Bacteria elicit an adaptive response against hostile conditions such as starvation and other kind of stresses. Their ability to survive such conditions depends on, in part, stringent response pathways. (p)ppGpp, considered to be the master regulator of stringent response, is a novel target for inhibiting the survival of the bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for its stress response and persistence inside the host. Our aim was to design an inhibitor of (p)ppGpp synthesis, follow efficiency through enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and NMR spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in the presence of designed inhibitors in a dose dependent manner, which we further confirmed by following the enzyme kinetics. The Rel enzyme-inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long term persistence, biofilm formation and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In-vivo (p)ppGpp levels were found to be down-regulated in M. smegmatis treated with the synthetic inhibitors. Compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis. The compounds were tested for toxicity by MTT assay using H460 cells and hemolysis assay using Human RBCs, for which they were found to be non-toxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.
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