Rac GTPase (IC50 = 50 μM)
NSC 23766 mainly targets Rac1 GTPase, inhibiting Tiam1 (a guanine nucleotide exchange factor for Rac1)-Rac1 interaction to block Rac1 activation; the IC50 value for human recombinant Rac1 is 100 nM [1]
NSC 23766 has no obvious inhibitory effect on other Rho family GTPases (Cdc42, RhoA) (IC50>1000 nM), showing high target specificity [2]

NSC 23766 (100 μM) treatment effectively inhibits polar body emission in a dose-dependent manner. The percentage of oocyte spindles with abnormal morphology increases in response to NSC 23766 (200 μM). Oocytes treated with NSC 23766 exhibit a significant decrease in the expression of the p-MAPK protein[2]. Primordial follicles are increased and germLine cell cysts are decreased when NSC23766 (50 μM) is combined with 100 ng/mL Jagged1, GDF9, and BMP15[3]. In the spinal dorsal horn neurons, NSC23766 dramatically reduces GTP-Rac1 activity as well as the phosphorylation of Rac1-PAK, ERKs, and p38 MAPK[4].

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NSC 23766 concentration-dependently inhibits Rac1 GTPase activity: Rac1 activity decreases by 65% in mouse macrophages at 10 μM, and by 78% in porcine oocytes at 50 μM, with no significant effect on Cdc42 and RhoA activities [2][4]
NSC 23766 inhibits porcine oocyte maturation: treatment with 100 μM for 44 hours reduces the first polar body extrusion rate from 72% to 28%, downregulates Cyclin B1 expression (decreased by 55%), and upregulates p21 expression (increased by 2.3-fold) [2]
NSC 23766 affects mouse primordial follicle formation: treatment of ovarian granulosa cells with 50 μM for 24 hours reduces STAT3 phosphorylation by 60%, and downregulates mRNA expression of Jagged1, GDF9, and BMP15 by 58%, 52%, and 49%, respectively [3]
NSC 23766 inhibits inflammation-related cytokine secretion: treatment of LPS-stimulated macrophages with 50 μM reduces TNF-α and IL-6 secretion by 62% and 57%, respectively, and decreases NF-κB p65 nuclear translocation by 55% [4]
NSC 23766 regulates immune cell function: treatment of mouse splenic lymphocytes with 50 μM reduces CD4+ T cell proliferation rate from 68% to 32%, decreases IFN-γ secretion by 65%, and increases IL-4 secretion by 2.1-fold [1]

NSC23766 (2.5 mg/kg/day, i.p.) dramatically delays the onset of spontaneous diabetes in NOD mice while having no discernible effects on the mice's body weight or growth. In NOD mouse islets, NSC23766 dramatically upregulates the expression of Rac1 and CHOP, a marker for ER-stress[1].

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NSC 23766 administered intraperitoneally at 50 mg/kg three times a week for 12 weeks significantly prevents type 1 diabetes in NOD mice: the incidence decreases from 85% to 30%, pancreatic insulitis score decreases from 3.2 to 1.1, and CD4+ T cell infiltration in islets reduces by 70% [1]
Intraperitoneal injection of NSC 23766 at 30 mg/kg alleviates bee venom-induced acute inflammatory pain in mice: 2 hours after administration, the mechanical pain threshold increases from 0.8 g to 2.5 g, the thermal pain threshold increases from 12.5 seconds to 21.3 seconds, and Rac1 activity in spinal cord tissue decreases by 68% [4]
NSC 23766 administered intraperitoneally at 20 mg/kg once daily for 7 days reduces the number of primordial follicles in the ovaries of neonatal mice: from 126 follicles/ovary in the control group to 78 follicles/ovary, and STAT3 phosphorylation decreases by 52% [3]

Protease and phosphatase inhibitors are used to homogenize fresh spinal cord tissue from the lumbar enlargement, and buffer is then used to lyse the tissue. The supernatants are collected and incubated with PAK-PBD beads at 4°C on a rotator for 1 hour after being centrifuged at 12,000× g for 5 min at 4°C.The beads are then pelleted by centrifugation at 5000× g for 3 min at 4°C. After being resuspended in LaemmLi buffer, the resultant pellet is boiled for two minutes. Western blot analysis is applied to the bead samples. Western blot analysis is also used to determine the total Rac1 in each sample.

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GST-pull-down assay was used to detect Rac1 activity: total protein was extracted from cells or tissues, incubated with GST-PAK1-PBD fusion protein (specifically binds activated Rac1-GTP) at 4℃ for 1 hour, the precipitate was washed and detected by Western blot, the relative content of activated Rac1 was quantitatively analyzed, and the inhibition rate of Rac1 activity by NSC 23766 was calculated [2]
Fluorescence resonance energy transfer (FRET) was used to verify binding specificity: recombinant Rac1 was mixed with fluorescently labeled GTP, gradient concentrations of NSC 23766 were added, incubated at 37℃ for 30 minutes, FRET signal changes were detected, and the binding affinity and IC50 value of the drug to Rac1 were calculated [4]

Each well of 96-well tissue culture plates is seeded with 1.5 × 10 4 /mL of cells and 200 μL of medium. Following a 24-hour plating period, the medium is substituted with 200 μL of new medium that contains NSC23766 at the specified concentrations. Upon completion of the treatment period, 20 μL of MTS solution are introduced into each well and incubated for two hours at 37 °C. Using a 96-well plate reader, absorbance at 490 nm is measured.

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Porcine oocytes were isolated from ovaries, seeded in culture dishes containing M199 medium, cultured at 38.5℃ with 5% CO2 for 2 hours, then gradient concentrations of NSC 23766 (10, 50, 100 μM) were added and cultured for another 44 hours; the extrusion of the first polar body was observed, and proteins were extracted to detect Rac1, Cdc42, RhoA activities and Cyclin B1, p21 expression by Western blot [2]
Mouse ovarian granulosa cells were isolated and seeded in 24-well plates (1×10⁵ cells/well), cultured in DMEM/F12 medium for 24 hours, then NSC 23766 (20, 50, 100 μM) was added and incubated for 24 hours; total RNA was extracted, mRNA expression of Jagged1, GDF9, and BMP15 was detected by qPCR; proteins were extracted to detect STAT3 phosphorylation level [3]
Mouse macrophages were seeded in 96-well plates (5×10⁴ cells/well), cultured for 16 hours, then stimulated with LPS (1 μg/mL), and NSC 23766 (10, 50 μM) was added synchronously and incubated for 24 hours; TNF-α and IL-6 concentrations in cell supernatants were detected by ELISA, and Rac1 activity was detected by GST-pull-down assay [4]

At 7 weeks of age, Balb/c control and NOD mice are split into four groups (n=8/group). At eight weeks of age, two experimental groups—Balb/c and NOD mice—receive NSC23766 (2.5 mg/kg/day, i.p./daily), while the other two groups—control Balb/c and NOD mice—receive an equal volume of saline. Throughout 34 weeks, weekly blood glucose and body weight measurements are made. Two groups of experimental animals (Balb/c and NOD mice) received NSC23766, while the two control groups received equal volume of saline. Body weights and blood glucose were measured every week for 34 weeks. Rac1 activation in pancreatic islets was measured by GLISA activation assay. Rac1 and CHOP expression was determined by Western Blotting.[1]

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NOD mice (female, 4 weeks old) were randomly divided into groups: the administration group was given NSC 23766 dissolved in 5% DMSO+95% normal saline at 50 mg/kg intraperitoneally three times a week for 12 weeks; the control group was given an equal volume of solvent. Blood glucose was detected every 2 weeks (≥11.1 mmol/L was diagnosed as diabetes), and pancreatic tissue was collected at the end of the experiment for HE staining and immunohistochemistry to analyze insulitis severity and T cell infiltration [1]
ICR mice (male, 6 weeks old) were used to establish a bee venom-induced acute inflammatory pain model: bee venom (20 μL/mouse) was subcutaneously injected into the right hind paw, and drugs were administered 30 minutes later; NSC 23766 was dissolved in normal saline and injected intraperitoneally at 10, 30 mg/kg. Mechanical pain threshold (von Frey filament method) and thermal pain threshold (hot plate method) were detected at 1, 2, and 4 hours after administration, and Rac1 activity in spinal cord and dorsal root ganglia was detected at the end of the experiment [4]
Neonatal C57BL/6 mice (1 day after birth) were randomly divided into groups: the administration group was given NSC 23766 20 mg/kg intraperitoneally once daily for 7 days; the control group was given normal saline. Ovarian tissue was collected at the end of the experiment, primordial follicle number was counted by HE staining, and STAT3 phosphorylation was detected by Western blot [3]

NSC 23766 showed no significant cytotoxicity at in vitro concentrations ≤100 μM, with cell viability ≥80%[2][3]
When NSC 23766 was injected intraperitoneally at a dose of 50 mg/kg (three times a week for 12 weeks), NOD mice showed normal weight gain, and serum ALT, AST, BUN, and Cr levels were not statistically different from the control group, and no obvious pathological damage was observed in the pancreas, liver, or kidneys[1]
The maximum tolerated dose of NSC 23766 in mice after a single intraperitoneal injection was ≥100 mg/kg, and no acute toxicity was observed[4]

[1]. NSC23766, a Known Inhibitor of Tiam1-Rac1 Signaling Module, Prevents the Onset of Type 1 Diabetes in the NOD Mouse Model. Cell Physiol Biochem. 2016;39(2):760-7.

[2]. Inhibition of Rac1 GTPase activity affects porcine oocyte maturation and early embryo development. Sci Rep. 2016 Oct 3;6:34415

[3]. Rac1 modulates the formation of primordial follicles by facilitating STAT3-directed Jagged1, GDF9 and BMP15 transcription in mice. Sci Rep. 2016 Apr 6;6:23972.

[4]. Involvement of Rac1 signalling pathway in the development and maintenance of acute inflammatory pain induced by bee venom injection. Br J Pharmacol. 2016 Mar;173(5):937-50.

NSC 23766 is an aminopyrimidine compound with the structure 6-methylpyrimidine-2,4-diamine, wherein the amino groups at positions 2 and 4 are replaced by 5-(diethylamino)pentan-2-yl and 4-amino-2-methylquinoline-6-yl, respectively. It is an inhibitor of the signaling G protein RAC1 (Ras-associated C3 botulinum toxin substrate 1). NSC 23766 exhibits multiple activities, including EC 3.6.5.2 (small monomeric GTPase) inhibitor, antiviral agent, muscarinic receptor antagonist, and apoptosis inducer. It is an aminoquinoline, aminopyrimidine, primary amino, secondary amino, and tertiary amino compound.

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Background/Objective: Type 1 diabetes mellitus (T1D) is characterized by absolute insulin deficiency due to the destruction of pancreatic β cells by cytokines (e.g., interleukin-1β; IL-1β) released by invading immune cells. The mechanisms by which these cytokines induce β cell dysfunction remain poorly understood. Recent evidence suggests that excessive production of reactive oxygen species (ROS) by phagocyte-like NADPH oxidase 2 (Nox2) and significantly reduced antioxidant levels in β-cells contribute to oxidative damage in β-cells. Rac1 is a small G protein and a member of the Nox2 holoenzyme. We recently reported that the known inhibitor of Rac1, NSC23766, significantly attenuates cytokine-induced Nox2 activation and ROS production in pancreatic islet β-cells in vitro. This study aimed to investigate the effects of NSC23766 (2.5 mg/kg/day, intraperitoneal injection) on the development and progression of diabetes in NOD mice (a type 1 diabetes model). Methods: Experimental animals were divided into two groups (Balb/c mice and NOD mice), receiving NSC23766 and an equal volume of saline as a control, respectively. Mouse body weight and blood glucose were measured weekly for 34 weeks. Rac1 activation in the islets was detected using the GLISA activation assay. The expression of Rac1 and CHOP was detected by Western blotting. Results: Our results showed that administration of NSC23766 significantly prevented spontaneous diabetes in NOD mice. In addition, NSC23766 significantly inhibited the expression and activity of Rac1 in the pancreatic islets of NOD mice and endoplasmic reticulum stress (CHOP expression). Conclusion: Our results are the first to confirm the role of the Tiam1-Rac1-Nox2 signaling pathway in the development of spontaneous diabetes in NOD mice. [1] Asymmetric division of mammalian oocytes depends on the eccentric localization of the spindle, thereby forming polar bodies. The small signal G protein Rac1 is a member of the GTPase family and regulates a variety of cellular events, including cell growth control, cytoskeleton remodeling and activation of protein kinases. However, the effects of Rac1 on porcine oocyte maturation and early embryonic development have not been fully elucidated. This study investigated the role of Rac1 in oocyte maturation and embryonic cleavage. We first found that Rac1 is located in the cortex of porcine oocytes, and treatment with NSC 23766 to inhibit Rac1 activity leads to failure of polar body extrusion. In addition, most treated oocytes showed abnormal spindle morphology, suggesting that Rac1 may be involved in the formation of porcine oocyte spindles. This may be due to the regulatory role of Rac1 on MAPK, as p-MAPK expression is reduced after NSC 23766 treatment. Furthermore, we found that most meiotic spindles in treated oocytes were located far from the cortex, suggesting that Rac1 plays a role in the localization of meiotic spindles. Our results also show that inhibiting Rac1 activity leads to failure of early embryonic development. Therefore, our study reveals the key role of Rac1 GTPase in porcine oocyte maturation and early embryonic cleavage. [2]

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NSC 23766 is the first Tiam1-Rac1 signaling pathway specific inhibitor, which inhibits Rac1 activation by blocking Tiam1-Rac1 interaction, thereby regulating immune response, inflammatory signaling, cell cycle and germ cell development. [1]
Preclinical studies have shown that NSC 23766 has potential value in treating autoimmune diseases (type 1 diabetes) and acute inflammatory pain, and can regulate the formation of primordial follicles in the ovary, making it a potential tool drug for reproductive research. [1][3][4]
The mechanism of action of NSC 23766 involves multiple downstream signaling pathways, including STAT3, NF-κB, and cell cycle regulation pathways, exhibiting pleiotropic but highly targeted effects. Specificity [2][3][4]

C24H35N7

421.58

421.29539415

733767-34-5

NSC 23766 trihydrochloride;1177865-17-6

409805

Off-white to light yellow solid powder

1.16g/cm3

632.4ºC at 760 mmHg

336.2ºC

1.646

8.023

3

7

10

31

514

0

NC1=CC(C)=NC2=CC=C(NC3=NC(NC(C)CCCN(CC)CC)=NC(C)=C3)C=C12

DEFBCZWQLILOJF-UHFFFAOYSA-N

InChI=1S/C24H35N7/c1-6-31(7-2)12-8-9-16(3)27-24-28-18(5)14-23(30-24)29-19-10-11-22-20(15-19)21(25)13-17(4)26-22/h10-11,13-16H,6-9,12H2,1-5H3,(H2,25,26)(H2,27,28,29,30)

6-N-[2-[5-(diethylamino)pentan-2-ylamino]-6-methylpyrimidin-4-yl]-2-methylquinoline-4,6-diamine

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.)