.

Not reported. Pharmacokinetic data for Z62954982 is not available in the literature. As a small molecule (MW 375.42), its absorption, distribution, metabolism, and excretion properties are not characterized, but it is typically administered by intraperitoneal injection for in vivo studies, suggesting it may have limited oral bioavailability.

Not reported. No formal toxicology data is available for this research compound. On-target toxicity could include disruption of essential Rac1 functions in various tissues, as Rac1 is critical for processes such as cell migration, adhesion, and reactive oxygen species production.

[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 involved in multiple processes of atherosclerotic plaque formation and is a potential new pharmacological target for cardiovascular disease. This article describes a pharmacophore virtual screening method followed by molecular docking calculations, which ultimately identified five new Rac1 inhibitors. These compounds were more effective than the reference compound NSC23766 in reducing intracellular Rac1-GTP levels, thus supporting the application of this method in the development of novel Rac1 inhibitors. [1]

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Impaired endothelial barrier function is a hallmark of inflammation. Rho family GTPases are crucial in regulating endothelial barrier function, but their exact role, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remains unclear. We used fusion cultures of human umbilical vein endothelial cells (HUVECs) or human dermal microvascular endothelial cells (HDMECs) to simulate the endothelial barrier. Transendothelial resistance (TER) was measured using an electrocellular-matrix impedance sensor (ECIS) to assess barrier function. The roles of Rac1 and RhoA in S1P-induced barrier enhancement were investigated. The results showed that pharmacological inhibition of Rac1 using Z62954982 failed to block S1P-induced barrier enhancement. Similarly, expression of the dominant-negative form of Rac1 or knockdown of native Rac1 using siRNA also failed to block S1P-induced TER elevation. Conversely, combined blocking of RhoA using Rhosin and Y16 inhibitors significantly reduced S1P-induced TER elevation. Real-time assessment of RhoA activation using a fluorescence resonance energy transfer (FRET) biosensor indicated that S1P primarily increased RhoA activation near cell edges and junctions. Furthermore, increased phosphorylation of myosin light chain-2 at cell edges and increased F-actin and focal adhesion proteins near intercellular junctions also promoted this process, and these changes could be blocked by pharmacological inhibition of RhoA. These results suggest that S1P can activate RhoA in the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, thereby enhancing endothelial barrier function. However, S1P-induced Rac1 activation does not appear to play a significant role in this process. [2]

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Background: Bcr and Abr are GTPase activators that specifically downregulate the activity of the small GTPase Rac in certain cell types in vivo. Rac1 is expressed in smooth muscle cells, which are a key cell type in the pathogenesis of pulmonary hypertension. The molecular mechanisms of hypoxia-associated pulmonary hypertension remain unclear. Methods/Main Findings: This study compared the occurrence of pulmonary hypertension in Bcr and Abr knockout mice with wild-type control mice after hypoxia exposure. In addition, pulmonary artery smooth muscle cells from these mice were cultured and their proliferation, p38 activation, and IL-6 production were examined under hypoxic conditions. The results showed that Bcr or Abr knockout mice exhibited increased right ventricular pressure, myocardial hypertrophy, and pulmonary vascular remodeling after hypoxia exposure. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxic conditions, reactive oxygen species (ROS) production by bcr-/- and abr-/- macrophages was increased. Consistent with the role of inflammation and oxidative stress in pulmonary hypertension-related vascular injury, Bcr and Abr knockout mice exhibited increased endothelial cell leakage after hypoxia exposure. The proliferation rate of pulmonary artery smooth muscle cells from Bcr or Abr-deficient mice under hypoxia treatment was also faster than that from wild-type mice. In addition, in the absence of Bcr or Abr, the levels of activated Rac1, phosphorylated p38, and interleukin-6 were elevated in these cells. Inhibition of Rac1 activation using the novel Rac inhibitor Z62954982 reduced the proliferation, p38 phosphorylation, and IL-6 levels of pulmonary artery smooth muscle cells under hypoxia conditions. Conclusion: Bcr and Abr play a key role in downregulating hypoxia-induced pulmonary hypertension by inhibiting Rac1 activity, thereby reducing oxidative stress generated by leukocytes and p38 phosphorylation, IL-6 production, and proliferation of pulmonary artery smooth muscle cells. [3]

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Z62954982 is a second-generation Rac1 inhibitor that is more potent and selective than the widely used NSC23766. Its development has been crucial for studying the specific role of Rac1 in diverse biological processes, including cardiovascular disease (e.g., pulmonary hypertension), neurobiology (neuronal growth and branching), and cancer (cell migration and metastasis). Its high selectivity for Rac1 over related GTPases (Cdc42, RhoA) makes it a superior chemical tool for deconvoluting the specific contributions of Rac1 in complex signaling networks.

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

CC1=C(C=C(C=C1)NC(=O)C2=CC(=CC=C2)OCC3=C(ON=C3C)C)S(=O)(=O)N

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.)