SRPIN340 (SRPK inhibitor)

SRPK1/serine arginine protein kinase 1 (Ki = 0.89 μM)

体外活性：SRPIN340 在 Flp-In293 细胞中抑制 SRPK 的 SR 磷酸化，并以剂量依赖性方式促进 SRp75 降解，从而抑制 HIV 产生。 SRPIN340 剂量 (5 mg/mL) 不会导致 CHO 细胞的染色体结构和染色体数量出现异常。 SRPIN340 在体外以剂量依赖性方式抑制 HCV 亚基因组复制子的表达和 HCV-JFH1 克隆的复制。细胞分析：SRPIN340 已被证明可以抑制 HIV-1 和其他需要 SR 蛋白依赖性 RNA 加工才能在 HIV-1 转染或感染的 Flp-In293 细胞系中繁殖的病毒的复制。此外，据报道，SRPIN340 可显着抑制 SRPK1 和 SRPK2 激酶活性，但不会有效抑制其他 SRPK，例如 Clk1 和 Clk4，SRPK1 的 Ki 值为 0.89μM。此外，SRPIN340 已被证明可以剂量依赖性地促进 SRp75 的降解，而 SRp75 是 HIV 表达所必需的。此外，SRPIN340 显示出对 Sindbis 病毒增殖的抑制作用，IC50 为 60μM，并能防止 Sindbis 病毒的细胞病变作用。

SRPIN340 在体内以剂量依赖性方式抑制 CNV 形成。 SRPIN340 显着降低 VEGF、MCP-1、ICAM-1 的蛋白水平，从而抑制巨噬细胞浸润。

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为了确定SRPK阻断是否抑制CNV的形成，我们量化了服用或不服用SRPIN340的RPE脉络膜复合物平片中的CNV大小。激光损伤后7天，与赋形剂治疗的动物（30737±3758μm2，n=31，p<0.05；图2A，B）相比，用2 pmolSRPIN340治疗的动物的平均CNV大小（19870±1935μm2）显著减小（n=33；n表示CNV损伤的数量）。此外，与2 pmol SRPIN340治疗的动物相比，更高剂量的SRPIN340给药（20 pmol；n=23）显著降低了CNV大小（15649±1803μm2，p<0.01），而较低剂量的给药（0.2 pmol；n=17）没有显著抑制CNV的形成（21741±3695μm2，p=0.10；图2A，B）。单独接受激光损伤的小鼠与接受激光和玻璃体内注射赋形剂溶液的小鼠之间，CNV大小没有显著差异。数据表明，SRPK阻断以剂量依赖的方式抑制CNV生长。[3]

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为了研究SRPIN340对Vegf亚型的影响，使用实时PCR分析了总Vegf和含外显子8a的Vegf亚基的mRNA表达。与用0.1%DMSO处理的小鼠（n=8；n表示眼睛数量）相比，用20pmol SRPIN340（n=6）处理的小鼠RPE脉络膜复合物中总Vegf的mRNA表达显著降低了56%（p<0.05；图3A）。同样，含Vegf外显子8a的mRNA表达显著降低了57%（p<0.05；图3B）。此外，用20pmol SRPIN340治疗的小鼠的总VEGF浓度（209.2±10.9 pg/mg，n=8）明显低于用0.1%DMSO治疗的小鼠（274.2±17.9 pg/mg）（n=10，p<0.01；图3C）。[3]

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SRPIN340以30 mg ml−1的浓度溶解在丙二醇（80%）和DMSO（20%）（但不是20%DMSO/80%水）中。通过卵圆管饲法以单次100μl剂量（100 mg kg−1）给药，并对血液和组织进行取样和质谱分析。在血浆中，1小时后检测到SRPIN340的浓度为1.55±0.91μg ml−1，4小时和8小时后分别降至0.43±0.19和0.77±0.2μg ml-1。24小时后，SRPIN340的血浆浓度为0.2±0.06μg ml−1。进行了单相指数衰减曲线拟合，SRPIN340在血浆中的持续半衰期（从1小时开始拟合的曲线）为13.49小时。然而，在递送的3 mg SRPIN中，血浆SRPIN340总量估计为2.0μg（30 g小鼠，45%红细胞压积，80 ml kg−1血容量），而胃中的浓度为100μg ml-1，表明药物吸收不良（补充图1）。此外，全身给药需要高浓度的DMSO（20%）。因此，我们尝试在体内局部注射SRPIN340以避免全身治疗。将未转导的A375细胞皮下注射并使其形成肿瘤。与DMSO（1%）对照注射的肿瘤相比，在靠近肿瘤部位的100μl 1×PBS中每天皮下注射2μg SRPIN340可显著降低肿瘤生长（P<0.001；单因素方差分析-Bonferroni事后；图5C）。肿瘤后分析显示，SRPIN340治疗的肿瘤中VEGF总表达降低（P<0.05，Student非配对t检验），抗血管生成VEGFxxxb亚型的检测没有差异，似乎不受治疗的影响（图5D）。在这项研究中，与敲除不同，肿瘤的大小足以切片和染色CD31作为微血管密度（MVD）的指标。与载体治疗的肿瘤相比，SRPIN340显著降低了MVD（图5E）。

体外激酶抑制活性和动力学分析。[1]
His6标记的mSRPK1、mSRPK2、mClk1和mClk4在大肠杆菌（BL21）中表达，并按所述纯化。酶和底物与指定浓度的ATP和SRPIN340一起孵育（如图3所示）。如所述测量SRPK1激酶活性。 使用SigmaPlot软件通过Lineweaver–Burk Plot分析抑制活性。[1]

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酶联免疫吸附试验[3]
每只眼睛放置四个激光损伤，并玻璃体内注射1μl 0.1%DMSO或SRPIN340。根据制造商的方案，使用酶联免疫吸附测定试剂盒测定上清液中VEGF、单核细胞趋化蛋白（MCP）-1和细胞间粘附分子（ICAM）-1的蛋白质水平，并将其标准化为总蛋白。

在 96 孔板中，接种白血病细胞（5 × 10⁴ 细胞/孔）和分离的 PBMC（8 × 10⁴ 细胞/孔）。每个孔装有 100 μL 完整 RPMI 培养基和 100 μL SRPIN340 溶液，其浓度各不相同。将 10% 胎牛血清和 0.4% DMSO (v/v) 添加到 RPMI 培养基中以稀释化合物。培养 48 小时（3 小时，37°C）后，将 MTT (5 mg/mL) 添加到孔中。室温下 30 分钟并以 500 × g 离心后，从板中除去 MTT 溶液，并添加 100 μL/孔的 DMSO 以溶解甲臜。使用酶标仪测量 540 nm 处的吸光度。每个实验方案都运行三次[2]。

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细胞活力测定细胞增殖通过两种方法测定。将每孔30000个A375细胞接种在24孔板上，这些细胞用打乱的shRNA、SRPK1-shRNA转导或未转导，并用SRPIN340处理。每24小时对细胞进行胰蛋白酶处理，并进行细胞计数。接种在盖玻片上的细胞也被Ki67染色。对于划痕试验，细胞在24孔板中生长至融合，并沿孔的中心线从板上刮下1mm厚的细胞线。在时间零点、12小时后和24小时后对每个孔进行成像。将划痕的覆盖百分比确定为伤口闭合百分比的衡量标准。[4]

mouse model with choroidal neovascularization (CNV)
~20 pmol
i.v.

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SRPIN340 (50 mM in 100% dimethyl sulfoxide, DMSO) was diluted with phosphate buffered saline (PBS, potassium chloride, 2.68 mM; potassium phosphate monobasic, 1.47 mM; sodium chloride, 136.89 mM; sodium phosphate dibasic, 8.10 mM) to various concentrations in 0.1% DMSO before treatment. Mice were divided into five groups: CNV induction alone (the control group) and CNV induction with 1 μl intravitreal injection of either 0.1% DMSO, 0.2 pmol, 2 pmol, or 20 pmol SRPIN340. Intravitreal injection was performed using a 33-gauge needle immediately after laser photocoagulation.[3]

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In vivo tumour model All animal experiments were carried out under a UK Home Office License after approval by the University of Bristol Ethical Review Group. A375, A375 shRNA control and A375 shRNA SRPK1 knockdown cells were cultured in T75 flasks to 80% confluence. Trypsinised cells were counted using a haemocytometer, and 2 million cells of A375 shRNA control and A375 shRNA SRPK1 were injected subcutaneously either into the left and right flanks of nude mice, or a single injection of untransduced A375 cells. Tumour-bearing mice (>3 mm) were weighed and tumours were measured by caliper bi-weekly. Mice bearing A375-untransfected tumours were treated with either 100 μl of 20 μg ml−1 SRPIN340 (diluted 100 × in PBS from 2 mg ml−1 stock in DMSO), or 100 μl of 1% DMSO vehicle control injected daily into the peritumoral space. [4]

[1]. Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11329-33.

[2]. Antimicrob Agents Chemother. 2010 Aug;54(8):3179-86.

[3]. Mol Vis. 2013:19:536-43.

[4]. Br J Cancer. 2014 Jul 29;111(3):477-85.

Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.[1]

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Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.[2]

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Purpose: To investigate the applicability of serine/arginine-rich protein kinase (SRPK)-specific inhibitor, SRPIN340, for attenuation of choroidal neovascularization (CNV) formation using a mouse model.[3]
Methods: Laser photocoagulation was performed to induce CNV in C57BL/6J mice, followed by intravitreal injection of SRPIN340 or vehicle. Seven days after the treatment, the CNV size was evaluated using a flatmount technique. Protein levels of vascular endothelial growth factor (VEGF) and inflammation-associated molecules, such as monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1, in the retinal pigment epithelium-choroid complex were measured with enzyme-linked immunosorbent assay. Expression levels of total Vegf, exon 8a-containing Vegf isoforms, and F4/80 (a specific marker for macrophage) were assessed using real-time PCR.[3]
Results: SRPIN340 inhibited CNV formation in a dose-dependent manner. Compared with the vehicle, SRPIN340 significantly decreased the protein levels of VEGF, MCP-1, ICAM-1, and consequently inhibited macrophage infiltration. Furthermore, SRPIN340 suppressed the gene expression levels of total Vegf and exon 8a-containing Vegf isoforms.[3]
Conclusions: SRPIN340, a specific inhibitor of SRPK, suppressed Vegf expression and attenuated CNV formation. Our data suggest the possibility that SRPIN340 is applicable for neovascular age-related macular degeneration as a novel chemical therapeutics.[3]

C18H18F3N3O

349.35

349.14

C, 61.88; H, 5.19; F, 16.31; N, 12.03; O, 4.58

218156-96-8

2797577

White to light yellow solid powder

1.3±0.1 g/cm3

395.9±42.0 °C at 760 mmHg

193.3±27.9 °C

0.0±0.9 mmHg at 25°C

1.578

4.15

45.23

1

6

3

25

445

0

FC(C1C([H])=C([H])C(=C(C=1[H])N([H])C(C1C([H])=C([H])N=C([H])C=1[H])=O)N1C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])(F)F

DWFGGOFPIISJIT-UHFFFAOYSA-N

InChI=1S/C18H18F3N3O/c19-18(20,21)14-4-5-16(24-10-2-1-3-11-24)15(12-14)23-17(25)13-6-8-22-9-7-13/h4-9,12H,1-3,10-11H2,(H,23,25)

N-[2-piperidin-1-yl-5-(trifluoromethyl)phenyl]pyridine-4-carboxamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.16 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 8 mg/mL

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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;
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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。