Discussion
Most serious public health problem of last decade has been due to the increasing resistance to clinically available useful antibiotics resulting in prevalence of tuberculosis.9 Thus, there is an urgent necessity to look for better anti-bacterials for which the new drug targets need to be identified. Against which a large pool of compound library can be screened using structure-based drug screening approach. Of late, an extensively evaluated bacterial target for the development of drugs is the protein of shikimate pathway. The fifth enzyme of the pathway, SK plays fundamental role in viability of microorganism. Due to absence of its homolog in mammals, it can serve as a promising target for therapeutic intervention.
Orthologs of SK are present in all the Mtb complex bacteria. Figure 2A shows pairwise alignment of proteins among the three domains of SK namely CD, SBD and LD between Mtb and MS. CD is the only domain which is conserved in Mtb and in MS whereas SBD and LD are semi-conserved.
In the current study to find the specific inhibitor for SK, structure based virtual screening of the in-house library was achieved. Out of which 44 hits had the highest docking score and were further evaluated in in vitro study against Mtb using MABA and MGIT. The result indicated 4 compounds to be active against Mtb . Specific activity of these 4 compounds against SK was assessed through ADP-Glo kinase assay where S-014-0149 was found to be the most-inhibitory compound. Cytoxicity of the four compounds was also checked in THP-1 human macrophages and the only compound which was found to be non-toxic was S-014-1049 (Table 2). In previous study from our group10this compound was already found active against drug resistant and drug susceptibleMtb strains and also shown synergistic effect with first line drugs of tuberculosis. In the same study10 this compound shown reduction in cfu of murine model infected withMtb. Efficacy of this compound was checked against H37Ra at different concentrations of MIC (0.5X, 1X and 2X) using rottlerin as a positive control.>50% inhibition of expression was observed at all the three concentrations in western blot assay (Figure 1E).
The 3 domains of SK were analysed by creating mutants at important residue in each domain. Assessing the kinase activity of the three mutants demonstrated that CD is most important among the three in defining the activity. Activity of compound S-014-1049 was also checked against the mutant proteins using ADP-Glo assay and the result showed specific binding of this compound to the CD. Docking study of the complex between SK and S-014-1049 also showed that LYS15 residue of CD forms hydrogen bond with fluorine atom (F3-N) of ligand at 3.2Å and nearby residues such as GLY14, THR17 and SER16 also form hydrogen bond with fluorine atoms (F1, F2 and F3). ARG117 also makes hydrogen bond with fluorine atom as well as pi-cation interaction with benzene ring. ASP34 forms pi-anion interaction with 3.84Å distance (Figure 1C). Hence, both in vitro and docking studies indicate binding of S-014-1049 to CD of SK Compound S-014-1049 is a polyphenol and derivative of curcumin. In some of the recent studies few of the polyphenols were found to be active against Mtb and are known to have many health benefits. The biological and pharmacological properties make polyphenols a valuable druggable molecule.4, 5. Therefore, this compound can be a promising drug candidate in future to cope with AMR.