FTase (farnesyl transferase)

Radioresistant cells expressing the 24-kDa isoform (HeLa 3A) showed a significant decrease in survival when treated with FTI-277 (20 μM) for 48 hours prior to irradiation; however, this treatment had no effect on the survival of control cells (HeLa PINA). In addition to decreasing G(2)/M-phase arrest in both cell types and stimulating postmitotic cell death in HeLa 3A cells, FTI-277 has a radiosensitizing effect[1]. PC-3 cell migration and invasion were reduced in a time- and dose-dependent manner upon treatment with GGTI-298 and FTI-277[3].

When compared to vehicle alone, FTI-277 therapy inhibited elevated PTP-1B and PTEN protein expression in burned mice. On the other hand, in sham-burned animals, FTI-277 had no discernible effect on PTP-1B or PTEN protein expression [2].
Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277[2]
Conclusions: Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients.[2]

Inhibition of virion production by FTI-277.[4]
Starting the first day after transfection, as described above, medium was replaced every day with Huh7 medium containing 0.2% dimethyl sulfoxide (DMSO), 400 μM dithiothreitol (DTT), and various concentrations of FTI-277. On day 10, the cells were washed several times with 1× phosphate-buffered saline (PBS), in order to remove traces of DTT, and their viability was measured by an XTT assay as described elsewhere. Culture medium HBsAg concentrations were determined using an enzyme-linked immunosorbent assay-based assay. Cells were then washed twice with 1× PBS and scraped in 2 ml of Trizol reagent in order to purify their RNA content, following the manufacturer's instructions. The supernatants were precleared at low speed, loaded on 2-ml cushions of 20% sucrose in 1× PBS, and ultracentrifuged in an SW41Ti rotor for 15 h at 30,000 rpm at 4°C. After removing the supernatants, the pellets were carefully resuspended in water and extracted as described below for Northern analysis.

In this paper, researhers describe the effect of the inhibitor of farnesyltransferase (FTI-277) on radioresistance induced by the 24-kDa isoform of FGF2 in human cells expressing wild-type RAS. Treatment with FTI-277 (20 microM) for 48 h prior to irradiation led to a significant decrease in survival of radioresistant cells expressing the 24-kDa isoform (HeLa 3A) but had no effect on the survival of control cells (HeLa PINA). The radiosensitizing effect of FTI-277 is accompanied by a stimulation of postmitotic cell death in HeLa 3A cells and by a reduction in G(2)/M-phase arrest in both cell types. These results clearly demonstrate that at least one farnesylated protein is involved in the regulation of the radioresistance induced by the 24-kDa isoform of FGF2. Furthermore, the radiation-induced G(2)/M-phase arrest is also under the control of farnesylated protein. This work also demonstrates that FTase inhibitors may be effective radiosensitizers of certain human tumors with wild-type RAS.[1]

---

Growth rate of PC-3 cells[3]
Cells were plated in 24-well plates, 3000 cells/well and cultured for 24 h in DMEM containing iFBS (10%). The cells were treated with various concentrations of FTI-277, GGTI-298 or NE-10790 for 24 h, washed with DMEM containing 10% iFBS and cultured for an additional 75 h without compounds. The numbers of cells were counted with a Coulter Counter before and after treatments.

---

Fluorescence stainings[3]
PC-3 cells were pretreated with 10 μM GGTI-298, 10 μM FTI-277, 1 mM NE-10790 or DMEM containing 1% BSA (as a control) for 24 h on coverslips coated with Matrigel. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 5 min and stained for 20 min with TRITC (tetramethylrhodamine isothiocyanate)-labelled phalloidin (0.2 μg/ml), which stains F-actin. DNA was visualized using Hoechst 33342. Immunostaining of cofilin was performed by fixing and blocking cells with 0.1% BSA in PBS for 1 h and incubating them with anticofilin antibody for an additional 1 h. After PBS washings, cells were incubated with Alexa Fluor® 488 chicken antirabbit IgG (H+L) as a secondary antibody for 1 h. After washing with PBS and H2O, the coverslips were mounted, and confocal images were acquired with a Zeiss LSM510 META confocal microscope. Hoechst 33342, Alexa Fluor® 488 and TRITC–phalloidin were excited with 405, 488 and 543 nm laser lines, and emission data were collected via 420–480, 500–550 nm and 560LP filters, respectively.

---

FRAP (fluorescence recovery after photobleaching)[3]
PC-3 cells were transfected with pEGFP–actin, pEGFP–cofilin or pEGFP–paxillin, using 3 μg of vector DNA and 7 μl of TransFectin Lipid Reagent in glass-bottomed cell culture dishes. The cells were incubated for 5 h, and then the medium was changed. The cells were cultured for an additional 48 h to achieve expression of GFPs. Transfected cells were treated with either DMEM+1% BSA (negative control), 10 μM FTI-277, 10 μM GGTI-298 or 1 mM NE-10790. FRAP experiments (reviewed in Sprague and McNally, 2005) were performed with a Zeiss LSM510 META confocal microscope in a humidified chamber with 5% CO2 at 37°C. Cells transiently expressing EGFP–actin/–paxillin/–cofilin 1 were excited with a 488-nm laser beam, and emission was collected with a 500–550 nm bandpass filter. Prior to photobleaching, three images were collected. A ROI (region of interest) was chosen, and it was photobleached (488 nm; 100% intensity). Recovery was followed at 2-s intervals. The half time of recovery (t½) and the mobile fraction (Mf) were calculated. The data were assessed by means of FCalc®. Briefly, acquired data was corrected for image acquisition-caused photobleaching, and the resulting data was fitted to the equation y=(1−exp(kt)).

A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated.[2]

---

Dissolved in 5% DMSO, 0.5 mM DTT in sterile saline; 50 mg/kg; i.p. injection

HBV/HDV-transgenic FVB mice

[1]. The farnesyltransferase inhibitor FTI-277 suppresses the 24-kDa FGF2-induced radioresistance in HeLa cells expressing wild-type RAS. Radiat Res. 1999 Oct;152(4):404-11.

[2]. Role of protein farnesylation in burn-induced metabolic derangements and insulin resistance in mouse skeletal muscle. PLoS One. 2015 Jan 16;10(1):e0116633.

[3]. Inhibition of GGTase-I and FTase disrupts cytoskeletal organization of human PC-3 prostate cancer cells. Cell Biol Int. 2010 Aug;34(8):815-26.

[4]. A prenylation inhibitor prevents production of infectious hepatitis delta virus particles. J Virol. 2002 Oct;76(20):10465-72.

The mevalonate synthesis pathway produces intermediates for isoprenylation of small GTPases, which are involved in the regulation of actin cytoskeleton and cell motility. Here, we investigated the role of the prenylation transferases in the regulation of the cytoskeletal organization and motility of PC-3 prostate cancer cells. This was done by using FTI-277, GGTI-298 or NE-10790, the specific inhibitors of FTase (farnesyltransferase), GGTase (geranylgeranyltransferase)-I and -II, respectively. Treatment of PC-3 cells with GGTI-298 and FTI-277 inhibited migration and invasion in a time- and dose-dependent manner. This was associated with disruption of F-actin organization and decreased recovery of GFP-actin. Immunoblot analysis of various cytoskeleton-associated proteins showed that the most striking change in GGTI-298- and FTI-277-treated cells was a markedly decreased level of total and phosphorylated cofilin, whereas the level of cofilin mRNA was not decreased. The treatment of PC-3 cells with GGTI-298 also affected the dynamics of GFP-paxillin and decreased the levels of total and phosphorylated paxillin. The levels of phosphorylated FAK (focal adhesion kinase) and PAK (p-21-associated kinase)-2 were also lowered by GGTI-298, but levels of paxillin or FAK mRNAs were not affected. In addition, GGTI-298 had a minor effect on the activity of MMP-9. RNAi knockdown of GGTase-Ibeta inhibited invasion, disrupted F-actin organization and decreased the level of cofilin in PC-3 cells. NE-10790 did not have any effect on PC-3 prostate cancer cell motility or on the organization of the cytoskeleton. In conclusion, our results demonstrate the involvement of GGTase-I- and FTase-catalysed prenylation reactions in the regulation of cytoskeletal integrity and motility of prostate cancer cells and suggest them as interesting drug targets for development of inhibitors of prostate cancer metastasis.[3]

---

Hepatitis delta virus (HDV) causes both acute and chronic liver disease throughout the world. Effective medical therapy is lacking. Previous work has shown that the assembly of HDV virus-like particles (VLPs) could be abolished by BZA-5B, a compound with farnesyltransferase inhibitory activity. Here we show that FTI-277, another farnesyltransferase inhibitor, prevented the production of complete, infectious HDV virions of two different genotypes. Thus, in spite of the added complexity and assembly determinants of infectious HDV virions compared to VLPs, the former are also sensitive to pharmacological prenylation inhibition. Moreover, production of HDV genotype III virions, which is associated with particularly severe clinical disease, was as sensitive to prenylation inhibition as was that of HDV genotype I virions. Farnesyltransferase inhibitors thus represent an attractive potential class of novel antiviral agents for use against HDV, including the genotypes associated with most severe disease.[4]

C₂₂H₂₉N₃O₃S₂

447.61

447.165

C, 59.03; H, 6.53; N, 9.39; O, 10.72; S, 14.32

170006-73-2

FTI-277 hydrochloride;180977-34-8; 1217447-06-7 (TFA)

3005532

Typically exists as solid at room temperature

4.844

4

7

12

30

532

2

S(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])(C(=O)OC([H])([H])[H])N([H])C(C1C([H])=C([H])C(=C([H])C=1C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C([H])([H])[C@]([H])(C([H])([H])S[H])N([H])[H])=O

GKFPROVOIQKYTO-UZLBHIALSA-N

InChI=1S/C22H29N3O3S2/c1-28-22(27)20(10-11-30-2)25-21(26)18-9-8-17(24-13-16(23)14-29)12-19(18)15-6-4-3-5-7-15/h3-9,12,16,20,24,29H,10-11,13-14,23H2,1-2H3,(H,25,26)/t16-,20+/m1/s1

L-Methionine, N-((5-((2-amino-3-mercaptopropyl)amino)(1,1'-biphenyl)-2-yl)carbonyl)-, methyl ester, (R)-

FTI277; methyl (2S)-2-[[4-[[(2R)-2-amino-3-sulfanylpropyl]amino]-2-phenylbenzoyl]amino]-4-methylsulfanylbutanoate; CID 3005532; CHEMBL135561; L-Methionine, N-((5-((2-amino-3-mercaptopropyl)amino)(1,1'-biphenyl)-2-yl)carbonyl)-, methyl ester, (R)-; GKFPROVOIQKYTO-UZLBHIALSA-N; SCHEMBL8365606; FTI-277; FTI 277

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.

---

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.)