| Size | Price | Stock | Qty |
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| 5mg |
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| Targets |
PKR[1]
Pyruvate kinase R (PKR) (activator) |
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| ln Vitro |
PKR activator 4 is a potent activator of pyruvate kinase R (PKR), the red blood cell-specific isoform of pyruvate kinase. PKR catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, generating ATP in the process. Activation of PKR increases ATP levels and decreases 2,3-DPG levels in red blood cells. In vitro assays demonstrate that PKR activator 4 increases PKR enzymatic activity in a concentration-dependent manner. No specific EC50 value is publicly available, but the compound is described as a "potent" activator. PKR activator 4 was disclosed in patent WO2019035864A1 as an example 7A, indicating it is one of a series of compounds with PKR activating activity.
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| ln Vivo |
No specific in vivo data for PKR activator 4; however, as a potent PKR activator, it has potential for in vivo efficacy in mouse models of sickle cell disease (e.g., Berkeley SCD mice or Townes SCD mice). PKR activation in vivo would be expected to reduce red blood cell sickling, improve red blood cell survival, decrease hemolysis, and reduce markers of inflammation and vaso-occlusion. Based on the patent description, PKR activator 4 has potential for the research of blood disorders, suggesting that in vivo studies may have been conducted but not publicly detailed. In vivo studies would involve oral administration to SCD mouse models, measurement of red blood cell PKR activity ex vivo, and assessment of hematological parameters (hemoglobin, hematocrit, reticulocyte count), hemolysis markers (bilirubin, LDH), and red blood cell sickling under hypoxic conditions.
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| Enzyme Assay |
Recombinant human pyruvate kinase-R (PKR) enzyme is expressed in E. coli or insect cells and purified. The PKR activity assay measures the conversion of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP, coupled to NADH oxidation via lactate dehydrogenase (LDH). The reaction mixture contains Tris-HCl buffer (pH 7.4), KCl (100 mM), MgCl2 (10 mM), PEP (0.5-5 mM), ADP (0.5-5 mM), NADH (0.2-0.5 mM), and LDH (10 U/mL). The reaction is initiated by adding PKR enzyme (10-50 ng). The decrease in absorbance at 340 nm (NADH oxidation) is monitored over 5-10 minutes at 37degC using a spectrophotometer or plate reader. The initial velocity (V0) is calculated. For activator screening, compounds are tested at varying concentrations (0.001-100 uM) and the fold-increase in activity relative to control (DMSO vehicle) is calculated. EC50 values are determined from dose-response curves (concentration giving 50% of maximal activation). PKR activator 4 is tested in this assay to confirm PKR activation. The compound was identified as example 7A in patent WO2019035864A1. For detailed protocols, refer to the patent.
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| Cell Assay |
For cellular PKR activation studies, human erythroblast cell lines (e.g., BEL-A or HUDEP-2) or primary human red blood cells are used. Cells are cultured in appropriate media (e.g., StemSpan medium with growth factors). Cells are treated with PKR activator 4 (0.01-100 uM) for 24-72 hours. Red blood cell PKR activity is measured in cell lysates using the PKR activity assay described above. Intracellular ATP levels are measured using a bioluminescence assay (e.g., CellTiter-Glo or ATP bioluminescence kit). 2,3-DPG levels are measured by an enzymatic colorimetric assay (2,3-DPG assay kit). The ratio of ATP to 2,3-DPG is calculated. Hemoglobin oxygen affinity is measured using a Hemox Analyzer or by assessing the P50 value (partial pressure of oxygen at which hemoglobin is 50% saturated). For sickling assays, red blood cells from sickle cell disease patients are treated with PKR activator 4 (0.1-10 uM) and exposed to hypoxic conditions (e.g., 2% O2 or sodium metabisulfite). The percentage of sickled red blood cells is quantified by light microscopy. Cell viability is assessed by MTT or by counting cells with trypan blue. Apoptosis (Annexin V/PI staining) and red blood cell morphology are also assessed. No specific cellular data for PKR activator 4 is publicly available; these are generic protocols for PKR activators.
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| Animal Protocol |
No published in vivo animal study for PKR activator 4. Based on typical protocols for PKR activators, in vivo efficacy studies can be conducted in mouse models of sickle cell disease (e.g., Berkeley SCD mice or Townes SCD mice). The compound is formulated in a suitable vehicle (e.g., 10% DMSO + 90% corn oil or 0.5% methylcellulose) and administered orally (gavage) once or twice daily at doses ranging from 1-50 mg/kg for 2-8 weeks. Endpoints include: red blood cell PKR activity (ex vivo assay), ATP and 2,3-DPG levels in red blood cells, complete blood count (CBC) (hemoglobin, hematocrit, red blood cell count, reticulocyte count), markers of hemolysis (plasma bilirubin, lactate dehydrogenase LDH, haptoglobin), red blood cell survival (biotinylation labeling method), histopathological examination of spleen and liver for extramedullary hematopoiesis and iron deposition, and assessment of red blood cell sickling under hypoxic ex vivo conditions. Vaso-occlusion studies (mesenteric microcirculation) may also be performed. PKR activator 4 has potential for the research of blood disorders, so such studies may be ongoing or planned. For detailed in vivo protocols, refer to the patent WO2019035864A1. These protocols are generic and not validated for PKR activator 4 specifically.
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| ADME/Pharmacokinetics |
No specific pharmacokinetic data for PKR activator 4. Based on its molecular properties (MW 416.57, LogP ~3-4 from structure), the compound is moderately lipophilic and would be expected to have moderate to good oral bioavailability. The compound contains a silane group (Si), which is unusual for drug molecules and may affect metabolism. The silane moiety may be labile in biological fluids, potentially leading to rapid clearance. Plasma half-life, Cmax, AUC, clearance, volume of distribution, and protein binding have not been publicly reported. Solubility: DMSO 50 mg/mL (120.03 mM) with ultrasonic and warming. In vivo formulation: 10% DMSO + 90% corn oil (≥5 mg/mL, 12.00 mM). Storage: powder at -20degC for 3 years; in solvent at -80degC for 6 months.
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| Toxicity/Toxicokinetics |
No specific toxicity data for PKR activator 4. As a PKR activator, the primary expected on-target effect is modulation of red blood cell metabolism, which is the intended therapeutic mechanism for sickle cell disease. Off-target activation of other pyruvate kinase isoforms (PKM1, PKM2, PKL) could potentially affect metabolism in other tissues, leading to adverse effects. The compound contains a silane group (Si), which may have unknown toxicity. In the patent WO2019035864A1, PKR activator 4 was selected as an example compound, suggesting it likely passed initial safety screening (e.g., cytotoxicity, hERG, CYP inhibition, genotoxicity). However, no specific toxicity data are publicly available. For research use only; handle with standard laboratory precautions (gloves, lab coat, eye protection). Not for human use.
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| References | |
| Additional Infomation |
PKR activator 4 (example 7A, CAS: 2283420-05-1) is a potent activator of pyruvate kinase R (PKR), the red blood cell-specific isoform of pyruvate kinase. PKR activation increases ATP production and reduces 2,3-DPG levels in red blood cells, shifting the hemoglobin oxygen dissociation curve to the left and increasing oxygen affinity. This has therapeutic potential for sickle cell disease (SCD) and other hemoglobinopathies, where increased hemoglobin oxygen affinity reduces red blood cell sickling under hypoxic conditions. PKR activator 4 was disclosed in patent WO2019035864A1 (Pyruvate kinase activators for use in treating blood disorders) as example 7A. Molecular formula: C18H24N6O2SSi (or C18H24N2O2SSi depending on source), molecular weight: 416.57. Solubility: DMSO 50 mg/mL (120.03 mM). Storage: powder at -20degC for 3 years. Not approved for clinical use. For research use only. Reference: Giovanni Cianchetta et al. Pyruvate kinase activators for use in treating blood disorders. WO2019035864A1.
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| Molecular Formula |
C18H24N6O2SSI
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|---|---|
| Molecular Weight |
416.572662353516
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| Exact Mass |
416.145
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| CAS # |
2283420-05-1
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| PubChem CID |
151335432
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| Appearance |
Off-white to light yellow solid powder
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
28
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| Complexity |
617
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN1C2=C(C=NNC2=O)C3=C1N=C(S3)CC4=NN(C=C4)COCC[Si](C)(C)C
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| InChi Key |
OJINPWVFOQGLEV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H24N6O2SSi/c1-23-15-13(10-19-21-18(15)25)16-17(23)20-14(27-16)9-12-5-6-24(22-12)11-26-7-8-28(2,3)4/h5-6,10H,7-9,11H2,1-4H3,(H,21,25)
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| Chemical Name |
7-methyl-4-[[1-(2-trimethylsilylethoxymethyl)pyrazol-3-yl]methyl]-3-thia-5,7,10,11-tetrazatricyclo[6.4.0.02,6]dodeca-1(8),2(6),4,11-tetraen-9-one
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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: 50 mg/mL (120.03 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (12.00 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4006 mL | 12.0028 mL | 24.0056 mL | |
| 5 mM | 0.4801 mL | 2.4006 mL | 4.8011 mL | |
| 10 mM | 0.2401 mL | 1.2003 mL | 2.4006 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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