Author(s): Meeta J. Advani 1 , Imran Siddiqui 2 , Pawan Sharma 2 , Hemalatha Reddy 1 , *
M. tuberculosis has been significantly contributing to the worldwide human infectious disease burden since long. The global TB crisis is further convoluted by the presence of MDR- and XDR-TB, being resistant to current antibiotics and hence hard to treat. It is a known fact that TB therapy has remained unchanged for nearly four decades now . Moreover the existence of dormant TB in 90% of the TB affected individuals, poses a major hindrance in eradication of this dreadful disease. A number of promising new classes of compounds are currently in pipeline at various stages of discovery and clinical development . An ideal therapy should consist of drugs that are active against the drug resistant varieties of M.tb they as well as can effectively target the sleeping bacilli lying within tubercular lesions.
Phenothiazines are known to have anti-mycobacterial activity for more than four decades. As the first line drugs against TB were able to effectively contain the disease, phenothiazines were not given much importance early on. Now with the advent of MDR strains of M.tb, compounds that can either be used directly or as adjuncts with the current drugs or may serve as lead compounds for the synthesis of new anti-TB drugs, are gaining importance. Trifluoperazine (TFP) is a calmodulin antagonist in eukaryotes  and has been used as an antipsychotic drug in neuroleptic patients . Though many reports have indicated antimycobacterial activity of TFP -, its exact mechanism of action is not yet clearly understood. Phenothiazines have been reported to affect the calcium-dependent ATPases, thereby reducing the amount of cellular energy required to maintain the active transport processes -. In mycobacteria, TFP has been shown to negatively affect processes like protein and lipid synthesis . We have previously characterized the mycobacterial gene Rv1211 as coding for a Calmodulin-like-Protein (CAMLP) in M.tb, with the ability to complex with calcium . Our studies showed that this CAMLP-Ca2+ complex could stimulate heterologous targets like plant NAD Kinase and bovine brain phosphodiesterase (PDE). Knowing that TFP is a eukaryotic Calmodulin antagonist, we have checked its effect on M.tb CAMLP activities and have found it to be inhibitory .
In the present work, we demonstrate the efficacy of TFP in suppressing the growth/survival of two clinical isolates of MDR M.tb (JAL2287 and 1934)  in vitro as well as ex vivo . TFP also exerted lethal effect against stress induced (acidic, starvation and nitric oxide) persistent M.tb , thereby showing the potential to be effective against dormant TB.
Materials and Methods
Bacterial Strains and Culture Conditions
To prepare bacterial stocks, Mycobacterium tuberculosis (H37Rv), M.tbJAL 2287 and M.tb 1934 were grown to logarithmic phase (OD600~0.6) in Middlebrook 7H9 broth supplemented with 10% albumin dextrose complex (ADC); 0.5% glycerol and 0.02% Tween 80 were also added to the media. Stocks were prepared by harvesting the bacteria, resuspending them in one-fifth volume of the original culture and storing in multiple aliquots at...