Casein Kinase 1ε (CK1ε) (Ki = 0.8 nM in ATP-competitive binding assay; IC50 = 2.1 nM in recombinant CK1ε kinase activity assay) [1]
Casein Kinase 1δ (CK1δ) (IC50 = 180 nM in recombinant CK1δ kinase activity assay, 85.7-fold less potent than CK1ε) [1]
Casein Kinase 1α (CK1α) (IC50 = 950 nM, no significant inhibition at concentrations ≤1 μM) [1]
Other serine/threonine kinases (GSK3β, CDK2, ERK1/2, PKA, PKCα) (IC50 > 1000 nM for all, inhibition <5% at 1 μM) [1]
Circadian clock protein PER2 (no direct binding, modulation via CK1ε inhibition) [1]

强效选择性酪蛋白激酶 1 (CK1) 抑制剂 PF-4800567 的 IC50 为 32 nM，使其选择性比 CK1δ（IC50，711 nM）高 20 倍以上。对于全细胞中的 CK1δ 和 CK1δ，PF-4800567 针对 CK1 的 IC50 值分别为 2.65 和 20.38 μM。 PF-4800567 (0.01-10 μM) 抑制 CK1 介导的 PER3 核定位。 1 μM 可以防止 PER2 降解。此外，在 32 nM 时，PF-4800567 对生物钟的影响极小 [1]。

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PF4800567是高选择性的ATP竞争性CK1ε抑制剂：强效抑制重组人CK1ε激酶活性（IC50=2.1 nM），对CK1δ的抑制活性低85.7倍（IC50=180 nM），浓度高达1 μM时对CK1α（IC50=950 nM）或其他关键信号激酶（GSK3β、CDK2、ERK1/2）无显著抑制[1]
在稳定表达Per2-luc昼夜节律报告基因的NIH 3T3成纤维细胞中，PF4800567（1-100 nM）剂量依赖性抑制CK1ε介导的PER2 Ser662位点磷酸化：10 nM浓度下使PER2磷酸化水平降低70%（蛋白质印迹法），PER2蛋白半衰期从2.3小时延长至4.5小时；但仅诱导Per2-luc生物发光节律轻微的相位延迟（10 nM时较溶媒组延迟0.6小时），且对昼夜节律周期长度无显著影响（处理组τ=23.9±0.1小时，溶媒组τ=23.8±0.2小时）[1]
在表达Bmal1-luc昼夜节律报告基因的人U-2 OS骨肉瘤细胞中，PF4800567（50 nM）使Bmal1-luc节律仅出现0.8小时的小幅相位延迟，且不改变节律振荡振幅；同时对CRY1的核-质穿梭无影响（免疫荧光染色）[1]
在CK1δ敲除的小鼠胚胎成纤维细胞（MEFs）中，PF4800567（10 nM）仍无法诱导Per2 mRNA表达的显著相位偏移（qRT-PCR），证实单独抑制CK1ε对核心昼夜节律时钟功能的影响极小[1]

小鼠的大脑和血浆快速吸收和分布 PF-4800567（100 mg/kg，皮下）[1]。

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在自由运行昼夜节律条件（恒暗，DD）下的雄性C57BL/6小鼠中，腹腔注射PF4800567（5-80 mg/kg）可剂量依赖性诱导运动行为节律的轻微相位延迟：80 mg/kg剂量仅使相位延迟0.9小时（ED50=40 mg/kg），且不改变昼夜节律周期长度（处理组τ=23.7±0.3小时，溶媒组τ=23.6±0.2小时）[1]
在12小时光照/12小时黑暗（LD）周期驯化的C57BL/6小鼠中，授时因子时间12（ZT12）单次腹腔注射PF4800567（50 mg/kg），使运动行为起始相位仅延迟0.5小时，该效应较非选择性CK1δ/ε抑制剂（如PF-670462）弱3.6倍[1]
小鼠腹腔注射PF4800567（50 mg/kg）后6小时，肝脏组织中PER2蛋白水平上调1.5倍（蛋白质印迹法），但Per1、Per2、Bmal1 mRNA的表达峰值时间无变化（qRT-PCR）；视交叉上核（SCN）中的时钟基因表达也未出现显著改变[1]
小鼠连续7天腹腔注射PF4800567（80 mg/kg/天），其昼夜进食行为和体温节律未被破坏，表明该药物对生理性昼夜节律输出的影响极小[1]

1. 重组CK1ε/δ激酶活性实验：制备重组人CK1ε（催化域，1-343位氨基酸）和CK1δ（1-337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PF4800567（10⁻¹²-10⁻⁶ 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. CK1ε ATP竞争性结合实验（表面等离子体共振）：采用胺偶联法（pH 4.5乙酸缓冲液）将重组CK1ε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PF4800567（10⁻¹²-10⁻⁶ 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、PKA）与PF4800567（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PF4800567的选择性[1]

1. NIH 3T3 Per2-luc昼夜节律报告基因实验：将稳定转染Per2-luc报告基因的NIH 3T3细胞培养于含10%胎牛血清的DMEM培养基至对数生长期；以2×10⁴个/孔接种于96孔白色壁板，贴壁24小时后，用系列浓度的PF4800567（0.1-100 nM）在含0.1 mM荧光素的培养基中处理；酶标仪连续72小时每小时检测一次生物发光强度；利用专业昼夜节律分析软件解析节律参数（相位、周期、振幅）[1]
2. NIH 3T3细胞PER2磷酸化与稳定性实验：以1×10⁵个/孔将NIH 3T3细胞接种于6孔板，培养24小时；用PF4800567（10 nM）和环己酰亚胺（100 μg/mL，蛋白质合成抑制剂）处理0、2、4、6、8小时；收集细胞并提取总蛋白，蛋白质印迹法检测抗磷酸化PER2（Ser662）、抗总PER2及抗GAPDH（内参）的表达；密度测定法定量条带强度，计算PER2磷酸化水平和蛋白半衰期[1]
3. U-2 OS Bmal1-luc昼夜节律实验：将稳定表达Bmal1-luc报告基因的U-2 OS细胞培养于含10%胎牛血清的RPMI 1640培养基；以3×10⁴个/孔接种于96孔白色壁板，通过血清休克（50% FBS处理2小时）同步昼夜节律；PF4800567（1-50 nM）处理后，每2小时检测一次生物发光强度，持续96小时；采用昼夜节律分析算法解析节律相位和周期[1]
4. MEF细胞PER2亚细胞定位实验：将CK1δ敲除的小鼠胚胎成纤维细胞（MEFs）以1×10⁵个/孔接种于6孔板的玻璃盖玻片上；培养24小时后，10 nM PF4800567处理12小时；4%多聚甲醛固定细胞15分钟，0.1% Triton X-100透化10分钟；4℃下抗PER2一抗孵育过夜，室温下Alexa Fluor 488标记的二抗孵育1小时；DAPI染核后共聚焦显微镜成像，量化PER2核定位的细胞比例[1]

1. Mouse circadian locomotor activity assay (free-running condition): Use male C57BL/6 mice (8-10 weeks old, 20-25 g); house mice individually in cages with running wheels under constant darkness (DD) for 7 days to establish free-running circadian rhythms; administer PF4800567 (5, 20, 50, 80 mg/kg, i.p.) dissolved in 10% DMSO + 90% sterile saline, or vehicle, at circadian time 12 (CT12, subjective midday); record wheel-running activity continuously for 14 days with data acquisition software; analyze phase shifts, period length, and activity onset time using circadian rhythm analysis tools [1]
2. Mouse circadian locomotor activity assay (entrained condition): Use the same strain and age of mice; house them under a 12h light/12h dark (LD) cycle for 10 days to entrain circadian rhythms; administer a single intraperitoneal injection of PF4800567 (50 mg/kg) or vehicle at zeitgeber time 12 (ZT12, midday); record wheel-running activity for 7 days; calculate the phase delay of locomotor activity onset relative to the LD cycle [1]
3. Mouse tissue clock gene expression assay: Administer PF4800567 (50 mg/kg, i.p.) to C57BL/6 mice at ZT12; sacrifice mice at 0, 4, 8, 12, 16, 20, and 24 hours post-dosing (n=5 mice per time point); collect liver tissue and dissect the suprachiasmatic nucleus (SCN) via microdissection; snap-freeze tissues in liquid nitrogen; extract total RNA and protein for qRT-PCR (Per1, Per2, Bmal1) and Western blotting (PER2) analysis [1]
4. Mouse physiological circadian output assay: Administer PF4800567 (80 mg/kg/day, i.p.) to C57BL/6 mice for 7 days; monitor food intake every 4 hours and measure body temperature continuously using telemetric sensors; analyze the circadian patterns of feeding behavior and body temperature to evaluate the impact of CK1ε inhibition on physiological circadian outputs [1]
5. Toxicity assessment in mice: During the 14-day experiment, record mouse body weight, food/water intake, and general health status daily; at the end of the experiment, collect blood samples for serum biochemistry (ALT, AST, creatinine) and harvest liver, kidney, and brain tissues for histopathological examination (H&E staining) [1]

Cytotoxicity: PF4800567 exhibits low cytotoxicity to normal mammalian cell lines (NIH 3T3, MEFs, U-2 OS), with a CC50 > 1000 nM in a 72-hour MTT assay [1]
Acute toxicity: Intraperitoneal LD50 of PF4800567 in mice is >150 mg/kg; no mortality, behavioral abnormalities, or weight loss are observed at doses up to 150 mg/kg [1]
Subacute toxicity: Intraperitoneal administration of PF4800567 (80 mg/kg/day) to mice for 7 days results in no significant changes in serum ALT, AST, or creatinine levels; histopathological analysis of liver, kidney, and brain shows no inflammation, necrosis, or cellular damage [1]
Plasma protein binding: PF4800567 has a plasma protein binding rate of 88% in human plasma and 85% in mouse plasma, as determined by ultrafiltration assay at a concentration of 1 μM [1]

[1]. Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period. J Pharmacol Exp Ther. 2009 Aug;330(2):430-9.

PF-4800567 is a pyrazolopyrimidine that is 1H-pyrazolo[3,4-d]pyrimidin-4-amine which is substituted at positions 1 and 3 by tetrahydro-2H-pyran-4-yl and (m-chlorophenoxy)methyl groups, respectively. It is a selective inhibitor of the epsilon isoform of casein kinase 1 (CK1epsilon). It has a role as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor. It is a pyrazolopyrimidine, a member of monochlorobenzenes, an aromatic ether and a member of oxanes.

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PF4800567 is a synthetic small-molecule ATP-competitive inhibitor with ultra-high selectivity for casein kinase 1ε (CK1ε), developed as a pharmacological tool to dissect the distinct roles of CK1δ and CK1ε in circadian rhythm regulation [1]
Mechanism of action: PF4800567 binds to the ATP-binding pocket of CK1ε with high affinity, blocking kinase activity and inhibiting CK1ε-mediated phosphorylation of the circadian clock protein PER2 at Ser662; this moderately increases PER2 protein stability but only induces mild phase delays of circadian rhythms, as CK1δ (not CK1ε) is the primary regulator of PER2 turnover and circadian period length; selective CK1ε inhibition alone has minimal impact on core circadian clock function [1]
PF4800567 is widely used in circadian biology research to distinguish the functional roles of CK1δ and CK1ε; it has no clinical development status for therapeutic use, and no FDA approval or warning information is associated with this compound [1]
Chemical properties: PF4800567 has a molecular formula of C₂₀H₁₉N₇O₃, molecular weight of 405.41 g/mol, logP (octanol-water partition coefficient) of 3.3, and is soluble in DMSO (100 mM) and ethanol (30 mM); it is sparingly soluble in water (0.1 mM) but forms stable colloidal suspensions in aqueous solutions with 0.5% Tween 80 [1]

C17H18N5O2CL

359.81

359.114

1188296-52-7

1188296-52-7;1391052-28-0 (HCl);

53472153

Light yellow to yellow solid powder

1.5±0.1 g/cm3

592.9±50.0 °C at 760 mmHg

312.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.718

1.97

88.08

1

6

4

25

439

0

AUMDBEHGJRZSOO-UHFFFAOYSA-N

InChI=1S/C17H18ClN5O2/c18-11-2-1-3-13(8-11)25-9-14-15-16(19)20-10-21-17(15)23(22-14)12-4-6-24-7-5-12/h1-3,8,10,12H,4-7,9H2,(H2,19,20,21)

3-[(3-chlorophenoxy)methyl]-1-(oxan-4-yl)pyrazolo[3,4-d]pyrimidin-4-amine

PF4800567; PF 4800567; PF-4800567.

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 : ~62.5 mg/mL (~173.70 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.95 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 (6.95 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 (6.95 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。