| Size | Price | Stock | Qty |
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| Targets |
CLOCK[1]
The primary target of CLK8 is the CLOCK protein. It binds to and interferes with CLOCK activity, specifically disrupting the protein-protein interaction between CLOCK and BMAL1. CLK8 does not affect the interaction of CLOCK with other mutants like CLOCK-F80A or K220A, confirming its selectivity for the native binding interface with BMAL1. |
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| ln Vitro |
In U2OS and NIH 3T3 cells, CLK8 (10-40 μM; 4-6 d) increases the amplitude of the Bmal1-dLuc signal in a dose-dependent manner without causing periodic variations [1]. In HEK293T cells, CLK8 (10–40 μM) diminishes the BMAL1–CLOCK connection while having no effect on the interactions between CLOCK–F80A, K220A, and BMAL1 [1]. In U2OS cells, CLOCK's nuclear localization is decreased by CLK8 (20 μM; 2 d) [1].
In vitro, CLK8 demonstrates its activity by modulating the amplitude of the circadian rhythm. In U2OS and NIH 3T3 cells, treatment with CLK8 at concentrations ranging from 10 to 40 microM for 4-6 days enhances the amplitude of the Bmal1-dLuc reporter signal in a dose-dependent manner, without altering the period of the oscillation. |
| ln Vivo |
In mouse liver whole cell lysates, CLK8 (25 mg/kg; a single ip) lowers CLOCK levels while leaving BMAL1 and CRY1 levels unaltered[1]. At dosages of 5 and 25 mg/kg, CLK8 (5-1000 mg/kg; ip) did not exhibit any mortality or clinical signs (dyspnea, hyporeflexia, decreased motor activity, piloerection, stooped posture, and corneal opacification)[1].
In vivo, CLK8 has been shown to effectively reduce CLOCK protein levels in mice. A single intraperitoneal (i.p.) injection of CLK8 at 25 mg/kg decreases CLOCK levels in whole-cell lysates of mouse livers. This reduction is specific, as the levels of other circadian proteins like BMAL1 and CRY1 remain unchanged. |
| Enzyme Assay |
A standard non-cellular assay for CLK8 has not been described in these documents, as its mechanism involves disrupting a protein-protein interaction. However, to study binding, one could perform a surface plasmon resonance (SPR) assay. Recombinant CLOCK and BMAL1 proteins would be immobilized on a chip, and various concentrations of CLK8 would be passed over to measure binding affinity and kinetics.
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| Cell Assay |
To measure the effect of CLK8 on the circadian clock, U2OS or NIH 3T3 cells stably expressing a Bmal1-dLuc reporter are used. Cells are synchronized with 100 nM dexamethasone for 2 hours. Then, media is replaced with fresh media containing varying concentrations of CLK8 (10-40 microM). Luminescence is recorded continuously for 4-6 days at 37degC in a luminometer to measure the effect on circadian amplitude and period.
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| Animal Protocol |
Animal/Disease Models: Male C57BL/6J mice (8 weeks, 18-24 g)[1]
Doses: 25 mg/kg Route of Administration: A single ip Experimental Results: A decrease in CLOCK levels was detected in whole cell lysates of the mouse livers, whereas the levels of BMAL1 and CRY1 were unaltered. diminished the abundance of CLOCK in the nucleus. The abundances of cytosolic and nuclear BMAL1 and CRY1 were unaltered. diminished Cry1 transcriptional level. An in vivo protocol for CLK8 involves administering the compound to C57BL/6J mice. A single dose of CLK8 at 25 mg/kg is prepared in a vehicle (e.g., 5% DMSO, 45% PEG300, and 50% water) and administered via intraperitoneal injection. After a specified time (e.g., 6-12 hours), mice are euthanized, and liver tissue is collected. CLOCK protein levels in liver lysates are then analyzed by Western blot. |
| ADME/Pharmacokinetics |
Specific pharmacokinetic data for CLK8 is not detailed. However, a toxicity study shows that it is well-tolerated. The fact that a single intraperitoneal dose of 25 mg/kg is sufficient to reduce CLOCK levels in the liver suggests that the compound is systemically bioavailable via this route of administration, but no detailed parameters like t1/2 or Cmax are provided.
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| Toxicity/Toxicokinetics |
A toxicity assessment was performed in mice. CLK8 was administered via intraperitoneal injection at doses ranging from 5 to 1000 mg/kg. At doses of 5 and 25 mg/kg, no mortality or clinical signs of toxicity were observed. Clinical signs that were monitored included dyspnea, reduced reflexes, decreased motor activity, piloerection, hunched posture, and corneal opacity.
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| References | |
| Additional Infomation |
CLK8 is a unique pharmacological tool because it targets the protein-protein interaction between CLOCK and BMAL1, rather than an enzymatic active site. This mechanism of action allows for the modulation of circadian rhythm amplitude without changing the period length, which could be beneficial for treating conditions associated with dampened circadian rhythms, such as certain sleep disorders or metabolic syndromes.
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| Molecular Formula |
C29H26N2O6
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|---|---|
| Molecular Weight |
498.526547908783
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| Exact Mass |
498.179
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| CAS # |
898920-65-5
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| PubChem CID |
4869040
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| Appearance |
White to off-white solid powder
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| LogP |
4.4
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
37
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| Complexity |
910
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(N)(=O)C1=CC=CC=C1NC(COC1=C2C(C3=CC=CC=C3)=CC(=O)OC2=C2CCC(C)(C)OC2=C1)=O
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| InChi Key |
SVELPNHVEDZZRQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H26N2O6/c1-29(2)13-12-19-22(37-29)15-23(35-16-24(32)31-21-11-7-6-10-18(21)28(30)34)26-20(14-25(33)36-27(19)26)17-8-4-3-5-9-17/h3-11,14-15H,12-13,16H2,1-2H3,(H2,30,34)(H,31,32)
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| Chemical Name |
2-[[2-[(8,8-dimethyl-2-oxo-4-phenyl-9,10-dihydropyrano[2,3-h]chromen-5-yl)oxy]acetyl]amino]benzamide
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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: 50 mg/mL (100.29 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0059 mL | 10.0295 mL | 20.0590 mL | |
| 5 mM | 0.4012 mL | 2.0059 mL | 4.0118 mL | |
| 10 mM | 0.2006 mL | 1.0029 mL | 2.0059 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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