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]
PknG (protein kinase G of Mycobacterium tuberculosis). AX20017 is a highly selective PknG inhibitor. It specifically targets PknG of Mycobacterium tuberculosis. The compound binds to PknG, with the main chain Glu233:O and Val235:NH forming hydrogen bonds with AX20017. Inhibition of PknG prevents the kinase from blocking lysosomal transfer and degradation of mycobacteria, leading to bacterial killing.

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)

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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]
In vitro, AX20017 inhibits PknG with an IC₅₀ of 0.39 μM. The compound has no effect on human kinases. AX20017 completely inactivates PknG-mediated blockage of lysosomal transfer and degradation of M. tuberculosis. Treatment with AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. These in vitro activities demonstrate the compound's potential as an antitubercular agent with a novel mechanism of action.

Detailed in vivo activity data for AX20017 are limited in publicly available sources. Based on its mechanism of action as a PknG inhibitor and its ability to promote mycobacterial killing in vitro, AX20017 is expected to demonstrate efficacy in animal models of tuberculosis. The compound's high selectivity for PknG over human kinases suggests a favorable safety profile for in vivo use.

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]
The PknG enzyme inhibition assay for AX20017 involves recombinant PknG incubated with ATP and a peptide substrate in the presence of varying concentrations of the compound. Kinase activity is measured by quantifying phosphorylated substrate using methods such as radioactive ATP incorporation, time-resolved fluorescence resonance energy transfer (TR-FRET), or luminescent kinase assays. IC₅₀ values are calculated from dose-response curves. Selectivity is confirmed by testing against a panel of human kinases.

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]
Mycobacterium tuberculosis cultures or infected macrophages are used for in vitro efficacy studies. M. tuberculosis is cultured in appropriate medium (e.g., Middlebrook 7H9 broth) and treated with increasing concentrations of AX20017. Bacterial viability is assessed by colony-forming unit (CFU) counting on agar plates. For macrophage infection studies, macrophages are infected with M. tuberculosis and treated with AX20017. Intracellular bacterial survival is assessed by CFU counting after lysis of macrophages. Lysosomal transfer and degradation of mycobacteria are assessed by fluorescence microscopy using lysosomal markers.

In vivo efficacy studies for AX20017 likely involve mouse models of tuberculosis. Mice are infected with M. tuberculosis via aerosol or intravenous injection. After infection is established, AX20017 is administered at various doses and schedules (typically oral or intraperitoneal). Bacterial burden in lungs and spleen is assessed by CFU counting at study termination. Histopathological analysis of lung tissues is performed to evaluate the extent of infection and inflammation.

AX20017 has a molecular weight of 264.34 g/mol and a molecular formula of C₁₃H₁₆N₂O₂S. The compound is soluble in DMSO at ≥32 mg/mL. Detailed pharmacokinetic parameters are not extensively published. As a small molecule with favorable physicochemical properties, AX20017 is expected to have acceptable oral bioavailability. The compound's selectivity for PknG over human kinases suggests a favorable pharmacokinetic and safety profile.

AX20017 showed no toxicity to J774 cells at concentrations up to 100 μM (no effect on protein synthesis, phagocytosis, or proliferation). [2]
Detailed toxicology data for AX20017 are limited in publicly available sources. The compound's high selectivity for PknG over human kinases suggests a low risk of off-target toxicity. Standard preclinical safety evaluation would include in vitro cytotoxicity profiling, hERG channel assessment, and in vivo toxicology studies in rodents. The compound's mechanism of action targeting a mycobacterial kinase provides a favorable therapeutic index.

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.

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]
AX20017 is a small-molecule PknG inhibitor with antitubercular activity. It specifically targets protein kinase G of Mycobacterium tuberculosis with an IC₅₀ of 0.39 μM and has no effect on human kinases. The compound promotes lysosomal transfer and killing of mycobacteria. AX20017 represents a promising lead compound for the development of novel antitubercular agents with a mechanism of action distinct from existing antibiotics. It has a molecular formula of C₁₃H₁₆N₂O₂S and a molecular weight of 264.34 g/mol.

C₁₃H₁₆N₂O₂S

264.34

268.12

C, 58.18; H, 7.51; N, 10.44; O, 11.92; S, 11.95

329221-38-7

673481

Solid powder

3.235

2

3

3

18

370

0

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

IEETZPYULYBOCV-UHFFFAOYSA-N

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)

2-(Cyclopropanecarbonylamino)-2,3,3a,4,5,6,7,7a-octahydro-1-benzothiophene-3-carboxamide

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 53~130 mg/mL ( 200.49~491.79 mM )
Ethanol : ~3 mg/mL

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.

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Solubility in Formulation 2: 10% DMSO+90% Corn Oil: ≥ 3.25 mg/mL (12.29 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)