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| Targets |
GSK-2556286 targets Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis. It acts as an agonist of Rv1625c, leading to increased intracellular cAMP levels and reduced cholesterol metabolism in the bacteria. The compound requires the presence of cholesterol for its activity, as it inhibits Mtb in a cholesterol-dependent manner. This unique mechanism distinguishes it from conventional anti-TB agents that target cell wall synthesis or protein synthesis. The compound also inhibits the growth of human macrophages with an IC50 of 0.07 μM.
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| ln Vitro |
In vitro, GSK-2556286 inhibits Mycobacterium tuberculosis inside infected macrophages with an IC50 of 0.07 μM in THP-1 cells in the presence of cholesterol. In axenic culture, the compound requires cholesterol to demonstrate activity, with IC50 values of 2.12 and 0.71 μM in H37Rv and Erdman strains respectively, whereas IC50 values in glucose medium are >125 and >50 μM respectively. At 5 μM for 24 hours, GSK-2556286 increases intracellular cAMP levels approximately 50-fold in Mtb lysates when Rv1625c is expressed. At 10 μM, it reduces 14CO2 release from 14C-cholesterol by 90% and decreases propionyl-CoA levels in cholesterol-cultured bacteria.
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| ln Vivo |
In vivo, GSK-2556286 shows significant bactericidal activity in mouse models of TB infection. In an acute TB infection mouse model, oral administration at 0.3-500 mg/kg for 8 consecutive days showed greatest effect at 200 mg/kg. In chronic TB infection models using BALB/c and C3HeB/FeJ mice, oral dosing at 10-200 mg/kg, 5 days per week for 4 weeks, demonstrated significant bactericidal activity, with maximal effect achieved at approximately 10 mg/kg. The compound also shows increased efficacy when coadministered with other agents. It is effective against MDR, XDR, and drug-sensitive TB strains.
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| Enzyme Assay |
In vitro enzyme/receptor binding assays for GSK-2556286 typically involve assessing its activity on Rv1625c, the membrane-bound adenylyl cyclase in Mycobacterium tuberculosis. Membrane fractions from Mtb expressing Rv1625c are incubated with the compound and ATP substrate. cAMP production is measured using a competitive immunoassay or radiometric assay. The EC50 for adenylyl cyclase activation can be determined from dose-response curves. Cholesterol dependence can be assessed by comparing activity in media with and without cholesterol supplementation. Binding affinity to Rv1625c can be evaluated using surface plasmon resonance or isothermal titration calorimetry.
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| Cell Assay |
In vitro cellular assays for GSK-2556286 use THP-1 human macrophage cells infected with Mycobacterium tuberculosis (H37Rv strain). Cells are cultured in RPMI-1640 medium with 10% fetal bovine serum and differentiated with phorbol 12-myristate 13-acetate (PMA). Infected macrophages are treated with various concentrations of the compound (typically 0.001-10 μM) for 3-7 days. Bacterial survival is assessed by colony-forming unit (CFU) counting after cell lysis, or by GFP fluorescence quantification. Cholesterol dependence is evaluated by comparing activity in cholesterol-supplemented versus glucose-only media. Cytotoxicity is assessed using LDH release or MTT assays.
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| Animal Protocol |
In vivo animal studies for GSK-2556286 use BALB/c and C3HeB/FeJ mouse models of TB infection. Mice are infected intranasally or intravenously with Mycobacterium tuberculosis. The compound is administered orally (p.o.) at doses ranging from 0.3 to 500 mg/kg, typically once daily or 5 days per week for 4-8 weeks. Efficacy is assessed by bacterial load (CFU) in lungs and spleen, histopathological analysis of granuloma formation, and survival rates. Pharmacodynamic endpoints include measurement of cAMP levels in lung tissue and assessment of cholesterol metabolism markers.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of GSK-2556286: The compound is orally active and shows good bioavailability. It is soluble in DMSO (3.33 mg/mL, 10.11 mM) with warming to 60°C. The compound acts via Rv1625c, increasing cAMP levels. In vivo PK parameters in mice: following oral administration, the compound shows dose-dependent exposure. Cmax and AUC increase with dose. The compound distributes to lung tissue, achieving concentrations above the MIC. Elimination half-life and metabolic stability data are reported in preclinical studies. The compound is metabolized primarily in the liver.
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| Toxicity/Toxicokinetics |
The toxicity profile of GSK-2556286 shows low cytotoxicity. In Vero cells, the IC50 is greater than 64 μg/mL, indicating a favorable selectivity index. In human macrophages, the compound inhibits growth with an IC50 of 0.07 μM. The compound is for research use only and not for human therapeutic use. Standard toxicity studies would include acute and subchronic oral toxicity in rodents, with monitoring of body weight, clinical signs, hematology, clinical chemistry, and histopathology. No specific genotoxicity or carcinogenicity data are reported in the available literature.
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| Additional Infomation |
GSK-2556286 (GSK286) is an orally active, cholesterol-dependent inhibitor of Mycobacterium tuberculosis with a novel mechanism of action targeting Rv1625c, a membrane-bound adenylyl cyclase. It was developed by GlaxoSmithKline as an investigational anti-tuberculosis agent. The compound shows efficacy against multidrug-resistant (MDR), extensively drug-resistant (XDR), and drug-sensitive TB strains. Its unique cholesterol-dependent activity and novel target make it a promising candidate for TB drug development. The compound is currently in preclinical or early clinical research stages and is not yet approved for clinical use. It is available for research use only.
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| Molecular Formula |
C18H23N3O3
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| Molecular Weight |
329.39
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| Exact Mass |
329.173
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| CAS # |
1210456-20-4
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| PubChem CID |
45833762
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| Appearance |
White to off-white solid powder
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| LogP |
1.8
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
24
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| Complexity |
512
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
AZOFJHATIPDIER-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H23N3O3/c1-12-4-3-5-16(13(12)2)24-15-6-8-21(9-7-15)11-14-10-17(22)20-18(23)19-14/h3-5,10,15H,6-9,11H2,1-2H3,(H2,19,20,22,23)
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| Chemical Name |
6-[[4-(2,3-dimethylphenoxy)piperidin-1-yl]methyl]-1H-pyrimidine-2,4-dione
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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 : ~3.33 mg/mL (~10.11 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 | 3.0359 mL | 15.1796 mL | 30.3591 mL | |
| 5 mM | 0.6072 mL | 3.0359 mL | 6.0718 mL | |
| 10 mM | 0.3036 mL | 1.5180 mL | 3.0359 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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