DNA/RNA methyltransferase

Sinefungin (0.5 or 1.0 μg/mL, 60 min) suppresses the elevation of histone H3K4 monomethylation in renal fibroblasts and renal epithelial cells and attenuates the increase in α-SMA caused by TGF-β1 [2].
Sinefungin Inhibits TGF-β1-Induced α-SMA-positive Myofibroblast Expression and TGF-β1-induced H3K4me1 in Renal Cells [2]
As shown in Figures 6 and 7, sinefungin inhibited H3K4me1 and ameliorated renal fibrosis induced by UUO. However, these results were from whole kidney lysates in UUO mice. To clarify the direct effect of sinefungin on TGF-β1-induced H3K4 methylation, we performed immunoblotting for H3K4 methylation in NRK-52E and NRK-49F cells. Pretreatment with sinefungin significantly reduced TGF-β1-induced α-SMA protein expression and inhibited H3K4me1 in a dose-dependent manner in both NRK-52E (Figure 8, A and B) and NRK-49F (Figure 9, A and B) cells. In contrast, sinefungin had no significant effect on H3K4me2 and H3K4me3 either in vivo in UUO kidneys or in vitro in epithelial cells (Figure 8, C and D) and fibroblasts (Figure 9, C and D). Finally, we examined whether TGF-β1 alters access located at H3K4me1-regulated sites using chromatin immunoprecipitation (ChIP) assays. We found that TGF-β1 increased H3K4me1 levels at collagen 1 (Col1a1), connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1) promoters in NRK-52E cells, and that sinefungin inhibited H3K4me1 levels (Figure 8E).

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Sinefungin (A9145) and a related metabolite, A9145C, were found to be potent inhibitors of Newcastle disease virion and vaccinia virion mRNA(guanine-7-)-methyltransferase and vaccinia virion mRNA(nucleoside-2'-)-methyltransferase. Both Sinefungin and A9145C were competitive inhibitors of these S-adenosyl-L-methionine-dependent enzymes having inhibition constants substantially less than S-adenosyl-L-homocysteine. These compounds also inhibited plaque formation by vaccinia virus in mouse L-cells [1].

For obstructive nephropathy, sinefungin (10 mg/kg daily) improves renal fibrosis right after UUO [2].
Sinefungin Ameliorates Renal Fibrosis in Obstructive Nephropathy [2]
In view of the need for developing new therapeutic agents for the treatment of renal fibrosis, we assessed the effect of a small molecule inhibitor for SET7/9, sinefungin, on the expression of mesenchymal markers and ECM proteins in UUO mice. We examined α-SMA and fibroblast-specific protein-1 (FSP-1) as mesenchymal markers, and collagen 1, collagen 3, and fibronectin as ECM proteins. Following the injection of sinefungin, UUO-induced mRNA of α-SMA, collagen 1 and collagen 3 were markedly suppressed in the kidney, both at 3 days and 7 days after UUO (Figure 6A). Western blot analysis also showed that sinefungin inhibited α-SMA protein expression (Figure 6B). Our immunohistochemical analysis revealed that staining for α-SMA, FSP-1, collagen 1, collagen 3, and fibronectin in the kidney tissues increased at 3 days, with a further increase at 7 days, after UUO. In contrast, sinefungin injection ameliorated those both at 3 and 7 days after UUO (Figure 6, C and D). However, injection of sinefungin did not affect UUO-induced TGF-β1 mRNA expression levels (Figure 6E).

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Sinefungin Inhibits H3K4me1 Simultaneously with the Amelioration of Renal Fibrosis in Obstructive Nephropathy [2]
It has previously been reported that increased levels of H3K4 methylation promote transcriptional activation of TGF-β1-induced fibrotic gene expression.19 To identify the effect of sinefungin on SET7/9 during the development of renal fibrosis, we tested whether sinefungin inhibits H3K4 methylation. Injection of sinefungin significantly inhibited UUO-induced H3K4 mono-methylation (H3K4me1) in mouse kidney (Figure 7A). In contrast, H3K4 di-methylation (H3K4me2) and H3K4 tri-methylation (H3K4me3) in the kidneys of UUO mice did not show a significant change after the injection of sinefungin (Figure 7, B and C).

Sinefungin (A9145) and a related metabolite, A9145C, were found to be potent inhibitors of Newcastle disease virion and vaccinia virion mRNA(guanine-7-)-methyltransferase and vaccinia virion mRNA(nucleoside-2'-)-methyltransferase. Both Sinefungin and A9145C were competitive inhibitors of these S-adenosyl-L-methionine-dependent enzymes having inhibition constants substantially less than S-adenosyl-L-homocysteine. These compounds also inhibited plaque formation by vaccinia virus in mouse L-cells [1].

Western Blot Analysis[2]
Cell Types: Renal epithelial cells.
Tested Concentrations: 0.5 or 1.0 μg/mL.
Incubation Duration: 60 minutes pretreatment before TGF-β1 (10 ng/mL).
Experimental Results: In NRK-52E and NRK-49F cells, TGF-β1-induced α-SMA protein expression was Dramatically diminished and H3K4me1 was inhibited in a dose-dependent manner.

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Cell Culture [2]
NRK-52E and NRK-49F cells were maintained in DMEM containing 5% FBS and penicillin/streptomycin. All cells were washed and growth was arrested for 24 hours in DMEM containing 0% FBS prior to each stimulation. Preincubation of Sinefungin was carried out 60 minutes before TGF-β1 (R&D Systems) stimulation. NRK-52E and NRK-49F cells were treated with TGF-β1 at the indicated dosage levels and times.

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siRNA Transfection In Vitro [2]
NRK-52E cells were plated in six-well culture dishes and were transfected 6 hours later (30% confluent) with Smad3 Silencer Select siRNA (si-Smad3, 12.5 nM) or Silencer Select Negative Control #1 siRNA (si-Neg) using lipofectamine 2000 according to the manufacturer’s instructions. After 6 hours, the transfected cells were washed, and fresh medium containing 0% FBS was added. The next day, cells were treated with or without TGF-β1, and processed for mRNA or protein extraction at the indicated time periods.

Animal/Disease Models: Male C57BL/6J mice (8 weeks old) [2].
Doses: 10 mg/kg
Route of Administration: UUO (prepared as a suspension in distilled water and 0.9% NaCl solution) is administered intraperitoneally (ip) (ip) immediately.
Experimental Results: Inhibition of α-SMA protein expression. These indicators (α-SMA, FSP-1, collagen 1, collagen 3) were improved on days 3 and 7 after UUO.

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Drug and siRNA Administration In Vivo [2]
Mice were administered with neutralizing anti-TGF-β1 antibody (1D11, 1.5 mg/kg), or normal mouse IgG1 (11711, 1.5 mg/kg) immediately after UUO by intraperitoneal injection. The same treatments were repeated every 48 hours until mice were killed as previously described.37 siRNA (In Vivo Pre-designed SET7/9-siRNA and In Vivo Negative Control #1 siRNA) and Invivofectamine 2.0 reagent (Invitrogen, Carlsbad, CA) complex (0.7 mg/mL) was prepared according to the manufacturer’s instructions. Immediately after right ureteral obstruction, 50 μL of SET7/9-siRNA solution (7 mg/kg) was injected retrogradely once into the right kidney via the ureter. Sinefungin was prepared as a suspension in distilled water and 0.9% NaCl solution, and administered intraperitoneally (0.1 mL per mouse) at a dose of 10 mg/kg per day immediately after UUO. The control group was administered an equal volume of vehicle (0.1 mL of distilled water and 0.9% NaCl solution) intraperitoneally. The same treatments were repeated every 24 hours until mice were killed. We selected the dose of Sinefungin based on described studies.

65482 Oral LD50 in mice: 1 gm/kg. CRC Handbook of Antibiotic Compounds, Vol. 1, Berdy, J., Boca Raton, FL, CRC Press, 1980, 5(298), 1981. 65482 Subcutaneous LD50 in mice: 185 mg/kg. Journal of Antibiotics, 26(463), 1973 [PMID:4792069]

[1]. Sinefungin, a potent inhibitor of virion mRNA(guanine-7-)-methyltransferase, mRNA(nucleoside-2'-)-methyltransferase, and viral multiplication. J Biol Chem. 1978 Jun 25;253(12):4075-7.

[2]. Inhibition of SET Domain-Containing Lysine Methyltransferase 7/9 Ameliorates Renal Fibrosis. J Am Soc Nephrol. 2016 Jan;27(1):203-15.

Sinefungin is an adenosine, a δ-(5'-adenosine) derivative of ornithine. It possesses antifungal and antibacterial activities. Sinefungin belongs to the adenosine class of compounds and is a non-protein α-amino acid. Functionally, it is associated with L-ornithine. Sinefungin is a solid. This compound belongs to the purine nucleoside and its analogues. These compounds are composed of purine bases linked to sugars. Proteins targeted by adenosine ornithine include RdmB, the methyltransferase TaqI, rRNA (adenine-N6-) methyltransferase, and the methyltransferase RsrI. Sinefungin has been reported in Streptomyces griseolus and Streptomyces halstedii, and relevant data exist. Sinefungin is a natural nucleoside associated with S-adenosine methionine, isolated from Streptomyces, and possesses antifungal, antiviral, and antiparasitic activities. Cifenimine inhibits DNA methyltransferases, thereby suppressing DNA synthesis. TGF-β1 activity induces methylation of histone H3 at lysine 4 (H3K4) by lysine methyltransferase 7/9 (SET7/9), which is crucial for the transcriptional activation of fibrosis genes in vitro. However, in vivo studies using experimental models of renal fibrosis are needed to develop therapeutics targeting SET7/9. This study investigated the signaling pathway by which TGF-β1 induces SET7/9 expression and whether inhibition of SET7/9 inhibits renal fibrosis in unilateral ureteral obstruction (UUO) mice and renal cell lines. Within the SET family, SET7/9 expression was upregulated on days 3 and 7 in UUO mice, and this upregulation could be inhibited by a TGF-β1 neutralizing antibody. TGF-β1 induces SET7/9 expression in normal rat kidney (NRK)-52E cells via Smad3. In renal biopsy specimens from patients diagnosed with IgA nephropathy and membranous nephropathy, SET7/9 expression was positively correlated with the degree of interstitial fibrosis (IgA nephropathy patients: r=0.59, P=0.001; membranous nephropathy patients: r=0.58, P<0.05). Furthermore, knockdown of SET7/9 expression mediated by small interfering RNA significantly alleviated renal fibrosis in UUO mice. The SET7/9 inhibitor cilifene also inhibited the expression of renal interstitial markers and extracellular matrix proteins in UUO mice and suppressed H3K4 monomethylation (H3K4me1). In addition, cilifene inhibited TGF-β1-induced α-smooth muscle actin expression and H3K4me1 formation in both NRK-52E and NRK-49F cells. In conclusion, cilifene, as a SET7/9 inhibitor, can improve renal fibrosis by inhibiting H3K4me1 and may be a potential therapeutic agent. [1]

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In summary, the expression of SET7/9 is regulated by the TGF-β1-Smad3 pathway and activates the transcription of fibrosis genes by increasing H3K4me1 levels. We confirmed the actual expression of SET7/9 in renal biopsy samples from patients with IgA nephropathy and membranous nephropathy and showed that the expression of SET7/9 is related to the area of fibrosis. Inhibition of SET7/9 not only reduced H3K4me1 levels but also improved renal fibrosis in a mouse model of renal fibrosis. In addition, the small molecule inhibitor of SET7/9, sinefungin, also showed a reduction in H3K4me1 levels and inhibited fibrosis in vivo and in vitro. In conclusion, we found that inhibition of SET7/9 is a therapeutic target for renal fibrosis and believe that sinefungin may be a candidate drug for the treatment of patients with chronic kidney disease (CKD). [1]

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Sinefungin is a small molecule drug that is currently in phase II clinical trials.

C15H23N7O5

381.38702

381.176

C, 47.24; H, 6.08; N, 25.71; O, 20.97

58944-73-3

65482

White to yellow solid

1.9±0.1 g/cm3

783.2±70.0 °C at 760 mmHg

427.5±35.7 °C

0.0±2.8 mmHg at 25°C

1.832

-1.33

6

11

7

27

529

6

C(C[C@@H](C(=O)O)N)[C@@H](C[C@@H]1[C@H]([C@H]([C@H](N2C=NC3=C(N)N=CN=C32)O1)O)O)N

LMXOHSDXUQEUSF-YECHIGJVSA-N

InChI=1S/C15H23N7O5/c16-6(1-2-7(17)15(25)26)3-8-10(23)11(24)14(27-8)22-5-21-9-12(18)19-4-20-13(9)22/h4-8,10-11,14,23-24H,1-3,16-17H2,(H,25,26)(H2,18,19,20)/t6-,7-,8+,10+,11+,14+/m0/s1

(2S,5S)-2,5-Diamino-6-[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]hexanoic acid

A 9145; Antibiotic A 9145;Antibiotic 32232RP; sinefungin; 58944-73-3; Sinefungina; Sinefungine; ADENOSYL-ORNITHINE; Compound 57926; Sinefunginum; RP 32232; Sinefungin; Adenosylornithine; Antibiotic A 9145

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)

H2O : ~100 mg/mL (~262.20 mM)

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.)