The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941) is Mycobacterium marinum and M. tuberculosis specific, which can withstand reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced stress. A library of over a million compounds was screened using in silico virtual ligand screening (VLS) to identify inhibitors against the modeled structure of MelF protein expressed by melF of mel2 locus so that M. marinum’s ability to withstand ROS/RNS stress could be reduced. The top ranked 1000 compounds were further screened to identify 178 compounds to maximize the scaffold diversity by manually evaluating the interaction of each compound with the target site. M. marinum melF was cloned, expressed and purified as maltose binding protein (MBP)-tagged recombinant protein in Escherichia coli. After establishing the flavin dependent oxidoreductase activity of MelF (~ 84 kDa), the inhibitors were screened for the inhibition of enzyme activity of whole cell lysate (WCL) and the purified MelF. Amongst these, 16 compounds could significantly inhibit the enzyme activity of purified MelF. For the six best inhibitory compounds, the minimal inhibitory concentration (MIC) was determined to be 3.4–19.4 μM and 13.5–38.8 μM for M. marinum and M. tuberculosis, respectively. Similarly, the minimal bactericidal concentration (MBC) was determined to be 6.8–38.8 μM and 27–38.8 μM against M. marinum and M. tuberculosis, respectively. One compound each in combination with isoniazid (INH) also showed synergistic inhibitory effect against M. marinum and M. tuberculosis with no cytotoxicity in HeLa cells. Interestingly, these inhibitors did not display any non-specific protein-structure destabilizing effect. Such inhibitors targeting the anti-ROS/RNS machinery may facilitate the efficient killing of replicating and nonreplicating mycobacteria inside the host cells.
Protein-protein interaction (PPI) network analysis is a powerful strategy to understand M. tuberculosis (Mtb) system level physiology in
the identification of hub proteins. In the present study, the PPI network of 79 Mtb toxin-antitoxin (TA) systems comprising of 167 nodes
and 234 edges was investigated. The topological properties of PPI network were examined by 'Network analyzer' a cytoscape plugin
app and STRING database. The key enriched biological processes and the molecular functions of Mtb TA systems were analyzed by
STRING. Manual curation of the PPI data identified four proteins (i.e. Rv2762c, VapB14, VapB42 and VapC42) to possess the highest
number of interacting partners. The top 15% hub proteins were identified in the PPI network by employing two statistical measures, i.e.
betweenness and radiality by employing cytohubba. Insights gained from the molecular protein models of VapC9 and VapC10 are also
documented.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.