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.