Chk1 (IC50 = 3 nM); CDK2 (IC50 = 0.16 μM)

SCH 900776 是一种效果较差的 CDK2 和 Chk2 抑制剂，IC50 值分别为 0.16 μM 和 1.5 μM。 SCH 900776 不会显着抑制细胞色素 P450 1A2、2C9、2C19、2D6 和 3A4 的人肝微粒体亚型。接触羟基脲 24 小时后，SCH 900776 会导致 DNA 复制能力出现剂量依赖性丧失。 SCH 900776 增强羟基脲、5-氟尿嘧啶和阿糖胞苷 γ-H2AX 反应。当 SCH 900776 与抗代谢物结合时，会导致 γ-H2AX 在不到两小时内积累，这是双链 DNA 断裂和复制叉崩溃的标志。此外，SCH 900776 剂量依赖性地抑制 Chk1 pS296 自磷酸化的积累。暴露于 SCH 900776 后，正常细胞的循环群体会诱导 Chk1 pS345 作为无效循环的一部分，可能由 AT 家族激酶和 DNA-PK 驱动。 Chk1 pS345 的这种快速、剂量依赖性积累与增殖的 WS1 细胞暴露于 SCH 900776 有关。[1]

当吉西他滨半小时后给予 SCH 900776 时，4 mg/kg 足以引起 γ-H2AX 生物标志物，8 mg/kg 比单独使用 SCH 900776 或吉西他滨产生更好的肿瘤药效学和消退反应。 SCH 900776 剂量的增加（16 mg/kg 和 32 mg/kg）导致肿瘤反应逐渐改善。至关重要的是，在 BALB/c 小鼠中，与强生物标志物激活和更好的肿瘤反应相关的 SCH 900776 剂量与吉西他滨对血液学参数的毒性增加无关。 [1]

以基于CDC25C的生物素化肽为底物，杆状病毒表达系统表达的重组His-Chk1为酶源的体外实验。在含有 50 mM Tris pH8.0、10 mM MgCl₂ 和 1 mM DTT 的激酶缓冲液中，His-Chk1 被稀释至 32 nM。将 CDC25C（CDC25 Ser216 C 末端生物素化肽）肽在激酶缓冲液中稀释至浓度 1.93 μM。为了在添加起始溶液后产生 6.2 nM Chk1、385 nM CDC25C 和 1% DMSO 的最终反应浓度，将 20 μL 32 nM Chk1 酶溶液和 20 μL 1.926 μM CDC25C 混合并与 10对于每个激酶反应，用 10% DMSO 稀释 μL SCH 900776。添加 50 μL 起始溶液（其中含有 2 μM ATP 和 0.2 μCi ³³P-ATP）启动反应，最终反应浓度为 1 μM ATP 和 0.2 μCi 每个反应 33 P-ATP。激酶反应在室温下运行 2 小时，并通过添加 100 μL 终止液来终止，终止液由 2 M NaCl、1% H₃PO₄ 和 5 组成。 mg/mL 链霉亲和素包被的 SPA 珠。 Filtermate 通用收集器与 96 孔 GF/B 过滤板组合用于收集 SPA 珠子。 2 M NaCl 和 2 M NaCl 加 1% 磷酸均用于洗珠两次。之后，用 TopCount 96 孔液体闪烁计数器测量信号。使用 SCH 900776 在八个点上连续稀释一式两份来创建剂量反应曲线。通过使用非线性回归分析，获得IC50值。

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激酶测定[1]
CHK1、CHK2和CDK激酶测定已在前面描述。Millipore激酶分析器服务用于生成SCH 900776对多种丝氨酸/苏氨酸和酪氨酸激酶的一般选择性数据。通常在两种浓度的SCH 900776（0.5和5μmol/L）和固定浓度的ATP（10μmol/L）下进行检测。数据以剩余活动百分比的形式提供，与未受抑制的对照组相比。

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使用温度依赖性荧光进行亲和力评估[1]
在白色96孔PCR板中，将1μmol/L CHK1重组激酶结构域蛋白（氨基酸残基2-274）与微摩尔浓度（通常为1-50μmol/L）的化合物在20μL测定缓冲液（25 mmol/L HEPES，pH 7.4，300 mmol/L NaCl，5 mmol/L二硫苏糖醇，2%二甲亚砜，Sypro Orange 5x）中混合。该板用透明条密封，并放置在热循环仪中。在25°C至95°C的熔化过程中，每0.5°C的增量监测一次荧光强度。数据被导出到Excel中，并经过专有的自定义曲线拟合算法（未公布），以得出温度依赖性荧光（TdF）Kd值。对于CHK1 TdF数据，通常使用双态结合模型（化合物结合天然和热展开的熔融球态）。与靶激酶的熔融球态结合的化合物通常比天然状态弱1000倍以上。由于结合焓变化的不确定性，所有TdF Kd值的误差幅度约为50%。

γ-H2AX测定[1]
简单地说，将细胞暴露于抗代谢物中以诱导CHK1的激活。对照人群不进行治疗。然后在2小时的暴露窗口（在抗代谢物存在的情况下）将 SCH 900776 滴定到细胞上。在SCH 900776共暴露2小时后，将细胞固定并渗透（70%乙醇），然后用异硫氰酸荧光素（FITC）偶联抗γ- h2ax单克隆抗体染色。细胞用碘化丙啶反染，随后使用流式细胞仪（Becton Dickinson LSR II）或Discovery 1免疫荧光平台进行分析。实验通常进行三次，数据以γ-H2AX阳性细胞的百分比表示，从而反映了γ-H2AX表型的总体外显率。

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用活性caspase>评价凋亡诱导 [1]
SCH 900776。然后清洗细胞，去除所有抗代谢物和SCH 900776。在这一点（T0或释放）评估Caspase活性，并在T + 24和T + 48小时进行进一步的检测。细胞随后用荧光标记的caspase底物孵育；细胞内底物的摄取和荧光与激活的半胱天冬酶水平相关。然后用流式细胞术测定表达活化半胱天冬酶的细胞百分比。

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溴脱氧尿苷掺入试验[1]
将细胞镀于10厘米的组织培养皿中，使其粘附。将细胞暴露于不同浓度的SCH 900776中超过2小时，无论事先是否有抗代谢物暴露。然后清洗细胞，并允许24小时尝试恢复s期。随后短暂（30分钟）暴露于溴脱氧尿苷（BrdU），以评估能够以活的方式重新进入细胞周期的细胞百分比。然后收获细胞，固定并渗透。随后进行酸变性步骤，以暴露基因组DNA中合并的BrdU表位，之后用fitc偶联的BrdU特异性单克隆抗体对样品进行免疫染色。然后用碘化丙啶对细胞进行反染色，以评估DNA含量并使用流式细胞术进行分析。BrdU阳性染色和碘化丙啶信号的双变量分析可以评估正在进行DNA合成的细胞数量和细胞系的整体细胞周期分布（G1， S， G2-M和亚G1）。每个浓度下每个种群的百分比

Female nude mice injected subcutaneously with A2780 or MiaPaCa2 cells
~50 mg/kg
Administered intraperitoneally

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In vivo tumor growth assessments, sampling, and skin biopsies [1]
For tumor implantation, specific cell lines were grown in vitro, washed once with PBS and resuspended in 50% Matrigel (BD Biosciences) in PBS to a final concentration of 4 × 107 to 5 × 107 cells per mL. Nude mice were injected with 0.1 mL of this suspension subcutaneously in the flank region. Tumor length (L), width (W), and height (H) were measured by a caliper twice a week on each mouse and then used to calculate tumor volume using the formula: (L × W × H)/2. Animals (N = 10) were randomized to treatment groups and treated intraperitoneally with either SCH 900776 (formulated in 20% hydroxypropyl β-cyclodextrin) or individual chemotherapeutic agents, formulated as recommended. Tumor volumes and body weights were measured during and after the treatment periods. Data were recorded as means ± SEM before being normalized to starting volume. Time to progression to 10x starting volume (TTP 10x) was monitored in some experiments. Animals were euthanized according to Institutional Animal Care and Use Committee guidelines. For pharmacodynamic marker analyses in mice, tumors and adjacent skin were collected at necropsy, fixed overnight in 10% formalin, and washed/stored in 70% ethanol. For skin punch biopsies, an area of approximately 4 square inches was shaved. Rats were anesthetized using inhaled isofluorane and dogs were locally anesthetized using subcutaneous administration of lidocaine. Samples were collected using a 4 mm biopsy punch. Skin punches were fixed in 10% formalin overnight before washing/storage in 70% ethanol.

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Pharmacokinetic determinations [1]
Plasma samples from test species were collected at various times after administration of SCH 900776. At each time-point, blood samples from 3 animals were combined and analyzed for SCH 900776 by LC/MS. Pharmacokinetic variables were estimated from the plasma concentration data. Cmax values (maximum plasma concentration) were taken directly from the plasma concentration-time profiles, and the area under the plasma concentration versus time curve area under curve (AUC) was calculated using the linear trapezoidal rule.

Furthermore, CHK1 pS345 positive cells were detected in skin punch biopsies taken from mice at SCH 900776 doses ≤25 mg/kg (75 mg/m2), in rats dosed IV at 5 and 10 mg/kg (30 and 60 mg/m2) and from dogs dosed IV at 2.5 and 5 mg/kg (45 and 89 mg/m2; Supplementary Fig. S10A to C). These data and the associated plasma exposures (pharmacokinetics) are summarized in Table 4 and comprise a pharmacological audit trail of SCH 900776 activity in three relevant preclinical species.

[1]. Mol Cancer Ther . 2011 Apr;10(4):591-602.

[2]. Mol Cancer Ther . 2012 Feb;11(2):427-38.

[3]. Clin Cancer Res . 2012 Oct 1;18(19):5364-73.

[4]. Nat Genet . 2021 Aug;53(8):1207-1220.

6-bromo-3-(1-methyl-4-pyrazolyl)-5-(3-piperidinyl)-7-pyrazolo[1,5-a]pyrimidinamine is a pyrazolopyrimidine.
See also: Mk-8776 (annotation moved to).

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Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds. [1]

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Many anticancer agents damage DNA and arrest cell-cycle progression primarily in S or G(2) phase of the cell cycle. Previous studies with the topoisomerase I inhibitor SN38 have shown the efficacy of the Chk1 inhibitor UCN-01 to overcome this arrest and induce mitotic catastrophe. UCN-01 was limited in clinical trials by unfavorable pharmacokinetics. SCH900776 is a novel and more selective Chk1 inhibitor that potently inhibits Chk1 and abrogates cell-cycle arrest induced by SN38. Like UCN-01, abrogation of SN38-induced arrest enhances the rate of cell death but does not increase overall cell death. In contrast, SCH900776 reduced the growth-inhibitory concentration of hydroxyurea by 20- to 70-fold. A similar magnitude of sensitization was observed with cytarabine. A 5- to 10-fold sensitization occurred with gemcitabine, but no sensitization occurred with cisplatin, 5-fluorouracil, or 6-thioguanine. Sensitization occurred at hydroxyurea concentrations that marginally slowed DNA replication without apparent activation of Chk1, but this led to dependence on Chk1 that increased with time. For example, when added 18 hours after hydroxyurea, SCH900776 induced DNA double-strand breaks consistent with rapid collapse of replication forks. In addition, some cell lines were highly sensitive to SCH900776 alone, and these cells required lower concentrations of SCH900776 to sensitize them to hydroxyurea. We conclude that some tumors may be very sensitive to the combination of SCH900776 and hydroxyurea. Delayed administration of SCH900776 may be more effective than concurrent treatment. SCH900776 is currently in phase I clinical trials, and these results provide the rationale and schedule for future clinical trials.[2]

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Purpose: Previous studies have shown that the replication checkpoint, which involves the kinases ataxia telangiectasia mutated and Rad3 related (ATR) and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical acute myelogenous leukemia (AML) during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design: AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA, and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results: Immunoblotting revealed increased Chk1 phosphorylation, a marker of checkpoint activation, in more than half of Chk1-containing AMLs after 48 hours of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S-phase arrest but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the antiproliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions: These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted.[3]

C₁₅H₁₈BRN₇

376.25

375.08

C, 47.88; H, 4.82; Br, 21.24; N, 26.06

891494-64-7

SCH900776;891494-63-6

16224745

white to off-white Solid powder

1.8±0.1 g/cm3

1.819

0.76

86.06

2

5

2

23

425

0

NC1=C(Br)C([C@@H]2CNCCC2)=NC2=C(C3=CN(C)N=C3)C=NN12

GMIZZEXBPRLVIV-VIFPVBQESA-N

InChI=1S/C15H18BrN7/c1-22-8-10(6-19-22)11-7-20-23-14(17)12(16)13(21-15(11)23)9-3-2-4-18-5-9/h6-9,18H,2-5,17H2,1H3/t9-/m0/s1

6-bromo-3-(1-methylpyrazol-4-yl)-5-[(3S)-piperidin-3-yl]pyrazolo[1,5-a]pyrimidin-7-amine

MK 8776; MK8776; MK-8776; SCH900776; 891494-64-7; SCH900776 S-isomer; SCH900776 (S-isomer); (S)-6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(piperidin-3-yl)pyrazolo[1,5-A]pyrimidin-7-amine; 6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-((3S)-piperidin-3-yl)pyrazolo(1,5-a)pyrimidin-7-amine; UNII-99Y1V29WVE; 99Y1V29WVE; MK-8776 S-isomer; SCH 900776; SCH-900776

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: ~75 mg/mL (~199.3 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.64 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 (6.64 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 (6.64 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 4% DMSO+30% propylene glycol: 5 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;
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。