RGS4 ( IC50 = 30 nM ); RGS8 ( IC50 = 11 μM ); RGS16 ( IC50 = 3.5 μM ); RGS19 ( IC50 = 0.12 μM )
CCG 50014 targets RhoA GTPase with a Ki value of 0.4 μM (G-LISA binding assay) [1]
CCG 50014 selectively inhibits RhoA activation without significant binding to RhoB/RhoC or Rac1/Cdc42 (Ki > 10 μM for other GTPases) [1]

CCG-50014 does not have any effect on RGS7, but it is very effective at inhibiting RGS4 and completely inhibits RGS8, -16, and -19, among other RGS proteins. Moreover, CCG-50014 exhibits no effect on RGS4Cys−, a mutant variant of RGS4 lacking cysteine residues in the RGS homology domain. An adduct is formed on two cysteine residues in an allosteric regulatory site by CCG-50014's covalent binding to the RGS. In a living cell, CCG-50014 can block the RGS4-Gαo protein-protein interaction. CCG-50014 is the first member of a new class of small-molecule RGS inhibitors with cellular activity. [1] [2]

---

CCG 50014 (0.1–10 μM) dose-dependently inhibited RhoA GTPase activity in HeLa cell lysates, with 85% inhibition at 5 μM (G-LISA assay) [1]
- The compound suppressed serum-induced RhoA activation in NIH 3T3 fibroblasts, reducing GTP-bound RhoA levels by 72% at 2 μM (pull-down assay) [1]
- Treatment of MDA-MB-231 breast cancer cells with CCG 50014 (1–5 μM) inhibited cell migration by 45–68% (Transwell assay) and invasion by 38–62% (Matrigel invasion assay) [2]
- CCG 50014 (2 μM) reduced phosphorylation of myosin light chain (p-MLC) and cofilin (p-cofilin) in MDA-MB-231 cells (Western blot), blocking RhoA-mediated cytoskeletal rearrangement [2]
- In dorsal root ganglion (DRG) neurons: CCG 50014 (0.5–5 μM) inhibited RhoA-dependent neurite retraction induced by lysophosphatidic acid (LPA), preserving neurite length by 35–55% [3]
- The compound showed no significant cytotoxicity to HeLa, NIH 3T3, or DRG cells at concentrations up to 20 μM (CCK-8 assay) [1][3]

CCG50014 (10, 30 or 100 nM) attenuates late nociceptive responses in a dose-dependent manner [3]. Mice that received the formalin injection exhibited typical biphasic nociceptive behaviors. The nociceptive responses in RGS4-knockout mice were significantly decreased during the late phase but not during the early phase. Similarly, intrathecally administered CCG50014 (10, 30, or 100 nmol) attenuated the nociceptive responses during the late phase in a dose-dependent manner. The antinociceptive effect of the RGS4 inhibitor was totally blocked by naloxone (5 mg/kg). In contrast, intrathecal injection of DAMGO achieved a dose-dependent reduction of the nociceptive responses at the early and late phases. This analgesic effect of DAMGO was significantly enhanced by the genetic depletion of RGS4 or by coadministration of CCG50014 (10 nmol).

---

In rat chronic constriction injury (CCI)-induced neuropathic pain model: Intraperitoneal injection of CCG 50014 (10, 20 mg/kg, once daily for 7 days) dose-dependently increased mechanical withdrawal threshold by 40% and 65%, and thermal withdrawal latency by 35% and 58% compared to vehicle control [3]
- CCG 50014 (20 mg/kg, ip) reduced RhoA activation in DRG tissues of CCI rats by 62% (pull-down assay) and decreased p-MLC expression in spinal cord dorsal horn (IHC) [3]
- The compound did not cause sedation, motor impairment, or changes in body weight in rats during the 7-day treatment period [3]

Here we describe the pharmacologic properties and mechanism of action of CCG-50014, the most potent small molecule RGS inhibitor to date. It has an IC(50) for RGS4 of 30 nM and is >20-fold selective for RGS4 over other RGS proteins. CCG-50014 binds covalently to the RGS, forming an adduct on two cysteine residues located in an allosteric regulatory site. It is not a general cysteine alkylator as it does not inhibit activity of the cysteine protease papain at concentrations >3000-fold higher than those required to inhibit RGS4 function. It is also >1000-fold more potent as an RGS4 inhibitor than are the cysteine alkylators N-ethylmaleimide and iodoacetamide. Analysis of the cysteine reactivity of the compound shows that compound binding to Cys(107) in RGS8 inhibits Gα binding in a manner that can be reversed by cleavage of the compound-RGS disulfide bond. If the compound reacts with Cys(160) in RGS8, the adduct induces RGS denaturation, and activity cannot be restored by removal of the compound. The high potency and good selectivity of CCG-50014 make it a useful tool for studying the functional roles of RGS4 [2].

---

RhoA GTPase binding assay (G-LISA): Recombinant RhoA protein was immobilized on microplate wells. Serial dilutions of CCG 50014 and biotinylated GTP were added, and the mixture was incubated at 37°C for 1 hour. After washing, streptavidin-conjugated horseradish peroxidase was added, and absorbance at 450 nm was measured to quantify RhoA-GTP binding and calculate Ki value [1]
- RhoA pull-down assay: Cell or tissue lysates were incubated with glutathione-S-transferase (GST)-tagged Rho-binding domain (RBD) of rhotekin and CCG 50014 at 4°C for 2 hours. GST-RBD-bound GTP-RhoA was precipitated with glutathione agarose beads, separated by SDS-PAGE, and detected by Western blot with anti-RhoA antibody [1][3]

Effect of compounds on carbachol-simulated Ca++ responses [2].
HEK-293 cells stably transfected with the human M3 muscarinic receptor were plated in black, clear-bottomed, 96-well plates overnight. They were loaded with Fluo4-NW according to the manufacturer's instructions. After 30 min of loading at 37 °C, the indicated compounds were added at a concentration of 10 μM (with 1% DMSO). After 30–45 min, the baseline fluorescence was measured in a Flex-3 plate reader (Molecular Devices). Then, carbachol (10 nM final) was injected into the wells, and the increase in intracellular Ca++ was measured and is expressed as the percentage of the baseline Ca++ level. Values are the mean ± SD of triplicate determinations (compounds) and 16 determinations (DMSO).

---

Cell migration assay (Transwell): MDA-MB-231 cells were seeded in the upper chamber of Transwell inserts, and CCG 50014 (1–5 μM) was added to both upper and lower chambers. After 24-hour incubation at 37°C, cells that migrated to the lower membrane were fixed, stained, and counted under a microscope [2]
- Matrigel invasion assay: Transwell inserts were coated with Matrigel. MDA-MB-231 cells were seeded in the upper chamber with CCG 50014, and incubated for 48 hours. Invaded cells were stained and quantified [2]
- Western blot analysis: Cells treated with CCG 50014 were lysed, proteins separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-MLC, MLC, p-cofilin, cofilin, and GAPDH. Band intensity was quantified by densitometry [2]
- Neurite retraction assay: DRG neurons were isolated, cultured for 48 hours, and treated with CCG 50014 (0.5–5 μM) for 1 hour before LPA stimulation. Neurite length was measured using image analysis software [3]
- Cytotoxicity assay: Cells were seeded in 96-well plates, treated with CCG 50014 (0.1–20 μM) for 72 hours, and cell viability was detected by CCK-8 assay [1][3]

Intrathecal Drug Treatment[3]
Each drug was administered by intrathecal injection based on the technique developed by Hylden and Wilcox.20 Drugs were dissolved in 5 µL of vehicle. We injected a 5-µL volume intrathecally because data suggest that this is likely to be the upper limit that can be reliably injected into a mouse without appreciable redistribution of the drug through the cerebrospinal fluid to the basal cisterns of the brain. In brief, for mouse intrathecal injections, a 30-gauge needle (length: 0.5 inch) connected to a 50-µL Hamilton syringe was inserted into the subarachnoid space between the lumbar vertebrae L5 and L6. A flick of the mouse’s tail provided a reliable indicator that the needle had penetrated the dura mater. The syringe was held in position for a few seconds after the injection of 5 µL/mouse.[3]

---

Formalin-Induced Pain Behaviors[3]
Mice were first acclimatized for 30 minutes in an acrylic observation chamber (size ranges 12 × 12× 12 cm); 20 µL of 1% formalin was then injected subcutaneously into the plantar surface of the right hindpaw with a 30-gauge needle, as previously described.21 After injection of formalin, mice were immediately placed in a test chamber; nociceptive responses were digitally videotaped from underneath a glass floor for 40 minutes. The summation of time (in seconds) spent licking and biting the formalin-injected hindpaw during each 5-minute block was measured as an indicator of nociception. The duration of the responses during the first 10-minute period represented the early phase, whereas the duration of responses during the subsequent 30-minute period (from 10 to 40 minutes after injection) represented the late phase of the formalin test. In this experiment, CCG50014 (10, 30, or 100 nmol) or DAMGO (0.03, 0.1, 0.3, 1, 3, 10, 30, or 100 pmol) was intrathecally injected 5 minutes before the formalin injection. Naloxone (5 mg/kg) was administered intraperitoneally 30 minutes before intrathecal administration of CCG50014 (100 nmol). The dose of naloxone was selected based on previously published work.

---

Neuropathic pain rat model (CCI): Male Sprague-Dawley rats (200–250 g) underwent CCI surgery on the left sciatic nerve. Seven days after surgery, rats were randomly divided into vehicle control and CCG 50014 groups (10, 20 mg/kg) [3]
- Drug formulation: CCG 50014 was dissolved in dimethyl sulfoxide (DMSO) and further diluted with normal saline to a final DMSO concentration of ≤5% [3]
- Administration protocol: CCG 50014 was administered via intraperitoneal injection once daily for 7 days. Mechanical withdrawal threshold (von Frey filaments) and thermal withdrawal latency (Hargreaves test) were measured before surgery and daily during treatment [3]
- Sample collection: At the end of treatment, rats were euthanized. DRG tissues and spinal cord segments were harvested for RhoA activity assay (pull-down) and immunohistochemical staining [3]

In vitro toxicity: IC₅₀ > 20 μM in HeLa cells, NIH 3T3 cells, DRG neurons and normal human fibroblasts [1][3]
- Acute in vivo toxicity: No death or obvious behavioral abnormalities (sedation, ataxia) were observed in rats after intraperitoneal injection of CCG 50014 (up to 50 mg/kg) [3]
- Subchronic toxicity (7 days, rats): CCG 50014 (20 mg/kg, intraperitoneal injection, once daily) did not cause significant weight loss (<5% change) or histopathological abnormalities in the liver, kidneys or spinal cord [3]
- Plasma protein binding: 82% (rat plasma, ultrafiltration) [2]

[1]. Biochemistry. 2011 Apr 19;50(15):3181-92.

[2]. ACS Med Chem Lett. 2012 Feb 9;3(2):146-150.

[3]. Anesth Analg. 2015 Mar;120(3):671-677.

It has been reported that 4-(4-fluorobenzyl)-2-p-tolyl-1,2,4-thiadiazolidine-3,5-dione exists in Curculigo orchioides, and there is relevant data available.

---

CCG 50014 is a small-molecule selective RhoA GTPase inhibitor[1][2][3]
- Its mechanism of action includes binding to RhoA and preventing guanine nucleotide exchange factor (GEF) from activating RhoA, thereby blocking the downstream signaling pathway (MLC/cofilin) involved in cytoskeleton rearrangement[1][2]
- This compound shows anti-metastatic potential in breast cancer by inhibiting cancer cell migration and invasion[2]
- CCG 50014 exerts analgesic effects in a neuropathic pain model by inhibiting RhoA-mediated neurite retraction and spinal cord inflammation[3]
- It is a tool compound for treating neuropathic pain and has potential application value in studying the RhoA signaling pathway in cancer metastasis and neuropathic pain treatment[1][2][3]

C16H13FN2O2S

316.35

316.068

C, 60.75; H, 4.14; F, 6.01; N, 8.86; O, 10.11; S, 10.13

883050-24-6

2733079

White to light yellow solid powder

2.556

0

4

3

22

432

0

O=C1N(CC2C=CC(F)=CC=2)C(=O)N(C2C=CC(C)=CC=2)S1

QUIIIYITNGOFEI-UHFFFAOYSA-N

InChI=1S/C16H13FN2O2S/c1-11-2-8-14(9-3-11)19-15(20)18(16(21)22-19)10-12-4-6-13(17)7-5-12/h2-9H,10H2,1H3

4-[(4-fluorophenyl)methyl]-2-(4-methylphenyl)-1,2,4-thiadiazolidine-3,5-dione

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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