| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| Other Sizes |
Purity: ≥98%
| Targets |
PknG (protein kinase G of Mycobacterium tuberculosis) – no IC50/Ki/EC50 values provided in these references; a radiometric ATP consumptive assay was performed but specific IC50 not reported. [2]
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| ln Vitro |
AX20017 is a highly selective low-molecular-weight inhibitor of protein kinase G (PknG) with an IC50 of 0.39 μM. The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. In the 2.4 A x-ray crystal structure of PknG in complex with AX20017, the compound AX20017 inhibitor is bound deep within a narrow pocket formed by the inter lobe cleft of the PknG domain. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine-binding site, targeting an active conformation of the kinase domain. Remarkably, although the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017-binding pocket is shaped by a unique set of amino acid side chains that are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compounds inhibitory potency.The results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of M. tuberculosis.
Kinase Assay: In vitro phosphorylation by PknG (0.5 μg) is in 25 mM Tris (pH 7.5), 2 mM MnCl2, and 0.5 μCi [γ-32P]ATP in the absence or presence of the reagents. To monitor kinase activity of PknGΔN, the protein is combined with equal amounts of the kinase-dead mutant of full-length PknG, PknG-K181M. To analyze kinase activity of PknG-I87S/A92S and PknG-C/S, the PknG-N-terminal fragment of PknG (2 μg) is included. Phosphorylated proteins are separated on 12.5% SDS/PAGE and analyzed by autoradiography or quantitated by PhosphorImage analysis. IC50 values are determined by using a radiometric ATP consumptive assay. Twelve concentrations of AX20017 in the range from 5 × 10-5M to 1.5 × 10-10 M are tested in each kinase assay. Cell Assay: Phagocytosis is analyzed after incubation of J774 cells for 30 min in the presence of the indicated concentration of AX20017 (0, 10, 20 μM), followed by incubating the cells for 2 h with latex beads at a ratio of 10:1 beads/cells in the continued presence of the inhibitor, followed by fixation in 3% paraformaldehyde as described. Cells are observed with a Axiophot using a ×63 objective. Proliferation of J774 cells is analyzed by incorporation of tritiated thymidine (0.1 μCi) for 12 h as described of cells that had been incubated for 48 h in the absence or presence of the AX20017(0, 10, 20 μM) AX20017 (25 μM) inhibited wild‑type PknG kinase activity in vitro; the PknG‑I87S/A92S double mutation resulted in >50% loss of inhibitory capacity (Figure 3C). [2] AX20017 at concentrations up to 100 μM showed no effect on protein synthesis, phagocytosis, or proliferation of J774 macrophage cells. [2] |
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| ln Vivo |
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| Enzyme Assay |
In vitro kinase assay: PknG (0.5 μg) was incubated in 25 mM Tris (pH 7.5), 2 mM MnCl₂, 0.5 μCi [γ‑³²P]ATP in the absence or presence of AX20017 (25 μM). Reaction mixtures were separated by 12.5% SDS‑PAGE and analyzed by autoradiography or PhosphorImage quantitation. For the PknG‑I87S/A92S mutant, the N‑terminal fragment of PknG (2 μg) was included. [2]
IC50 determination: A radiometric ATP consumptive assay (³³PanQinase Activity Assay) was used. Twelve concentrations of AX20017 from 5×10⁻⁵ M to 1.5×10⁻¹⁰ M were tested per kinase assay. (Specific IC50 values not reported). [2] Limited proteolysis and domain mapping were performed to identify PknG domains. [2] |
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| Cell Assay |
J774 mouse macrophage cells were used. Protein synthesis: cells labeled with 0.4 μCi of ³⁵S‑methionine/cysteine for 9 h in the presence of AX20017 (1, 10, 100 μM), lysed, and TCA‑precipitable counts measured. Phagocytosis: cells incubated with 1, 10, or 100 μM AX20017 for 30 min, then latex beads (10:1 beads/cell) for 2 h, fixed, and observed. Proliferation: cells incubated for 48 h with 1, 10, or 100 μM AX20017, then 0.1 μCi tritiated thymidine for 12 h, and incorporation measured. Even at 100 μM, no effect on protein synthesis, phagocytosis, or proliferation was observed. [2]
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| Animal Protocol |
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| Toxicity/Toxicokinetics |
AX20017 showed no toxicity to J774 cells at concentrations up to 100 μM (no effect on protein synthesis, phagocytosis, or proliferation). [2]
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| References |
Bioinformation.2011;7(1):1-4. Epub 2011 Aug 20.Proc Natl Acad Sci U S A.2007 Jul 17;104(29):12151-6. Epub 2007 Jul 6.
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| Additional Infomation |
X‑ray crystal structure of PknG in complex with AX20017 (PDB ID 2PZI, resolution 2.4 Å) reveals that the inhibitor binds deep within the adenosine‑binding pocket of the kinase domain. Key interacting residues include Glu233, Val235, Ile87, Ala92, and others. The combination of residues Ile165, Val179, Gly236, and Ile292 is unique to PknG and not found in any human kinase, explaining the high specificity of AX20017. [2]
In a molecular docking study (reference 1), AX20017 showed a GlideScore of –5.6 and formed hydrogen bonds with Glu233 and Val235 of PknG. [1] |
| Molecular Formula |
C₁₃H₁₆N₂O₂S
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| Molecular Weight |
264.34
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| Exact Mass |
268.12
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| Elemental Analysis |
C, 58.18; H, 7.51; N, 10.44; O, 11.92; S, 11.95
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| CAS # |
329221-38-7
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| Related CAS # |
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| PubChem CID |
673481
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| Appearance |
Solid powder
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| LogP |
3.235
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
18
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| Complexity |
370
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C(=C(C(N([H])[H])=O)C2=C1C([H])([H])C([H])([H])C([H])([H])C2([H])[H])N([H])C(C1([H])C([H])([H])C1([H])[H])=O
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| InChi Key |
IEETZPYULYBOCV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H20N2O2S/c14-11(16)10-8-3-1-2-4-9(8)18-13(10)15-12(17)7-5-6-7/h7-10,13H,1-6H2,(H2,14,16)(H,15,17)
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| Chemical Name |
2-(Cyclopropanecarbonylamino)-2,3,3a,4,5,6,7,7a-octahydro-1-benzothiophene-3-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| 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 : 53~130 mg/mL ( 200.49~491.79 mM )
Ethanol : ~3 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3.25 mg/mL (12.29 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 32.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: 10% DMSO+90% Corn Oil: ≥ 3.25 mg/mL (12.29 mM) (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.7830 mL | 18.9150 mL | 37.8301 mL | |
| 5 mM | 0.7566 mL | 3.7830 mL | 7.5660 mL | |
| 10 mM | 0.3783 mL | 1.8915 mL | 3.7830 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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