title: Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria.
abstract: Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using 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 nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsmin the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring 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 inMycobacterium smegmatis, where inhibition in each case was observed.In vivo, (p)ppGpp levels were found to be downregulated inM. smegmatistreated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogenMycobacterium tuberculosisThe compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.
authors.affiliation: Molecular Biophysics Unit, Division of Biological Sciences, Indian Institute of Science, Bangalore, India.
authors.forename: Kirtimaan
authors.initials: K
authors.lastname: Syal
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Kelly
authors.initials: K
authors.lastname: Flentie
authors.affiliation: Molecular Biophysics Unit, Division of Biological Sciences, Indian Institute of Science, Bangalore, India.
authors.forename: Neerupma
authors.initials: N
authors.lastname: Bhardwaj
authors.affiliation: Department of Organic Chemistry, Division of Chemical Sciences, Indian Institute of Science, Bangalore, India.
authors.forename: Krishnagopal
authors.initials: K
authors.lastname: Maiti
authors.affiliation: Department of Organic Chemistry, Division of Chemical Sciences, Indian Institute of Science, Bangalore, India.
authors.forename: Narayanaswamy
authors.initials: N
authors.lastname: Jayaraman
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Stallings
authors.affiliation: Molecular Biophysics Unit, Division of Biological Sciences, Indian Institute of Science, Bangalore, India dipankar@mbu.iisc.ernet.in.
authors.forename: Dipankar
authors.initials: D
authors.lastname: Chatterji
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D006158:Guanosine Pentaphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D009161:Mycobacterium / Q000187:drug effects / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D006158:Guanosine Pentaphosphate
keywords: (p)ppGpp; mycobacteria; stringent response