Sinefungin   中文名称: 西奈芬净

DNA/RNA methyltransferase

Sinefungin（0.5 或 1.0 μg/mL，60 分钟）抑制肾成纤维细胞和肾上皮细胞中组蛋白 H3K4 单甲基化的升高，并减弱 TGF-β1 引起的 α-SMA 的增加 [2]。
sininefungin抑制TGF-β1诱导的α- sma阳性肌成纤维细胞表达及TGF-β1诱导的肾细胞H3K4me1 [2]
如图6、7所示，sininefungin抑制H3K4me1，改善UUO诱导的肾纤维化。然而，这些结果来自UUO小鼠的全肾裂解物。为了明确sininefungin对TGF-β1诱导的H3K4甲基化的直接影响，我们在NRK-52E和NRK-49F细胞中进行了H3K4甲基化的免疫印迹。在NRK-52E（图8，a和B）和NRK-49F（图9，a和B）细胞中，sininefungin预处理显著降低TGF-β1诱导的α-SMA蛋白表达，并以剂量依赖性方式抑制H3K4me1。相比之下，无论在UUO肾脏体内还是体外上皮细胞（图8、C和D）和成纤维细胞（图9、C和D）中，丝霉素对H3K4me2和H3K4me3均无显著影响。最后，我们使用染色质免疫沉淀（ChIP）检测了TGF-β1是否会改变位于h3k4me1调节位点的通路。我们发现TGF-β1增加了NRK-52E细胞中胶原1 （Col1a1）、结缔组织生长因子（CTGF）和纤溶酶原激活物抑制剂-1 （PAI-1）启动子处的H3K4me1水平，而sininefungin抑制了H3K4me1水平（图8E）。

---

Sinefingin（A9145）和相关代谢产物A9145C被发现是新城疫病毒颗粒和痘苗病毒颗粒mRNA（鸟嘌呤-7-）-甲基转移酶和痘苗疫苗颗粒mRNA（核苷-2'-）-甲基移动酶的强效抑制剂。Sinefingn和A9145C都是这些S-腺苷-L-蛋氨酸依赖性酶的竞争性抑制剂，其抑制常数远小于S-腺苷-L-同型半胱氨酸。这些化合物还抑制了痘苗病毒在小鼠L细胞中的斑块形成[1]。

对于梗阻性肾病，Sinefungin/ 西奈芬净（每日 10 mg/kg）可在 UUO 治疗后立即改善肾纤维化 [2]。
Sinefungin/西奈芬净改善梗阻性肾病肾纤维化的作用机制[2]
鉴于开发肾纤维化新型治疗药物的需求，我们评估了SET7/9小分子抑制剂Sinefungin/西奈芬净对UUO小鼠间充质标志物及细胞外基质蛋白表达的影响。研究选取α-SMA和成纤维细胞特异性蛋白-1（FSP-1）作为间充质标志物，胶原蛋白1/3和纤连蛋白作为ECM指标。结果显示：在UUO术后3天和7天，Sinefungin/西奈芬净给药组肾脏中α-SMA、胶原蛋白1/3的mRNA表达均显著抑制（图6A）。Western blot分析证实辛伐汀可抑制α-SMA蛋白表达（图6B）。免疫组化显示UUO术后3天肾脏组织α-SMA、FSP-1、胶原蛋白1/3及纤连蛋白染色增强，7天时进一步加剧；而Sinefungin/西奈芬净治疗组这两个时间点的上述指标均显著改善（图6C、D）。但Sinefungin/西奈芬净对UUO诱导的TGF-β1 mRNA表达无显著影响（图6E）。

---

Sinefungin/西奈芬净通过抑制H3K4me1改善梗阻性肾病纤维化[2]
既往研究表明H3K4甲基化水平升高可促进TGF-β1诱导的纤维化基因转录激活。为明确Sinefungin/西奈芬净在肾纤维化进程中调控SET7/9的作用机制，我们检测了其对H3K4甲基化的影响。实验发现Sinefungin/西奈芬净能显著抑制UUO小鼠肾脏H3K4单甲基化（H3K4me1）（图7A），但对H3K4二甲基化（H3K4me2）和三甲基化（H3K4me3）水平无显著影响（图7B、C）。

Sinefungin和相关代谢产物A9145C被发现是新城疫病毒体和牛痘病毒体信使核糖核酸（鸟嘌呤-7-）-甲基转移酶和牛痘疫苗信使核糖核酸的有效抑制剂。Sinefingen和A9145C都是这些S-腺苷-L-蛋氨酸依赖性酶的竞争性抑制剂，其抑制常数显著小于S-腺苷-LH-半胱氨酸。这些化合物还抑制痘苗病毒在小鼠L细胞中形成斑块[1]。

蛋白质印迹分析[2]
细胞类型：肾上皮细胞。
测试浓度：0.5 或 1.0 µg/mL。
孵育时间：在 TGF-β1 (10 ng/mL) 之前预处理 60 分钟。
实验结果：在NRK-52E和NRK-49F细胞中，TGF-β1诱导的α-SMA蛋白表达显着减少，并且H3K4me1以剂量依赖性方式受到抑制。

---

细胞培养[2]
NRK-52E和NRK-49F细胞在含有5%FBS和青霉素/链霉素的DMEM中维持。在每次刺激之前，洗涤所有细胞，并在含有0%FBS的DMEM中抑制生长24小时。在TGF-β1（研发系统）刺激前60分钟，对Sinefingn进行预孵育。以指定的剂量水平和时间用TGF-β1处理NRK-52E和NRK-49F细胞。

---

siRNA体外转染[2]
将NRK-52E细胞铺在六孔培养皿中，6小时后（30%融合）根据制造商的说明，使用脂质体2000用Smad3 Silencer Select siRNA（si-Smad3，12.5 nM）或Silencer Select-阴性对照#1 siRNA（si-Neg）转染。6小时后，洗涤转染的细胞，并加入含有0%FBS的新鲜培养基。第二天，细胞用或不用TGF-β1处理，并在指定的时间段进行mRNA或蛋白质提取。

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.

---

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 mouse LD50 oral 1 gm/kg CRC Handbook of Antibiotic Compounds, Vols.1- , Berdy, J., Boca Raton, FL, CRC Press, 1980, 5(298), 1981
65482 mouse LD50 subcutaneous 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 that is the the delta-(5'-adenosyl) derivative of ornithine. It has a role as an antifungal agent and an antimicrobial agent. It is a member of adenosines and a non-proteinogenic alpha-amino acid. It is functionally related to a L-ornithine.
Sinefungin is a solid. This compound belongs to the purine nucleosides and analogues. These are compounds comprising a purine base attached to a sugar. The proteins that adenosyl-ornithine target include RdmB, modification methylase TaqI, rRNA (adenine-N6-)-methyltransferase, and modification methylase RsrI.
Sinefungin has been reported in Streptomyces griseolus and Streptomyces halstedii with data available.
Sinefungin is a natural nucleoside related to S-adenosylmethionine that has been isolated from Streptomyces species with antifungal, antiviral and antiparasitic activity. Sinefungin inhibits DNA methyltransferase which leads to an inhibition of DNA synthesis.

---

TGF-β1 activity results in methylation of lysine 4 of histone H3 (H3K4) through SET domain-containing lysine methyltransferase 7/9 (SET7/9) induction, which is important for the transcriptional activation of fibrotic genes in vitro. However, in vivo studies utilizing an experimental model of renal fibrosis are required to develop therapeutic interventions that target SET7/9. In this study, we investigated the signaling pathway of TGF-β1-induced SET7/9 expression and whether inhibition of SET7/9 suppresses renal fibrosis in unilateral ureteral obstruction (UUO) mice and kidney cell lines. Among the SET family, SET7/9 was upregulated on days 3 and 7 in UUO mice, and the upregulation was suppressed by TGF-β1 neutralizing antibody. TGF-β1 induced SET7/9 expression via Smad3 in normal rat kidney (NRK)-52E cells. In human kidney biopsy specimens from patients diagnosed with IgA nephropathy and membranous nephropathy, SET7/9 expression was positively correlated with the degree of interstitial fibrosis (r=0.59, P=0.001 in patients with IgA nephropathy; and r=0.58, P<0.05 in patients with membranous nephropathy). In addition, small interfering RNA-mediated knockdown of SET7/9 expression significantly attenuated renal fibrosis in UUO mice. Sinefungin, an inhibitor of SET7/9, also suppressed the expression of mesenchymal markers and extracellular matrix proteins and inhibited H3K4 mono-methylation (H3K4me1) in kidneys of UUO mice. Moreover, sinefungin had an inhibitory effect on TGF-β1-induced α-smooth muscle actin expression and H3K4me1 in both NRK-52E and NRK-49F cells. In conclusion, sinefungin, a SET7/9 inhibitor, ameliorates renal fibrosis by inhibiting H3K4me1 and may be a candidate therapeutic agent.[1]

---

In summary, SET7/9 expression is regulated by the TGF-β1–Smad3 pathway, leading to transcriptional activation of fibrotic genes through increased H3K4me1. We confirmed the actual expression of SET7/9 in renal biopsy samples from patients who were diagnosed with IgAN and MN, and showed that SET7/9 expression correlated with fibrotic areas. Inhibition of SET7/9 not only suppressed H3K4me1 levels but also ameliorated renal fibrosis in a mouse model of renal fibrosis. Furthermore, a small molecular inhibitor of SET7/9, sinefungin, also showed decreased H3K4me1 levels as well as suppressed fibrogenesis in vivo and in vitro. In conclusion, we identified inhibition of SET7/9 as a therapeutic target for kidney fibrosis, and suggest that sinefungin may be a candidate therapeutic agent for CKD patients.[1]

---

SINEFUNGIN is a small molecule drug with a maximum clinical trial phase of II.

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

208.65

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。