Rap1A ( pIC50 = 3 μM ); Histamine H1 receptor ( Ha-Ras > 20 μM )

Both RhoA parent (GGTI298) and ROCK parent (H1152) significantly reduce cAMP agonist-stimulated IK(ap), while night also reduces the colocalization of KCNN4c with the apical membrane marker malt agglutinin in T84WT cells [1]. Knockdown of NF-κB eliminates NF-κB activation, thereby sensitizing cells to activation of DR5 induced by the combined induction of GGTI298 and TRAIL. GGTI298/TRAIL activates NF-κB and inhibits Akt. Knocking out DR5 can prevent the reduction of IκBα and p-Akt induced by GGTI298/TRAIL, indicating that DR5 mediates the reduction of IκBα and p-Akt induced by GGTI298/TRAIL. In contrast, DR4 knockdown further promotes GGTI298/TRAIL-induced p-Akt reduction [2].

In vivo model ileal loop experiments showed that when TRAM-34, GGTI298 or H1152 were injected into the loop together with cholera toxin, fluid volume concentration was reduced in a dose-dependent manner [1].

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A major barrier towards the study of the effects of drugs on Giant Cell Tumor of Bone (GCT) has been the lack of an animal model. In this study, we created an animal model in which GCT stromal cells survived and functioned as proliferating neoplastic cells. A proliferative cell line of GCT stromal cells was used to create a stable and luciferase-transduced cell line, Luc-G33. The cell line was characterized and was found that there were no significant differences on cell proliferation rate and recruitment of monocytes when compared with the wild type GCT stromal cells. We delivered the Luc-G33 cells either subcutaneously on the back or to the tibiae of the nude mice. The presence of viable Luc-G33 cells was assessed using real-time live imaging by the IVIS 200 bioluminescent imaging (BLI) system. The tumor cells initially propagated and remained viable on site for 7 weeks in the subcutaneous tumor model. We also tested in vivo antitumor effects of Zoledronate (ZOL) and Geranylgeranyl transferase-I inhibitor (GGTI-298) alone or their combinations in Luc-G33-transplanted nude mice. ZOL alone at 400 µg/kg and the co-treatment of ZOL at 400 µg/kg and GGTI-298 at 1.16 mg/kg reduced tumor cell viability in the model. Furthermore, the anti-tumor effects by ZOL, GGTI-298 and the co-treatment in subcutaneous tumor model were also confirmed by immunohistochemical (IHC) staining. In conclusion, we established a nude mice model of GCT stromal cells which allows non-invasive, real-time assessments of tumor development and testing the in vivo effects of different adjuvants for treating GCT.[4]

The specified cells are lysed in Reporter Lysis Buffer and then put through a luciferase activity assay in a luminometer using the Luciferase Assay System. The relative luciferase activity is calibrated to the protein concentration.

In 96-well cell culture plates, cells are seeded, and the following day, they are treated with the specified agents. The sulforhodamine B assay is used to calculate the number of viable cells.

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Cell Survival Assay[2]
Cells were seeded in 96-well cell culture plates and treated the next day with the agents indicated. The viable cell number was determined using the sulforhodamine B assay, as previously described.

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Detection of Apoptosis[2]
Apoptosis was evaluated by Annexin V staining using Annexin V-PE apoptosis detection kit purchased commercially following the manufacturer's instructions. Caspase activation was also detected by Western blotting (as described below) as an additional indicator of apoptosis.

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Western Blot Analysis[2]
Whole-cell protein lysates were prepared and analyzed by Western blotting as described previously.

Mice aged 6 to 8 weeks are used in the ileal loop experiment, which uses a modified rabbit ileal loop assay. The animals are gut sterilized, fed only water ad libitum, and fasted for twenty-four hours before surgery. A combination of xylazine (5 mg/kg body weight) and ketamine (35 mg/kg body weight) is used to induce anesthesia. A laparotomy is done, and non-absorbable silk is used to tie the 5-cm-long experimental loops at the terminal ileum. Each loop is filled with the following fluids using a tuberculin syringe equipped with a disposable needle through the ligated end of the loop as the ligature is tightened: pure CT (1 μg; positive control), saline (negative control), CT (1 μg) + TRAM-34 (varying concentrations in μM as shown in Fig. 7), CT (1 μg) + H1152 (1 μM), and CT (1 μg) + GGTI298 (variable concentrations in μM), a specific inhibitor of Rap1A. The mice are sutured back into their cages and the intestine is reattached to the peritoneum. After six hours, the loops are removed and the animals are sacrificed by cervical dislocation. The fluid from every loop is collected, and the ratio of the fluid volume inside the loop to its length (fluid accumulation ratio in g/cm) is computed to show how effective different inhibitors are.

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Mouse Ileal Loop Experiment[1]
The ileal loop experiment was performed in 6–8-week-old mice by a modified rabbit ileal loop assay originally described by De and Chatterje. Following gut sterilization, the animals were kept fasted for 24 h prior to surgery and fed only water ad libitum. Anesthesia was induced by a mixture of ketamine (35 mg/kg of body weight) and xylazine (5 mg/kg of body weight). A laparotomy was performed, and the experimental loops of 5-cm length were constricted at the terminal ileum by tying with non-absorbable silk. The following fluids were instilled in each loop by means of a tuberculin syringe fitted with a disposable needle through the ligated end of the loop as the ligatured was tightened: pure CT (1 μg; positive control), saline (negative control), CT (1 μg) + TRAM-34 (different concentrations in μm as indicated in Fig. 7), CT (1 μg) + H1152 (1 μm), and CT (1 μg) + GGTI298 (different concentrations in μm), a specific inhibitor of Rap1A. The intestine was returned to the peritoneum, and the mice were sutured and returned to their cages. After 6 h, these animals were sacrificed by cervical dislocation, and the loops were excised. The fluid from each loop was collected, and the ratio of the amount of fluid contained in the loop with respect to the length of the loop (fluid accumulation ratio in g/cm) was calculated as a reflection of the efficacy of various inhibitors.

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dissolved in DMSO, dilute in saline; 1.16 mg/kg; s.c. injection

Nude mice

[1]. The Epac1 signaling pathway regulates Cl- secretion via modulation of apical KCNN4c channels in diarrhea. J Biol Chem. 2013 Jul 12;288(28):20404-15.

[2]. Dissecting the roles of DR4, DR5 and c-FLIP in the regulation of geranylgeranyltransferase I inhibition-mediated augmentation of TRAIL-induced apoptosis. Mol Cancer. 2010 Jan 29;9:23.

[3]. Platelet-derived growth factor receptor tyrosine phosphorylation requires protein geranylgeranylation but not farnesylation. J Biol Chem. 1996 Nov 1;271(44):27402-7.

[4]. A mouse model of luciferase-transfected stromal cells of giant cell tumor of bone. Connect Tissue Res. 2015 Nov;56(6):493-503.

(2S)-2-[[[4-[[(2R)-2-amino-3-mercaptopropyl]amino]-2-(1-naphthalenyl)phenyl]-oxomethyl]amino]-4-methylpentanoic acid methyl ester is a leucine derivative.

C27H33N3O3S

479.634

479.22

C, 67.61; H, 6.93; N, 8.76; O, 10.01; S, 6.69

180977-44-0

GGTI298 Trifluoroacetate; 1217457-86-7; 180977-44-0; 205590-41-6 (HCl)

9811606

White solid powder

6.474

4

6

11

34

661

2

S([H])C([H])([H])[C@@]([H])(C([H])([H])N([H])C1C([H])=C([H])C(C(N([H])[C@]([H])(C(=O)OC([H])([H])[H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=C(C=1[H])C1=C([H])C([H])=C([H])C2=C([H])C([H])=C([H])C([H])=C12)N([H])[H].FC(C(=O)O[H])(F)F

XVWPFYDMUFBHBF-CLOONOSVSA-N

InChI=1S/C27H33N3O3S/c1-17(2)13-25(27(32)33-3)30-26(31)23-12-11-20(29-15-19(28)16-34)14-24(23)22-10-6-8-18-7-4-5-9-21(18)22/h4-12,14,17,19,25,29,34H,13,15-16,28H2,1-3H3,(H,30,31)/t19-,25+/m1/s1

methyl (2S)-2-[[4-[[(2R)-2-amino-3-sulfanylpropyl]amino]-2-naphthalen-1-ylbenzoyl]amino]-4-methylpentanoate

GGTI 298; GGTI298; GGTI-298; GGTI298; 180977-44-0; Ggti 298; (S)-methyl 2-(4-(((R)-2-amino-3-mercaptopropyl)amino)-2-(naphthalen-1-yl)benzamido)-4-methylpentanoate; ELA97V8Q7P; CHEMBL282748; L-Leucine, N-[4-[[(2R)-2-amino-3-mercaptopropyl]amino]-2-(1-naphthalenyl)benzoyl]-, methyl ester; GGTI-298

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)