K02288

ALK1 (IC50 = 1.8 nM); ALK2 (IC50 = 1.1 nM); ALK3 (IC50 = 34.4 nM); ALK6 (IC50 = 6.3 nM); ALK4 (IC50 = 302 nM); ALK5 (IC50 = 321 nM)[1]

K02288 在减少 BMP4 刺激诱导的 Smad1/5/8 强磷酸化方面表现出 100 nM 的表观 IC50。 0.5 μM 的 K02288 几乎完全抑制 Smad2 磷酸化 [1]。 K02288 的激酶铰链上连接有两个氢键，因此与 ALK1 的结合方式类似于 ATP。在 HUVEC 中，K02288 还会引起过度萌发表型并抑制 BMP9-ALK1 信号传导 [2]。

K02288诱导斑马鱼胚胎背化[1]
为了进一步验证K02288作为药理学工具的作用，我们测试了它对Tg(BRE:mRFP)转基因斑马鱼胚胎的影响，斑马鱼胚胎在BMP反应元件的控制下表达单体红色荧光蛋白(mRFP)。完整的BMP信号已被证明是横跨背腹轴的组织祖细胞的适当规范所必需的。K02288以剂量依赖的方式诱导dorsalized表型(图5A)，正如之前dorsomorphin所示。在8-10µM浓度下观察到严重背侧化表型(图5B)，与mRFP蛋白表达缺失相关(图5C) [1]。

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K02288诱导鸡胚CAM模型血管生成功能障碍[2]
在鸡胚CAM模型中，K02288 (1 μM)促进血管生成缺陷[2]。K02288诱导的过度发芽效应与Notch通路破坏时观察到的效果相似[8]，这表明K02288增加了尖端细胞的规格，可能导致血管形成功能障碍。为了评估K02288是否会干扰体内血管生成，我们使用了鸡胚胎绒毛膜尿囊膜(CAM)模型，该模型可以方便地可视化和量化血管生成。同样，K02288的效果与ALK1-Fc相似(图4a)。两种治疗方法均观察到血管生成中断的两种不同表型。处理CAM模型的一个子集显示与3D培养模型一致的超芽。此外，血管新生芽周围可见阴影和光晕，提示血管功能不全。大部分CAM模型显示出低血管密度的独特表型，反映了过度发芽后发生的功能失调的血管生成(图4a)。因此，与ALK1-Fc类似，小分子抑制剂K02288在体内具有抑制血管生成的潜力。[2]

ALK1-6的体外激酶测定[1]
ALK1-6的激酶反应在室温下于96孔板中进行45分钟，混合2.5 nM激酶，0.5 mg/mL去磷酸化酪蛋白(Sigma)， 6µM ATP, 0.05µCi/µL [γ-32P]ATP, 10 mM MnCl2和0.2% BSA的激酶缓冲液。在激酶反应缓冲液中加入浓度为0至10µM的抑制剂，并进行三次试验。反应用磷酸淬灭，结合到96孔P81磷酸纤维素滤板上，用Microscint 20闪烁液使用Spectramax L发光计进行测定。将数据归一化为未处理的100%酶活性对照，并减去阴性对照作为背景。

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Kinase-Glo®测定[1]
按照制造商的说明，使用激酶- glo®进行ActRIIA (ACVR2)的激酶测定。简单地说，将以下物质在96孔板中混合并在室温下反应3小时，最终体积为100µL, 10 nM激酶(2小时时EC50)， 0.5 mg/mL去磷酸化酪蛋白，10µM ATP, 10 mM MnCl2和激酶缓冲液中的0.2% BSA。在激酶反应缓冲液中加入浓度为0至10µM的抑制剂，并重复测试。在15分钟、30分钟、1小时、2小时和3小时时，将20µL的反应混合物转移到384孔板上，加入20µL的激酶- glo®，静置10分钟以熄灭反应并产生光，使用Spectramax L发光计测量。2小时的时间点在反应的线性部分内，由于良好的信噪比，因此用于计算，并且与较早的时间点一致。将数据归一化为未处理的100%酶活性对照，并减去阴性对照作为背景。

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Kinase-wide Selectivity Profiling/激酶范围选择性分析[1]
通过Nanosyn (www.nanosyn.com)对200种人激酶在0.1和1µM抑制剂浓度下的选择性进行分析。

Sprouting assays/发芽试验[2]
按照Nakatsu等人的描述[23]，将HUVEC培养成球体(500个细胞/球体)，并包埋在纤维蛋白凝胶中。在EGM-2培养基中，将K02288或ALK1-Fc添加到凝胶的顶部。每2天更换一次介质。在添加抑制剂2天后对芽数和芽长进行量化，并在处理4天后获得图像。实验至少重复三次，误差条表示SEM。

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转染和双荧光素酶测定[2]
用脂质体LTX试剂转染800 ng RBPJ荧光素酶构建物和200 ng Renilla荧光素酶于10 cm培养皿中。24 h后，将细胞在低血清培养基中复制于包被sDll4或BSA的24孔板中。细胞附着5小时，K02288加药30分钟，10 ng/mL BMP9再加药16小时。根据制造商的方案进行双荧光素酶测定。

Chemical Inhibitor Treatment of Zebrafish[1]
Inhibitor stocks were diluted in DMSO and then further diluted in fish water to give the required inhibitor concentrations. To minimize the amount of inhibitors used, experiments were performed in 24-well plates with approximately 20 embryos per well in a volume of 1 mL. The chemical treatment was applied by immersing 8- to 16-cell stage Tg(BRE:mRFP) embryos (dorsalization experiment) or 12 hours old Tg(fli1a:eGFP) embryos (ISV experiment) in the fish water supplemented with DMSO or the chemical inhibitors. For the ISV experiment, embryos were manually dechorionated after bud stage before treatment. Embryos were scored and photographed at 26 or 48 hours.

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CAM assays[2]
Fertilised chicken eggs were incubated at 37 °C with a relative air humidity of 65 %. On embryo development day 3 (EDD 3), a hole of approximately 3 mm in diameter was opened in the eggshell, and on EDD 6, the hole in the shell was extended to a diameter of approximately 3 cm. A polyethylene ring was deposited on the CAM and 100 μL of either K02288, ALK1-Fc or PBS was pipetted inside the ring. After 4 more days (EDD 10), the vessels were visualised under a microscope and representative pictures acquired.

[1]. A new class of small molecule inhibitor of BMP signaling. PLoS One. 2013 Apr 30;8(4):e62721.

[2]. A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis. Angiogenesis. 2015 Apr;18(2):209-17.

Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-β signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development.[1]

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Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-β receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology.[2]

C20H20N2O4

352.3838

352.142

C, 68.17; H, 5.72; N, 7.95; O, 18.16

1431985-92-0

46173038

Off-white to yellow solid powder

1.2±0.1 g/cm3

522.2±50.0 °C at 760 mmHg

269.6±30.1 °C

0.0±1.4 mmHg at 25°C

1.614

3.13

86.83

2

6

5

26

424

0

O(C([H])([H])[H])C1C(=C(C([H])=C(C=1[H])C1=C(N([H])[H])N=C([H])C(C2C([H])=C([H])C([H])=C(C=2[H])O[H])=C1[H])OC([H])([H])[H])OC([H])([H])[H]

CJLMANFTWLNAKC-UHFFFAOYSA-N

InChI=1S/C20H20N2O4/c1-24-17-9-13(10-18(25-2)19(17)26-3)16-8-14(11-22-20(16)21)12-5-4-6-15(23)7-12/h4-11,23H,1-3H3,(H2,21,22)

3-(6-amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenol

K02288; K 02288; 3-[6-Amino-5-(3,4,5-Trimethoxyphenyl)pyridin-3-Yl]phenol; 3-(6-amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenol; A3F; CHEMBL1230714; K-02288

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 (7.09 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (7.09 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 3 中的溶解度: ≥ 2.5 mg/mL (7.09 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 储备液加入到 900 μL 玉米油中并混合均匀。

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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;
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。