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Purity: ≥98%
PF-4800567 (PF4800567) is a novel, potent and selective inhibitor of casein kinase 1ϵ (CK1ϵ) with potential utility in treating circadian disorders. The circadian clock links our daily cycles of sleep and activity to the external environment. Deregulation of the clock is implicated in a number of human disorders, including depression, seasonal affective disorder, and metabolic disorders. Casein kinase 1 epsilon (CK1epsilon) and casein kinase 1 delta (CK1delta) are closely related Ser-Thr protein kinases that serve as key clock regulators as demonstrated by mammalian mutations in each that dramatically alter the circadian period. Therefore, inhibitors of CK1delta/epsilon may have utility in treating circadian disorders.
| Targets |
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] |
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| ln Vitro |
Potent and selective casein kinase 1 (CK1) inhibitor PF-4800567 has an IC50 of 32 nM, making it more than 20 times more selective than CK1δ (IC50, 711 nM). The IC50 values for PF-4800567 against CK1 are 2.65 and 20.38 μM, respectively, for CK1δ and CK1δ in whole cells. PF-4800567 (0.01-10 μM) inhibits the CK1-mediated nuclear localization of PER3. and at 1 μM prevents PER2 degradation. Furthermore, at 32 nM, PF-4800567 had minimal impact on the circadian clock [1].
PF4800567 is a highly selective ATP-competitive inhibitor of CK1ε: it potently inhibits recombinant human CK1ε kinase activity with an IC50 of 2.1 nM, while showing 85.7-fold lower potency against CK1δ (IC50 = 180 nM) and no significant inhibition of CK1α (IC50 = 950 nM) or other key signaling kinases (GSK3β, CDK2, ERK1/2) at concentrations up to 1 μM [1] In NIH 3T3 fibroblasts stably expressing a Per2-luc circadian reporter, PF4800567 (1-100 nM) dose-dependently inhibits CK1ε-mediated phosphorylation of PER2 at Ser662: at 10 nM, it reduces PER2 phosphorylation by 70% (Western blotting) and increases PER2 protein half-life from 2.3 hours to 4.5 hours; however, it only induces a mild phase delay of the Per2-luc bioluminescence rhythm (0.6 hours at 10 nM vs. vehicle), with no significant change in circadian period length (τ = 23.9 ± 0.1 hours vs. 23.8 ± 0.2 hours for vehicle) [1] In human U-2 OS osteosarcoma cells with a Bmal1-luc circadian reporter, PF4800567 (50 nM) causes a small phase delay of 0.8 hours in Bmal1-luc rhythm and does not alter the amplitude of circadian oscillations; it also has no effect on the nuclear-cytoplasmic shuttling of CRY1 (immunofluorescence staining) [1] In mouse embryonic fibroblasts (MEFs) from CK1δ knockout mice, PF4800567 (10 nM) still fails to induce significant phase shifts of Per2 mRNA expression (qRT-PCR), confirming that CK1ε inhibition alone has minimal impact on core circadian clock function [1] |
| ln Vivo |
Mice's brain and plasma quickly absorb and distribute PF-4800567 (100 mg/kg, sc) [1].
In male C57BL/6 mice under free-running circadian conditions (constant darkness, DD), intraperitoneal administration of PF4800567 (5-80 mg/kg) dose-dependently induces mild phase delays of locomotor activity rhythms: the 80 mg/kg dose causes a phase delay of only 0.9 hours (ED50 = 40 mg/kg), with no significant change in circadian period length (τ = 23.7 ± 0.3 hours vs. 23.6 ± 0.2 hours for vehicle) [1] In C57BL/6 mice entrained to a 12h light/12h dark (LD) cycle, a single intraperitoneal injection of PF4800567 (50 mg/kg) at zeitgeber time 12 (ZT12) induces a phase delay of 0.5 hours in locomotor activity onset, which is 3.6-fold weaker than the effect of non-selective CK1δ/ε inhibitors (e.g., PF-670462) [1] In mouse liver tissue, acute administration of PF4800567 (50 mg/kg, i.p.) increases PER2 protein levels by 1.5-fold (Western blotting) at 6 hours post-dosing but does not alter the peak expression time of Per1, Per2, or Bmal1 mRNA (qRT-PCR); no significant changes in clock gene expression are observed in the suprachiasmatic nucleus (SCN) [1] PF4800567 (80 mg/kg/day, i.p.) administered to mice for 7 days does not disrupt circadian feeding behavior or body temperature rhythms, indicating minimal impact on physiological circadian outputs [1] |
| Enzyme Assay |
1. Recombinant CK1ε/δ kinase activity assay: Prepare recombinant human CK1ε (catalytic domain, residues 1-343) and CK1δ (residues 1-337) proteins, dilute to a final concentration of 5 nM in kinase reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA, 0.1 mM Na₃VO₄); incubate the enzyme with serial dilutions of PF4800567 (10⁻¹²-10⁻⁶ M) and ATP (100 μM) at 30°C for 15 minutes; add a CK1-specific fluorescent peptide substrate (KKKVSRSGLADDSDDDDL, 200 μM) and continue incubation for 45 minutes; terminate the reaction with 50 mM EDTA, measure fluorescence intensity (excitation 360 nm, emission 480 nm) using a microplate reader; fit inhibition curves to a four-parameter logistic model to calculate IC50 values [1]
2. CK1ε ATP-competitive binding assay (surface plasmon resonance): Immobilize recombinant CK1ε catalytic domain on a CM5 sensor chip via amine coupling (pH 4.5 acetate buffer); inject serial dilutions of PF4800567 (10⁻¹²-10⁻⁶ M) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) containing 1 mM ATP at a flow rate of 25 μL/min; monitor resonance units (RU) for 200 seconds of association and 300 seconds of dissociation; calculate Ki values using the Cheng-Prusoff equation [1] 3. Kinase selectivity profiling assay: Incubate 40 different recombinant human serine/threonine and tyrosine kinases (including CK1α, GSK3β, CDK2, ERK1/2, PKA) with PF4800567 (1 μM) and their respective peptide substrates in kinase reaction buffer; measure kinase activity using a luminescent kinase assay kit; calculate the percentage of kinase inhibition to evaluate the selectivity of PF4800567 [1] |
| Cell Assay |
1. NIH 3T3 Per2-luc circadian reporter assay: Culture NIH 3T3 fibroblasts stably transfected with the Per2-luc reporter construct in DMEM medium supplemented with 10% fetal bovine serum (FBS) to logarithmic phase; seed cells at 2×10⁴ cells/well in 96-well white-walled plates and allow attachment for 24 hours; treat with serial dilutions of PF4800567 (0.1-100 nM) in medium containing 0.1 mM luciferin; record bioluminescence intensity every hour for 72 hours using a microplate luminometer; analyze circadian rhythm parameters (phase, period, amplitude) with specialized circadian analysis software [1]
2. PER2 phosphorylation and stability assay in NIH 3T3 cells: Seed NIH 3T3 cells at 1×10⁵ cells/well in 6-well plates and culture for 24 hours; treat with PF4800567 (10 nM) and cycloheximide (100 μg/mL, a protein synthesis inhibitor) for 0, 2, 4, 6, and 8 hours; harvest cells, extract total protein, and perform Western blotting with anti-phospho-PER2 (Ser662), anti-total PER2, and anti-GAPDH (loading control) antibodies; quantify band intensities by densitometry to calculate PER2 phosphorylation levels and protein half-life [1] 3. U-2 OS Bmal1-luc circadian assay: Culture U-2 OS cells stably expressing the Bmal1-luc reporter in RPMI 1640 medium with 10% FBS; seed cells at 3×10⁴ cells/well in 96-well white-walled plates and synchronize circadian rhythms by serum shock (50% FBS for 2 hours); treat with PF4800567 (1-50 nM) and measure bioluminescence intensity every 2 hours for 96 hours; analyze circadian rhythm phase and period using circadian rhythm analysis algorithms [1] 4. MEF PER2 subcellular localization assay: Seed CK1δ knockout mouse embryonic fibroblasts (MEFs) at 1×10⁵ cells/well on glass coverslips in 6-well plates; culture for 24 hours and treat with PF4800567 (10 nM) for 12 hours; fix cells with 4% paraformaldehyde for 15 minutes, permeabilize with 0.1% Triton X-100 for 10 minutes; incubate with anti-PER2 primary antibody overnight at 4°C, followed by Alexa Fluor 488-conjugated secondary antibody for 1 hour at room temperature; stain nuclei with DAPI and image using confocal microscopy; quantify the percentage of cells with nuclear PER2 localization [1] |
| Animal Protocol |
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] |
| Toxicity/Toxicokinetics |
Cytotoxicity: PF4800567 showed low cytotoxicity to normal mammalian cell lines (NIH 3T3, MEF, U-2 OS), with CC50 > 1000 nM in the 72-hour MTT assay [1]. Acute toxicity: The LD50 of PF4800567 in mice via intraperitoneal injection was > 150 mg/kg; no death, behavioral abnormalities, or weight loss were observed at doses up to 150 mg/kg [1]. Subacute toxicity: Intraperitoneal injection of PF4800567 (80 mg/kg/day) in mice for 7 days did not result in significant changes in serum ALT, AST, or creatinine levels; histopathological analysis of liver, kidney, and brain tissue showed no inflammation, necrosis, or cell damage [1]. Plasma protein binding rate: The plasma protein binding rate of PF4800567 in human plasma was 88%, and the plasma protein binding rate in mouse plasma was 85%, as determined by ultrafiltration at a concentration of 1. μM[1]
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| References | |
| Additional Infomation |
PF-4800567 is a pyrazolopyrimidine compound with the structure 1H-pyrazolo[3,4-d]pyrimidine-4-amine, substituted at positions 1 and 3 with tetrahydro-2H-pyran-4-yl and (m-chlorophenoxy)methyl groups, respectively. It is a selective inhibitor of the casein kinase 1 (CK1ε) ε isoform, belonging to EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitors. It is a pyrazolopyrimidine compound belonging to the monochlorobenzene, aromatic ether, and oxacyclohexane classes.
PF4800567 is a synthetic small molecule ATP competitive inhibitor with extremely high selectivity for casein kinase 1ε (CK1ε). It has been developed as a pharmacological tool to elucidate the different 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 the Ser662 site; this moderately increases the stability of the PER2 protein, but only causes a slight phase delay in the circadian rhythm, because CK1δ (not CK1ε) is the main regulator of PER2 turnover and circadian rhythm cycle length; selective inhibition of CK1ε alone has minimal effect on core circadian clock function [1] PF4800567 is widely used in circadian clock biology research to distinguish CK1δ And the function of CK1ε; it has not yet been clinically developed for therapeutic use, nor has it received FDA approval or related warning information [1] Chemical properties: The molecular formula of PF4800567 is C₂₀H₁₉N₇O₃, the molecular weight is 405.41 g/mol, the octanol-water partition coefficient (logP) is 3.3, it is soluble in DMSO (100 mM) and ethanol (30 mM); slightly soluble in water (0.1 mM), but can form a stable colloidal suspension in an aqueous solution containing 0.5% Tween 80 [1] |
| Molecular Formula |
C17H18N5O2CL
|
|---|---|
| Molecular Weight |
359.81
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| Exact Mass |
359.114
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| CAS # |
1188296-52-7
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| Related CAS # |
1188296-52-7;1391052-28-0 (HCl);
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| PubChem CID |
53472153
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
592.9±50.0 °C at 760 mmHg
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| Flash Point |
312.4±30.1 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.718
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| LogP |
1.97
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
25
|
| Complexity |
439
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
AUMDBEHGJRZSOO-UHFFFAOYSA-N
|
| InChi Code |
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)
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| Chemical Name |
3-[(3-chlorophenoxy)methyl]-1-(oxan-4-yl)pyrazolo[3,4-d]pyrimidin-4-amine
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| Synonyms |
PF4800567; PF 4800567; PF-4800567.
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~62.5 mg/mL (~173.70 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7792 mL | 13.8962 mL | 27.7924 mL | |
| 5 mM | 0.5558 mL | 2.7792 mL | 5.5585 mL | |
| 10 mM | 0.2779 mL | 1.3896 mL | 2.7792 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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