AX20017 

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^[3].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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)^[3].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Walburger A, et al. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science. 2004 Jun 18;304(5678):1800-4.  [Content Brief]

  [2]. Santhi N, et al. Insights from the molecular docking of withanolide derivatives to the target protein PknG from Mycobacterium tuberculosis. Bioinformation. 2011;7(1):1-4.  [Content Brief]

  [3]. Scherr N, et al. Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):12151-6.  [Content Brief]