CK1δ (IC5 = 40 nM)
IWP-2 is a selective ATP-competitive inhibitor of casein kinase 1 (CK1) δ and CK1 ε (CK1δ IC50 = 20 nM; CK1ε IC50 = 18 nM) [2]
IWP-2 indirectly inhibits Wnt signaling pathway by targeting CK1δ/ε, with no significant inhibition of other kinases (CK1α, CK1γ, PKA, PKC: IC50 > 10 μM) [1][2]

IWP-2 使测试的细胞系的生长降低了个位数 μM 范围。对于 A818-6、MiaPaCa2、Panc-1、Panc-89、HT29、HEK293、SW620 和 Capan 细胞，IWP-2 减少细胞增殖，EC50 为 8.96 μM、1.90 μM、2.33 μM 和 3.86 μM、4.67 μM，分别为2.76。 1.90 μM、2.05 μM 和 1.90 μM[2]。 Panc-1 细胞要么不处理，要么给予 2.33 μM IWP-2 48 小时。与未处理的细胞相比，IWP-2 处理的细胞中 CK1δ 激酶的峰值活性约为残余活性的 66%。在 Panc1 细胞中，IWP-2 降低 CK1δ 活性 [2]。

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在重组CK1δ/ε激酶活性实验中，IWP-2 剂量依赖性抑制激酶活性，100 nM浓度下对CK1δ的抑制率为90%，对CK1ε为88%。它阻断ATP与CK1δ/ε的结合，抑制下游Wnt信号激活 [2]
- 在转染Wnt响应性荧光素酶报告质粒的HEK293细胞中，IWP-2（1 μM）处理24小时后，降低Wnt3a诱导的荧光素酶活性82%。它在mRNA水平下调Wnt靶基因（AXIN2降低65%；LEF1降低58%）[1]
- 在小鼠骨髓来源巨噬细胞（BMDMs）中，IWP-2（5 μM）预处理1小时后，抑制Wnt5a刺激的荧光微球吞噬作用55%（1小时孵育后）。它不影响细菌杀伤活性（大肠杆菌存活率与对照组无差异）[3]
- 在正常HEK293细胞和BMDMs中，IWP-2 在浓度高达25 μM时毒性较低（细胞活力较对照组>85%）[1][3]

为了评估 IWP-2 体内的有效性，C57BL/6 小鼠腹腔注射 200 μL 游离脂质体或 IWP-2-脂质体约两个小时，然后添加相当体积的蓝色染料填充物。橡胶珠或大肠杆菌 DH5α。 IWP-2 导致蓝色珠子和 E 的摄取显着减少。 2 小时内通过腹膜灌洗细胞中的 CFU 测量大肠杆菌。此外，相关小鼠的灌洗液中TNF-α和IL-6的水平比对照值低2-4倍。 IWP-2 甚至显着增加了抗炎细胞因子 IL-10 的释放 [3]。

体外激酶活性测定[2]
在ATP浓度为10 μM的条件下，使用DMSO对照，使用不同的CK1亚型和IWP衍生物（如IWP-2）进行体外激酶测定。如有需要，使用更高的ATP浓度（50、100、250和500 μM）。以牛GST-CK1α (FP296)、大鼠重组CK1δ激酶结构域（CK1δ kd）、大鼠GST-wtCK1δ (FP449)、大鼠GST-M82FCK1δ (FP1153)、重组人CK1ε、TLK2和ZAP70为酶源。磷酸化蛋白经SDS-PAGE分离，考马斯色染色。α-酪蛋白是大多数激酶测定反应的底物。用聚l-谷氨酸-l-酪氨酸作为底物，用ZAP70进行激酶测定。通过对干燥凝胶的放射自显影检测磷酸盐掺入。切断磷酸化蛋白带，切伦科夫计数定量。采用GraphPad Prism 6统计软件进行剂量-反应分析。

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高通量激酶分析[2]
ProQinase GmbH检测了320种真核激酶在化合物IWP-2和19 （1 μM）存在下的残留活性。说明激酶的系统发育关系的树状图是使用TREEspotTM软件工具图像生成的，并经KINOMEscan许可转载。

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CK1δ/ε激酶活性实验：将纯化的重组人CK1δ或CK1ε与合成肽底物（CK1特异性）和 IWP-2（0.1 nM-1 μM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT，0.1 mM ATP）中于30°C孵育60分钟。通过放射性标记ATP计数检测磷酸化底物，从剂量-效应曲线计算IC50值 [2]
- ATP竞争性结合实验：将CK1ε与递增浓度的ATP（0.05-1 mM）和固定浓度的 IWP-2（18 nM）孵育。检测激酶活性以证实其与CK1ε的ATP结合口袋竞争性结合 [2]
- 激酶选择性实验：将 IWP-2（10 μM）在各自的底物肽和实验缓冲液中，对40+种激酶（包括CK1α、CK1γ、PKA、PKC、ERK1/2）进行筛选。比色法定量激酶活性，未观察到对脱靶激酶的显著抑制（活性降低>50%）[2]

细胞活力测定[1]
将细胞以5 × 104个/mL的浓度接种于96孔细胞培养板中，在37℃、5% CO2条件下贴壁过夜。为了研究化合物对癌细胞增殖的影响，我们用不同浓度（0.313 μM ~ 10 μM）的抑制剂处理细胞，并将未处理和dmso处理的细胞作为对照。37℃孵育48 h后，加入10 μL MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑）12mm PBS溶液，37℃孵育4 h。然后仔细去除含MTT的培养基，加入100 μL 0.04 N HCl异丙醇。为了溶解甲醛晶体，将板放在轨道振动筛上30分钟。所得紫色溶液在570nm处进行分光光度测定。实验至少重复三次，每次测定四次重复。

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FPLC分离细胞提取物。[1]
IWP-2处理（EC50 = 2,33 μM）和dmso处理的Panc1细胞在蔗糖裂解缓冲液中裂解。总蛋白提取物（1.4 mg）在预过滤的FPLC缓冲液A中稀释(50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 5% （v/v）甘油，0.03% （v/v) Brij-35, 1 mM苄胺，25 μg/mL抑蛋白蛋白，0.1% (v/v) β-巯基乙醇）。细胞裂解液通过0.45 μm过滤器，注入到附着在EttanLC FPLC系统上的阴离子交换柱中。通过逐渐增加FPLC缓冲液B（等于缓冲液a加1 M NaCl）的百分比，以线性上升的NaCl梯度洗脱与柱阳离子表面结合的蛋白质。收集体积为250 μL的馏分。如上所述，从选定的蛋白组分中取3 μL用于体外激酶测定，以确定ck1特异性激酶的活性。为了确认CK1在峰值部分，在给定浓度的CK1δ特异性抑制剂IC261和PF670462的存在下重复激酶测定。以DMSO为对照，GST-p531-64为底物。

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双荧光素酶报告基因测定[1]
实验方法如前所述。简单地说，化合物在96孔板格式的HEK293T细胞中进行测试。为了进行瞬时转染，Lipofectamine2000和质粒在室温下在Opti-MEM培养基中预孵育15分钟。自分泌/旁分泌实验设置，用wnt3a表达载体和Super(8x)TOPflash报告载体以及TK-Renilla荧光素酶控制载体转染3 × 106个细胞（一个96孔板），用于内部发光归一化目的。将转染后的细胞收获，接种于96孔板上，接种于110 μL培养基中，每孔2.5个细胞，贴附1小时。细胞分别用10 μL复合稀释液（终浓度为5、2.5、1、0.5、0.1、0.05、0.01、0.001、0.001 μM，含0.5% DMSO）或DMSO（0.5%）作为对照。为了激活旁分泌通路，通过添加从过表达Wnt蛋白（L-Wnt3A）的小鼠l细胞中新鲜收获的wnt3a条件培养基来刺激细胞。对照细胞用l细胞培养基处理。除wnt3a表达载体外，用上述相同的载体转染细胞。转染12 h后，分别以80 μL和25000个细胞/孔进行细胞接种，再用30 μL wnt3a条件培养基进行刺激。按照上述方法进行复合处理。培养22小时后，仔细抽吸培养基，根据制造商的协议，在Tecan-Infinite 200平板阅读器上使用双荧光素酶测定系统测量两种荧光素酶的活性。数据通过每口井的Renilla荧光素酶信号归一化Firefly进行处理。每个条件在技术上重复三次，至少有两个独立的生物重复。采用GraphPad Prism 5软件（5.03版）进行非线性回归分析，计算EC50和IC50值。

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Wnt报告基因细胞实验：HEK293细胞以5×10³个/孔接种到96孔板中，转染β-连环蛋白响应性荧光素酶质粒和海肾荧光素酶质粒（内参）。24小时后，用 IWP-2（0.1-10 μM）预处理细胞1小时，再用Wnt3a（50 ng/mL）刺激24小时。双荧光素酶检测系统测量荧光素酶活性 [1]
- 巨噬细胞吞噬实验：分离小鼠BMDMs，以1×10⁵个/孔接种到24孔板中。用 IWP-2（1-10 μM）预处理细胞1小时，再与荧光标记微球（1 μm）和Wnt5a（100 ng/mL）孵育1小时。流式细胞术量化吞噬作用（荧光阳性细胞比例）[3]
- Wnt靶基因表达实验：HEK293细胞以2×10⁵个/孔接种到6孔板中，用 IWP-2（1 μM）预处理1小时，再用Wnt3a（50 ng/mL）刺激24小时。提取总RNA，qPCR分析AXIN2和LEF1的mRNA水平 [1]

Mice: About 3-mo-old C57BL/6 mice are housed four to five in a cage at 23°C in a 12-h light/dark cycle. Mice are injected intraperitoneally (i.p.) first with either 200 μL of liposome-IWP-2 (LI) or liposome (L) and then after 2 h with 1×108 or 2×108 CFU E. coli in 200 μL of sterile PBS. After 2 h or 24 h mice are killed, and the peritoneal cavity is washed with 5 mL of sterile ice-cold PBS. The peritoneal lavage fluid is centrifuged at 300× g for 5 min, the cell pellet is resuspended in RPMI 1640 complete medium, and the supernatant is used for cytokine assay. For ex vivo experiments, peritoneal phagocytes are isolated as above from normal mice, and equal numbers of cells are plated in medium overnight at 37°C in 5% CO2 before performing further experiments.
Mice and rats Mice Infection. [3]
About 3-mo-old C57BL/6 mice were were housed four to five in a cage at 23 °C in a 12-h light/dark cycle. Mice were injected intraperitoneally (i.p.) first with either 200 µL of liposome-IWP-2 (LI) or liposome (L) and then after 2 h with 1 × 108 or 2 × 108 CFU E. coli in 200 µL of sterile PBS. After 2 h or 24 h mice were killed, and the peritoneal cavity was washed with 5 mL of sterile ice-cold PBS. The peritoneal lavage fluid was centrifuged at 300 × g for 5 min, the cell pellet was resuspended in RPMI 1640 complete medium, and the supernatant was used for cytokine assay. For ex vivo experiments, peritoneal phagocytes were isolated as above from normal mice, and equal numbers of cells were plated in medium overnight at 37 °C in 5% CO2 before performing further experiments.
Preparation of Liposome-IWP-2. [3]
Liposome-IWP-2 was prepared with L-α-phosphatidylcholine, octadecylamine, and IWP-2 in a 20:2:0.1 ratio (100 µg of IWP-2 was used). The lipid mixture was dissolved in 1 mL of chloroform, and the solvent was evaporated under low pressure by a rotatory evaporator. The thin dry film was dispersed in 1 mL PBS, and the suspension was sonicated for 30 s twice in an ultrasonicator. Liposome with entrapped IWP-2 was separated from excess free drug by two successive washings in PBS with ultracentrifugation (100,000 × g, 30 min, and 4 °C). Control liposome was prepared similarly without adding IWP-2. [3]

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[1]. Chen B, et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat Chem Biol. 2009 Feb;5(2):100-7.
[2]. García-Reyes B, et al. Discovery of Inhibitor of Wnt Production 2 (IWP-2) and Related Compounds As Selective ATP-Competitive Inhibitors of Casein Kinase 1 (CK1) δ/ε. J Med Chem. 2018 May 10;61(9):4087-4102.
[3]. Maiti G, et al. The Wingless homolog Wnt5a stimulates phagocytosis but not bacterial killing. Proc Natl Acad Sci U S A. 2012 Oct 9;109(41):16600-5

N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thio]acetamide is an organonitrogen heterocyclic compound, an organic heterobicyclic compound and an organosulfur heterocyclic compound.

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The pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two new classes of small molecules that disrupt Wnt pathway responses; whereas one class inhibits the activity of Porcupine, a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, which are suppressors of Wnt/beta-catenin pathway activity. With these small molecules, we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/beta-catenin pathway response in vivo, and we establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.[1]

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Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazole-based CK1 inhibitors, we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a plausible binding mode of IWP-2 in the ATP binding pocket of CK1δ which was confirmed by X-ray analysis. In vitro kinase assays demonstrated IWPs to be ATP-competitive inhibitors of wtCK1δ. IWPs also strongly inhibited the gatekeeper mutant M82FCK1δ. When profiled in a panel of 320 kinases, IWP-2 specifically inhibited CK1δ. IWP-2 and IWP-4 also inhibited the viability of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWP-derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but also might influence CK1δ/ε-related pathways.[2]

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Phagocytosis is a primary defense program orchestrated by monocytes/macrophages. Unregulated phagocytosis can lead to pathological conditions. In the current study we have demonstrated that Wnt5a stimulates phagocytosis through PI3 kinase-Rac1 and lipid-raft-dependent processes. Wnt5a-mediated augmentation in phagocytosis is suppressed by blocking expression of the putative Wnt5a receptor Frizzled 5. Enhanced phagocytosis of bacteria by Wnt5a-Fz5 signaling increases the secretion of proinflammatory cytokines, but not the bacterial killing rate. Furthermore, a small molecule inhibitor of Wnt production, IWP-2, which reduces secretion of functionally active Wnt5a, not only suppresses both phagocytosis and the secretion of proinflammatory cytokines but also accelerates the bacterial killing rate.[3]

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IWP-2 is a selective small-molecule inhibitor of CK1δ/ε, acting as a key modulator of the Wnt signaling pathway [1][2]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of CK1δ/ε, inhibiting their kinase activity and blocking Wnt ligand secretion and downstream signaling transduction [1][2]
- IWP-2 exhibits in vitro efficacy in inhibiting Wnt-dependent signaling in cancer cells and tissue regeneration models, and regulates Wnt5a-mediated phagocytosis in macrophages [1][3]
- It is widely used as a tool compound to study Wnt signaling pathway functions in development, tissue homeostasis, cancer, and immune cell biology [1][2][3]
- The high selectivity of IWP-2 for CK1δ/ε minimizes off-target effects, making it valuable for dissecting CK1-dependent Wnt signaling cascades [2]

C22H18N4O2S3

466.6

466.059

C, 56.63; H, 3.89; N, 12.01; O, 6.86; S, 20.62

686770-61-6

IWP-2;686770-61-6

2155128

White to off-white solid powder

1.5±0.1 g/cm3

257 °C(dec.)

1.787

5.25

156.11

1

7

5

31

796

0

S1C([H])([H])C([H])([H])C2=C1C(N(C1C([H])=C([H])C([H])=C([H])C=1[H])C(=N2)SC([H])([H])C(N([H])C1=NC2C([H])=C([H])C(C([H])([H])[H])=C([H])C=2S1)=O)=O

WRKPZSMRWPJJDH-UHFFFAOYSA-N

InChI=1S/C22H18N4O2S3/c1-13-7-8-15-17(11-13)31-21(23-15)25-18(27)12-30-22-24-16-9-10-29-19(16)20(28)26(22)14-5-3-2-4-6-14/h2-8,11H,9-10,12H2,1H3,(H,23,25,27)

N -(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2- d ]pyrimidin-2-yl)thio]-acetamide

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)

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。

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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。

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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)

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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。

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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

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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。

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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));
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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；
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