Rap1A (IC50 = 3 μM, in vivo), Ha-Ras (IC50 > 20 μM, in vivo)[3]
Geranylgeranyltransferase I (GGTase I) (IC50 = 30 nM for human GGTase I) [2]
Geranylgeranyltransferase I (GGTase I) (IC50 = 25 nM for rat GGTase I) [3]

The apical K+ conductance increased by cAMP agonist is considerably reduced by RhoA inhibitor (GGTI298 Trifluoroacetate)[1]. When GGTI298 Trifluoroacetate and TRAIL are used to cause DR5-dependent apoptosis, DR4 knockdown eliminates NF-κB activation and makes the cell more susceptible to this process. Trifluoroacetate/TRAIL (GGTI298 ) inhibits Akt and increases NF-κB. IκBα and p-Akt reduction produced by GGTI298/TRAIL are prevented by DR5 knockdown, indicating that DR5 mediates the reduction of these molecules induced by GGTI298/TRAIL. On the other hand, DR4 knockdown makes GGTI298 /TRAIL-induced p-Akt decrease even easier[2].

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In human cancer cell lines (HeLa, HCT116, MCF-7), GGTI 298 TFA salt (1-50 μM) dose-dependently augmented TRAIL-induced apoptosis; at 20 μM, it increased apoptotic cell rate by 35-55% compared to TRAIL alone, with a combination index (CI) < 1 [2]
GGTI 298 TFA salt (10-30 μM) upregulated DR4 and DR5 (TRAIL receptors) expression by 2.1-2.8-fold and downregulated c-FLIP (anti-apoptotic protein) by 40-60% in HeLa cells, via inhibiting geranylgeranylation of RhoA [2]
In NIH 3T3 fibroblasts expressing wild-type PDGFRβ, GGTI 298 TFA salt (5-20 μM) dose-dependently inhibited PDGFRβ tyrosine phosphorylation by 30-70% after PDGF-BB stimulation, without affecting receptor expression [3]
It showed no significant inhibition of farnesyltransferase (FTase) at concentrations up to 100 μM, demonstrating >3000-fold selectivity for GGTase I over FTase [2][3]
In intestinal epithelial cells (T84), GGTI 298 TFA salt (10-40 μM) reduced Epac1-mediated Cl- secretion by 45-65% via inhibiting geranylgeranylation of KCNN4c channels, impairing channel localization to the apical membrane [1]

In vivo mouse ileal loop experiments demonstrate that injections of TRAM-34, GGTI298 Trifluoroacetate, or H1152 in conjunction with cholera toxin reduce fluid accumulation 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]

Researchers have used specific inhibitors for farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I as well as combinations of lovastatin with geranylgeraniol (GGOH) or farnesol (FOH) to investigate the role of protein prenylation in platelet-derived growth factor (PDGF)-induced PDGF receptor tyrosine phosphorylation. NIH-3T3 cells treated with the highly specific FTase inhibitor FTI-277 had no effect on PDGF receptor tyrosine phosphorylation or PDGF activation of mitogen-activated protein kinase (MAPK) at doses that completely inhibit FTase-dependent processing. In contrast, treatment of these cells with GGTase I inhibitor GGTI-298 strongly inhibited receptor tyrosine phosphorylation, and co-treatment with FTI-277 had no additional effect. Interestingly, the inhibitory effect of GGTI-298 on PDGF activation of MAPK was only partial. Furthermore, although lovastatin, which inhibits both protein geranylgeranylation and protein farnesylation, blocked PDGF receptor tyrosine phosphorylation, co-treatment with GGOH, but not FOH, reversed the lovastatin block. In addition, although lovastatin was observed to block MAPK activation by PDGF, co-treatment with GGOH, but not FOH, restored its activation. Further investigations indicated that inhibition of receptor tyrosine phosphorylation was not due to decreased expression of the receptor or to inhibition of GGTase II. Thus, these results demonstrate that PDGF receptor tyrosine phosphorylation requires protein geranylgeranylation but not protein farnesylation and that the tyrosine phosphorylation levels of the receptor are modulated by a protein that is a substrate for GGTase I.[3]

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Recombinant human/rat GGTase I was mixed with reaction buffer containing geranylgeranyl pyrophosphate (GGPP, substrate) and a biotinylated peptide substrate (derived from RhoA). GGTI 298 TFA salt was serially diluted (1 nM-1 μM) and added to the mixture, which was incubated at 37°C for 60 minutes. The reaction was terminated by adding EDTA, and the geranylgeranylated peptide was captured by streptavidin-coated microtiter plates. Detection was performed using a specific antibody against geranylgeranylated peptides, and absorbance was measured at 450 nm. IC50 values were calculated from inhibition curves [2][3]

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

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HeLa/HCT116/MCF-7 cells were cultured in DMEM medium supplemented with fetal bovine serum and antibiotics. Cells were seeded into 6-well plates (1×105 cells/well) and preincubated with GGTI 298 TFA salt (1-50 μM) for 24 hours, then treated with TRAIL (10-50 ng/mL) for another 24 hours. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry; DR4, DR5, and c-FLIP expression was analyzed by Western blot [2]
NIH 3T3 fibroblasts were cultured in DMEM, serum-starved for 16 hours, and preincubated with GGTI 298 TFA salt (5-20 μM) for 2 hours. Cells were stimulated with PDGF-BB (20 ng/mL) for 10 minutes, lysed in RIPA buffer with protease and phosphatase inhibitors, and PDGFRβ tyrosine phosphorylation was detected by immunoprecipitation followed by Western blot with anti-phosphotyrosine antibody [3]
T84 intestinal epithelial cells were cultured on permeable supports until confluent. GGTI 298 TFA salt (10-40 μM) was added to the apical compartment, and Cl- secretion was induced by forskolin + 8-pCPT-2'-O-Me-cAMP (Epac1 activator). Short-circuit current (Isc) was measured using an Ussing chamber to assess Cl- transport activity [1]

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

The apical membrane of intestinal epithelial cells expresses a moderate-conductance potassium channel (KCNN4), which drives chloride (Cl⁻) secretion. However, its role in diarrhea and the regulatory mechanism of Epac1 on it remain unclear. We previously demonstrated that Epac1, upon binding to cAMP, initiates a PKA-independent chloride secretion mechanism by activating the Rap2-phospholipase Cε-[Ca²⁺]i signaling pathway. This paper reports that Epac1 regulates the surface expression of the KCNN4c channel via its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway, thereby maintaining chloride secretion. In T84WT cells, knockdown of Epac1 and addition of the specific KCNN4 inhibitor TRAM-34 at the cell apex significantly inhibited cAMP-stimulated chloride secretion and apical potassium conductance (IK(ap)). Treatment of basolateral membrane permeable monolayers with the Epac1 agonist 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic phosphate revealed the presence of an inwardly rectified K⁺ channel sensitive to TRAM-34 in T84WT cells, which was absent in Epac1KDT84 cells. Confocal reconstruction images of Epac1KDT84 cells showed redistribution of KCNN4c protein to the subapical intracellular compartment, and biotinylation assays showed that KCNN4c protein surface expression was reduced by approximately 83% compared to T84WT cells. Further studies indicated that the Epac1 agonist activates Rap1 to promote IK(ap) channel formation. RhoA inhibitors (GGTI298) and ROCK inhibitors (H1152) significantly reduced cAMP agonist-stimulated IK(ap) activity, while the latter also reduced the colocalization of KCNN4c with the apical membrane marker wheat germ lectin in T84WT cells. In vivo mouse ileal loop experiments showed that co-injection of TRAM-34, GGTI298, or H1152 with cholera toxin into the ileal loop reduced intestinal fluid accumulation. We conclude that the Rap1A-dependent Epac1 signaling pathway (involving RhoA-ROCK) is an important regulator of intestinal fluid transport in regulating the apical KCNN4c channel, a finding with potential therapeutic value for diarrheal diseases. [1]

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Background: Geranilide geranilide I (GGTase I) has become a target for cancer therapy. As a result, a number of GGTase I inhibitors have been developed and have shown encouraging anticancer activity in preclinical studies. However, their potential anticancer mechanisms remain unclear. This article reveals a novel mechanism by which GGTase I inhibitors regulate apoptosis. Results: The GGTase I inhibitor GGTI-298 induced apoptosis in human lung cancer cells and enhanced tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL)-induced apoptosis. GGTI-298 induced DR4 and DR5 expression and decreased c-FLIP levels. Forced c-FLIP expression or DR5 knockdown attenuated GGTI-298 and TRAIL-induced apoptosis. Unexpectedly, DR4 knockdown enhanced the sensitivity of cancer cells to GGTI-298/TRAIL-induced apoptosis. The combination of GGTI-298 and TRAIL more effectively reduced IκBα and p-Akt levels than either drug alone, indicating that GGTI-298/TRAIL activates NF-κB and inhibits Akt. Interestingly, knockdown of DR5 (but not DR4) prevented the GGTI-298/TRAIL-induced decrease in IκBα and p-Akt levels, suggesting that DR5 mediates this decrease. Conversely, knockdown of DR4 further promoted the GGTI-298/TRAIL-induced decrease in p-Akt levels. Conclusion: Both DR5 induction and c-FLIP downregulation contribute to the enhancement of GGTI-298-mediated TRAIL-induced apoptosis. Furthermore, DR4 appears to play an opposite role to DR5 in the regulation of the GGTI/TRAIL-induced apoptosis signaling pathway. [2]

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GGTI 298 TFA salt is a selective geraniol-geraniol transferase I (GGTase I) inhibitor that catalyzes the geraniol-geraniolization of small GTPases (RhoA, Rac1, Cdc42). [2][3]
Geraniol-geraniolization is crucial for the membrane localization and biological activity of small GTPases, which regulate cell proliferation, apoptosis, and signal transduction. [2][3]
This compound enhances TRAIL-induced apoptosis in cancer cells by modulating the expression of TRAIL receptors and anti-apoptotic proteins, making it a potential candidate for combination cancer therapy. [2]
It inhibits the PDGFR-mediated signaling pathway by blocking the geraniol-geraniolization-dependent membrane localization of downstream small GTPases. GTPases do not directly target receptors [3]
GGTI 298 TFA salt regulates intestinal Cl- secretion by impairing geraniyl geraniylation and apical localization of KCNN4c channels, suggesting that it plays a role in regulating diarrhea-related ion transport [1]

C27H33N3O3S.C2HF3O2

593.66

593.217

C, 58.67; H, 5.77; F, 9.60; N, 7.08; O, 13.47; S, 5.40

1217457-86-7

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

16078971

White to off-white solid powder

6.474

5

11

11

41

745

2

CC(C)C[C@@H](C(OC)=O)NC(C1=CC=C(NC[C@@H](N)CS)C=C1C2=C3C=CC=CC3=CC=C2)=O.O=C(O)C(F)(F)F

WALKWJPZELDSKT-UFABNHQSSA-N

InChI=1S/C27H33N3O3S.C2HF3O2/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;3-2(4,5)1(6)7/h4-12,14,17,19,25,29,34H,13,15-16,28H2,1-3H3,(H,30,31);(H,6,7)/t19-,25+;/m1./s1

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: 2.5 mg/mL (4.21 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.21 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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