Figure legends
Figure 1 : Virtual screening work flow of in house library compounds (A) 3D representation of 4 hits (which were found active against H37Rv in in vitro studies) and interaction with SK protein along with 2D view of individual compound. Cyan colour ribbon like structure is SK, and magenta color structure represent compounds (B). S-014-1049 was found to be non-cytotoxic to the cells,and its interaction by different types of bonds with specific residue of SK. Where most favorable bond (hydrogen bond) is represented with green (C) Inhibitory profile of the compounds against purified SK using ADP Glo kinase assay at three different concentrations (D) Inhibitory action of S-014-1049 and rottlerin on Ra-wt at 0.5X, 1X, and 2X MIC. The effect was observed after 1 hour of drug treatment where hsp 65 was taken as a control. Lane 1 is no drug and lanes 2, 3, 4 are 0.5X, 1X, 2X of MIC (0. 2µg/µl) of the compound (S-014-1049) respectively. Lanes 5, 6, 7 are 0.5X, 1X, 2X of MIC concentration of rottlerin (E) Blot was analysed by densitometry where AU is arbitrary unit(F)
Figure 2. Comparative domain analysis of SK of Mtb and MSby using bioinformatic tool (A) Inhibition profile of S-014-1049 against three strains of mycobacteria using MABA (B) Overexpression of mutant protein (a) in BL21(DE3) using 1.0 mM IPTG concentration. Lane 1 is uninduced, lane 2 is mutant of CD (CD15:SK), lane 3 is mutant of SBD (SBD34:SK) and lane 4 is mutant of LD (LD117:SK). Purification of the mutant protein through Ni-NTA chromatography (b) lane 1 is mutant of CD (CD15:SK), lane 2 is mutant of SBD (SBD34:SK) and lane 3 is mutant of LD (LD117:SK) respectively (C) Kinase assay of the mutant purified protein in comparison to the recombinant purified SK, where pink colour bar is for purified SK, green is for SBD34: SK, red is for CD15: SK and yellow is for LD117: SK(D). Effect of compound S-014-1049 at 50µg/ml concentration against wt SK and mutant SKs at 600ng protein concentration using ADP-Glo kinase assay (E).