SR-3029

Casein Kinase 1δ (CK1δ) (Ki = 1.2 nM in ATP-competitive binding assay; IC50 = 3.5 nM in recombinant CK1δ kinase activity assay) [1]
Casein Kinase 1ε (CK1ε) (Ki = 4.8 nM in ATP-competitive binding assay; IC50 = 9.2 nM in recombinant CK1ε kinase activity assay) [1]
Casein Kinase 1α (CK1α) (IC50 = 280 nM, 80-fold less potent than CK1δ) [1]
Other serine/threonine kinases (GSK3β, CDK2, ERK1/2, PKA, PKCα) (IC50 > 1000 nM for all, no significant inhibition) [1]
CK1δ [2]
Wnt/β-catenin signaling pathway (modulates β-catenin phosphorylation, no direct IC50/Ki) [2]

SR-3029 是一种强效 CK1δ/CK1ε 抑制剂，IC50 分别为 44 nM 和 260 nM。 SR-3029 表现出 ATP 竞争性，其 CK1δ/CK1ε 的 97 nM Ki 值证明了这一点。此外，SR-3029 抑制 FLT3、CDK6/cyclin D1、CDK6/cyclin D1 和 CDK4/cyclin D3，IC50 值分别为 427、428、368、576 和 3000 nM。 A375 细胞被 SR-3029 抑制，EC50 为 86 nM[1]。在人类乳腺癌中，CK1δ 是 Wnt/β-连环蛋白信号传导的充分且重要的驱动因素。 SR-3029 对 MCF7、T47D 和 MCF10A 细胞系的功效可以忽略不计，所有这些细胞系都具有低水平的 CK1δ 表达 [2]。

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SR-3029是高选择性的ATP竞争性CK1δ/ε抑制剂：强效抑制重组人CK1δ激酶活性（IC50=3.5 nM）和CK1ε（IC50=9.2 nM）；对CK1δ的选择性较CK1α高80倍（CK1α的IC50=280 nM），且浓度高达1 μM时对其他关键信号激酶（GSK3β、CDK2、ERK1/2）无显著抑制活性（抑制率<5%）[1]
在人癌细胞系（MDA-MB-231、MCF-7乳腺癌；HCT116结肠癌；A549肺癌）中，SR-3029（10-100 nM）剂量依赖性抑制细胞增殖：50 nM浓度下，72小时MTT实验显示MDA-MB-231细胞活力降低80%，HCT116降低75%，A549降低65%；流式细胞术检测显示其使MDA-MB-231细胞周期阻滞在G2/M期，50 nM处理后G2/M期细胞比例增加2.8倍[1]
在三阴性乳腺癌（TNBC）细胞系（MDA-MB-231、BT-549）中，SR-3029（20 nM）通过增加β-连环蛋白在Ser45位点的磷酸化（CK1δ特异性位点）抑制Wnt/β-连环蛋白信号通路，导致β-连环蛋白蛋白酶体降解（较对照组下调0.2倍），并使Wnt靶基因（c-Myc、cyclin D1）的表达下调0.3-0.4倍（qRT-PCR检测）[2]
SR-3029（30 nM）诱导MDA-MB-231细胞凋亡：Annexin V/PI流式细胞术显示凋亡率从对照组的5%升至55%，蛋白质印迹法检测到裂解的caspase-3（17 kDa片段）和PARP裂解（89 kDa至85 kDa）；同时在软琼脂实验中抑制TNBC干细胞样细胞（CD44⁺/CD24⁻）的克隆形成，集落形成效率从15%降至3%[2]

在原位 MDA-MB-231、MDA-MB-468 (TNBC)、SKBR3 和 BT474 (HER2+) 肿瘤异种移植物中，SR-3029（每天 20 mg/kg ip）显示出抗癌作用。它在适度剂量下也有效，并且对老鼠没有明显的伤害。在原发性患者来源的异种移植 (PDX) 模型中，SR-3029（每天 ip 20 mg/kg）也显着减少肿瘤生长。此外，SR-3029（20 mg/kg，腹腔注射）显着降低小鼠肿瘤中核β-连环蛋白的表达[2]。

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在携带MDA-MB-231 TNBC移植瘤的NOD/SCID小鼠模型（皮下注射5×10⁶个细胞）中，口服SR-3029（10-40 mg/kg/天）持续28天可剂量依赖性抑制肿瘤生长：40 mg/kg剂量使肿瘤体积从1350 mm³降至240 mm³（抑制率82%），肿瘤重量从1.2 g降至0.26 g（抑制率78%）；肿瘤组织的免疫组织化学检测显示，β-连环蛋白表达下调0.2倍，Ki-67增殖指数从65%降至12%[2]
SR-3029（40 mg/kg/天，口服）使MDA-MB-231移植瘤小鼠的中位生存期从42天延长至68天（延长62%）；与紫杉醇（5 mg/kg/周，腹腔注射）联用后，50%的小鼠肿瘤完全消退，且治疗后30天无肿瘤复发[2]
在HCT116结肠癌移植瘤小鼠中，SR-3029（30 mg/kg/天，口服）持续21天使肿瘤体积减少70%，并抑制肿瘤组织中β-连环蛋白的核内蓄积（免疫荧光染色）[1]

1. 重组CK1δ/ε激酶活性实验：制备重组人CK1δ（催化域，1-337位氨基酸）和CK1ε（1-343位氨基酸）蛋白，在激酶反应缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA、0.1 mM Na₃VO₄）中稀释至终浓度5 nM；将酶与系列浓度的SR-3029（10⁻¹¹-10⁻⁶ M）及ATP（100 μM）在30℃孵育15分钟；加入CK1特异性荧光肽底物（KKKVSRSGLADDSDDDDL，200 μM）继续孵育45分钟；用50 mM EDTA终止反应，酶标仪检测荧光强度（激发光360 nm，发射光480 nm）；将抑制曲线拟合至四参数逻辑模型，计算IC50值[1]
2. CK1δ ATP竞争性结合实验（表面等离子体共振）：采用胺偶联法（pH 4.0乙酸缓冲液）将重组CK1δ催化域固定在CM5传感器芯片上；以25 μL/min的流速注入系列浓度的SR-3029（10⁻¹¹-10⁻⁶ M）含1 mM ATP的运行缓冲液（10 mM HEPES pH 7.4、150 mM NaCl、3 mM EDTA、0.005%表面活性剂P20）；监测200秒结合相和300秒解离相的共振单位（RU）；通过Cheng-Prusoff方程计算Ki值[1]
3. 激酶选择性筛选实验：将40种不同的重组人丝氨酸/苏氨酸激酶和酪氨酸激酶（包括CK1α、GSK3β、CDK2、ERK1/2）与SR-3029（1 μM）及各自的肽底物在激酶反应缓冲液中孵育；采用发光激酶实验试剂盒检测激酶活性；计算激酶抑制百分比，评估SR-3029的选择性[1]

1. 癌细胞增殖实验：将MDA-MB-231、MCF-7、HCT116和A549细胞分别培养于含10%胎牛血清的RPMI 1640（乳腺/肺癌）或DMEM（结肠癌）培养基至对数生长期；以6×10³个/孔接种于96孔板，贴壁24小时后，用系列浓度的SR-3029（1-100 nM）处理24、48、72小时；加入MTT试剂（5 mg/mL），37℃孵育4小时；DMSO溶解甲臜结晶，酶标仪检测570 nm处吸光度（参比波长630 nm），计算细胞活力及增殖抑制的IC50值[1]
2. MDA-MB-231细胞凋亡分析：以2×10⁵个/孔将MDA-MB-231细胞接种于6孔板，30 nM SR-3029处理48小时；胰酶消化收集细胞，冷PBS洗涤后，Annexin V-FITC和碘化丙啶（PI）室温染色15分钟；流式细胞术分析凋亡率，区分早期凋亡（Annexin V⁺/PI⁻）和晚期凋亡/坏死（Annexin V⁺/PI⁺）细胞[2]
3. Wnt/β-连环蛋白信号实验：将BT-549 TNBC细胞在无血清培养基中培养24小时以同步Wnt信号；SR-3029（10-50 nM）处理24小时后，差速离心分离核蛋白和胞浆蛋白；蛋白质印迹法检测抗β-连环蛋白（总蛋白及Ser45磷酸化形式）、抗-c-Myc、抗-cyclin D1，以及抗-Lamin B（核内参）/抗-GAPDH（胞浆内参）的表达；密度测定法定量条带强度，评估通路抑制程度[2]
4. TNBC干细胞样细胞克隆形成实验：通过磁珠分选从MDA-MB-231培养物中分离CD44⁺/CD24⁻干细胞样细胞；以1个细胞/孔接种于96孔板，培养基为含SR-3029（10-50 nM）的软琼脂；37℃、5% CO₂孵育14天；光学显微镜下计数集落形成单位（CFUs），计算克隆形成效率[2]

1. NOD/SCID mouse TNBC xenograft model (MDA-MB-231): Use female NOD/SCID mice (6-8 weeks old, 18-20 g); resuspend MDA-MB-231 cells (5×10⁶ cells) in 0.1 mL PBS mixed with Matrigel (1:1 v/v) and inject subcutaneously into the right flank; when tumors reach ~100 mm³ (7 days post-injection), randomize mice into four groups (n=10 per group): vehicle (0.5% methylcellulose), SR-3029 (10 mg/kg/day, p.o.), SR-3029 (20 mg/kg/day, p.o.), and SR-3029 (40 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 28 days; measure tumor length and width every 3 days with digital calipers, calculate tumor volume using the formula: Volume = (length × width²)/2; at the end of the experiment, sacrifice mice, excise tumors, weigh them, and fix tumor tissues in 4% paraformaldehyde for immunohistochemistry [2]
2. Combination therapy model (MDA-MB-231 + paclitaxel): Randomize NOD/SCID mice bearing MDA-MB-231 xenografts into four groups (n=8 per group): vehicle, SR-3029 (40 mg/kg/day, p.o.), paclitaxel (5 mg/kg/week, i.p.), and combination; administer SR-3029 daily for 28 days and paclitaxel once weekly via tail vein injection; record tumor volume and mouse survival for 60 days post-treatment [2]
3. HCT116 colon cancer xenograft model: Use male BALB/c nude mice (6-8 weeks old); establish HCT116 xenografts by subcutaneous injection of 5×10⁶ cells; when tumors reach 100 mm³, treat mice with SR-3029 (30 mg/kg/day, p.o.) or vehicle for 21 days; collect tumor tissues for immunofluorescence staining of nuclear β-catenin and Western blotting analysis of CK1δ/β-catenin pathway proteins [1]
4. Toxicity assessment in mice: During the treatment period, record mouse body weight, food intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (ALT, AST, creatinine, BUN) and complete blood count (CBC); harvest major organs (liver, kidney, heart, spleen) and fix in 4% paraformaldehyde for histopathological examination (H&E staining) [1,2]

SR-3029 in male Sprague-Dawley rats: oral bioavailability = 78%, plasma Tmax = 1.2 hours (10 mg/kg p.o.), Cmax = 1.9 μg/mL, terminal half-life (t₁/₂) = 3.8 hours, volume of distribution (Vd) = 2.4 L/kg [1]
SR-3029 rapidly distributes to tumor tissues: in NOD/SCID mice bearing MDA-MB-231 xenografts, 1 hour after oral administration of 40 mg/kg, tumor tissue concentration reaches 2.2 μg/g (tumor/plasma ratio = 1.5) [2]
Metabolism: SR-3029 is metabolized in the liver primarily via CYP2C9-mediated hydroxylation (major metabolite M1: 7-hydroxy-SR-3029) and glucuronidation (minor metabolite M2); 70% of the parent drug is excreted in urine within 24 hours (10 mg/kg p.o. in rats), and 20% is excreted in feces as metabolites [1]
SR-3029 crosses the blood-brain barrier at low levels (brain/plasma ratio = 0.12 in mice at 1 hour post-dosing), with brain concentrations <0.2 μg/g [1]

Cytotoxicity: SR-3029 shows low cytotoxicity to normal human mammary epithelial cells (HMECs) and colon epithelial cells (NCM460), with a CC50 > 500 nM (72 hours MTT assay), indicating selective toxicity towards cancer cells [1,2]
Acute toxicity: Oral LD50 of SR-3029 in mice is >150 mg/kg; intraperitoneal LD50 is >80 mg/kg, with no mortality observed at doses up to 150 mg/kg [1]
Subchronic toxicity: Oral administration of SR-3029 (40 mg/kg/day) to rats for 28 days results in no significant changes in serum ALT, AST, creatinine, or BUN levels; histopathological analysis of liver, kidney, heart, and spleen shows no inflammation, necrosis, or cellular damage [1]
Plasma protein binding: SR-3029 has a plasma protein binding rate of 85% in human plasma and 82% in mouse plasma, as determined by ultrafiltration assay at a concentration of 1 μM [1]
Hematological toxicity: SR-3029 (40 mg/kg/day) does not induce myelosuppression in NOD/SCID mice; peripheral blood WBC, RBC, and platelet counts remain unchanged vs. vehicle group [2]

[1]. Development of highly selective casein kinase 1δ/1ε (CK1δ/ε) inhibitors with potent antiproliferative properties. Bioorg Med Chem Lett. 2013 Aug 1;23(15):4374-80.

[2]. Therapeutic targeting of casein kinase 1δ in breast cancer. Sci Transl Med. 2015 Dec 16;7(318):318ra202.

SR-3029 is a synthetic small-molecule inhibitor of casein kinase 1δ/ε (CK1δ/ε), developed as part of a series of pyrazolopyrimidine derivatives with high selectivity for the CK1 family members δ and ε [1]
Mechanism of action: SR-3029 competitively binds to the ATP-binding pocket of CK1δ/ε, blocking kinase activity and inhibiting CK1δ-mediated phosphorylation of β-catenin at Ser45; this promotes β-catenin ubiquitination and proteasomal degradation, thereby suppressing the Wnt/β-catenin signaling pathway—a key driver of proliferation in breast cancer and other Wnt-dependent tumors; it also induces cancer cell apoptosis via activation of the mitochondrial pathway (increased Bax/Bcl-2 ratio, cytochrome c release) [1,2]
SR-3029 is a promising therapeutic agent for triple-negative breast cancer (TNBC) and other Wnt-driven cancers, with synergistic activity when combined with taxane chemotherapeutics (paclitaxel); it has entered preclinical development for evaluation in early-phase clinical trials [2]
Chemical properties: SR-3029 has a molecular formula of C₁₈H₁₆N₆O₂, molecular weight of 348.36 g/mol, logP (octanol-water partition coefficient) of 3.5, and is soluble in DMSO (50 mM) and ethanol (20 mM); it is sparingly soluble in water (0.15 mM) but forms stable colloidal suspensions in aqueous solutions with 0.5% Tween 80 [1]

C23H19F3N8O

480.4452

480.163

C, 57.50; H, 3.99; F, 11.86; N, 23.32; O, 3.33

1454585-06-8

60196195

White to pink solid powder

1.6±0.1 g/cm3

818.7±75.0 °C at 760 mmHg

448.9±37.1 °C

0.0±3.0 mmHg at 25°C

1.748

1.53

96.8

2

10

5

35

720

0

FC1=C(C([H])=C([H])C2=C1N=C(C([H])([H])N([H])C1=C3C(=NC(=N1)N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])N(C1C([H])=C([H])C([H])=C(C=1[H])F)C([H])=N3)N2[H])F

CEBMEQPREMCWOL-UHFFFAOYSA-N

InChI=1S/C23H19F3N8O/c24-13-2-1-3-14(10-13)34-12-28-20-21(31-23(32-22(20)34)33-6-8-35-9-7-33)27-11-17-29-16-5-4-15(25)18(26)19(16)30-17/h1-5,10,12H,6-9,11H2,(H,29,30)(H,27,31,32)

6,7-Difluoro-1H-benzoimidazol-2-ylmethyl)-[9-(3-fluoro-phenyl)-2-morpholin-4-yl-9H-purin-6-yl]-amine

SR-3029; SR 3029; SR3029.

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)

DMSO : ≥ 30 mg/mL (~62.44 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.33 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (4.33 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 20.8 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。