FTase (farnesyl transferase)

HeLa 3A, a kind of radioresistant cell that expresses the 24-kDa isoform, showed a substantial decrease in survival after 48 hours of treatment with FTI-277 (20 microM), while control cells (HeLa PINA) showed no significant change in survival. FTI-277's radiosensitizing action is followed by a decrease in G(2)/M-phase arrest in both cell types as well as an increase in postmitotic cell death in HeLa 3A cells [1]. In a dose- and time-dependent manner, PC-3 cells treated with GGTI-298 and FTI-277 were unable to migrate or invade [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]

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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.

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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.

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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]

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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]

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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]

C22H30CLN3O3S2

484.07

483.142

C, 54.59; H, 6.25; Cl, 7.32; N, 8.68; O, 9.92; S, 13.25

180977-34-8

FTI-277;170006-73-2; 1217447-06-7 (TFA)

88309922

White to off-white solid powder

5.013

5

7

12

31

532

2

COC(=O)[C@H](CCSC)NC(=O)C1=C(C=C(C=C1)NC[C@H](CS)N)C2=CC=CC=C2.Cl

PIAFFJUUNXEDEW-PXPMWPIZSA-N

InChI=1S/C22H29N3O3S2.ClH/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);1H/t16-,20+;/m1./s1

methyl (5-(((R)-2-amino-3-mercaptopropyl)amino)-[1,1-biphenyl]-2-carbonyl)-L-methioninate hydrochloride

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 (5.16 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 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 (5.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 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 (5.16 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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Solubility in Formulation 4: 33.33 mg/mL (68.85 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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