My research group focuses on the development of NCE against infectious diseases with particular emphasis on tuberculosis. Our expertise involves in developing new protocols for high throughput screening of synthetic as well as natural product inhibitors against novel targets as well as whole cells; study the mode of action of inhibitors, mechanism of enzyme-inhibitor interaction. Our major interest is to understand the latent stage of the tuberculosis bacilli and its reactivation into actively growing stage. For these we are using biochemical, proteomics and molecular biology approach on different in vitro dormancy models. We also do screening against different mammalian cell lines to identify anti-proliferative, anti-apoptotic, cytotoxic activities of inhibitors.

Predoctoral  Period  (1988-1990)

 

                      I started  my research work in 1988 with the study on the antitumer activity of methylglyoxal . This study showed that methylglyoxal specifically inhibits the respiration of malignant cells without inhibiting the respiration of normal cells. Simultaneously I have purified and characterized an enzyme named as aminoacetone synthase from goat liver mitochondria [Biochemical . J. (1991) 275, 575-579].

 

Doctoral Period  (1990-1994)

 

      In 1990, when I was selected for National Fellowship towards the Ph.D. degree, I decided to do my Ph.D. work with a protozoan pathogen L.donovani that is the causative agent for kala-azar, a serious health hazard in many parts of the tropical world, including India. It was my desire to work on some problem of national interest. This protozoan pathogen has a digenic life cycle. The flagellated promastigote or vector form transforms in liver macrophages to flagellated pathogenic amastigote.

               In my Ph.D. work, I have shown that Leishmania donovani promastigotes do not require extracellular Ca²⁺ for its growth and division but amastigote to promastigote transformation is strictly dependent on extracellular Ca²⁺. Disruption of Ca²⁺ homeostasis also affects the transformation process [Prop. Indian National Sci. Acad.  B58 No.5, pp 281-288 (1992). With the help of permeabilization technique, I have shown that along with endoplasmic reticulum and mitochondria Leishmania sp. possess a third organelle as major intracellular Ca²⁺ pool (unpublished). I have also tried to investigate the energy metabolism in permeabilized Leishmania donovani promastigote cells. With the help of fluorimetric techniques I have shown that temperature shift acts as a signal to elevate cytoplasmic free Ca²⁺. Accumulated results, both on promastigote and amastigote stage in different pathogenic and nonpathogenic strains indicates the possibility of the involvement of temperature sensitive Ca²⁺ pool in the pathogenicity of the organism [FEBS LETTS 375, pp83-86 (1995)].

      In AstraZeneca (1996-2002)  

                   Most of the period I spent here developing assays for primary and secondary screens in HTS programs.  I was also involved in elucidating the catalytic pathways and mechanism of action of the enzyme Hypoxanthine-Guanine-Xanthine phosphoribosyl transferase from Plasmodium falciparam. From kinetic studies it is shown that this enzyme follows a Ping-Pong type mechanism. I have also shown by chemical modification and site directed mutagenesis that a critical histidine residue is involved in xanthine selectivity of the enzyme [Mol. Biochem. Parasitol (2004) 137(2) pp 267-276].  After that I was assigned to different projects with different kind of responsibilities. In brief, my major achievements are (1) development of an HTS assay (bacterial RNA polymerase) and its transfer to HTS facility at Aldreley Park UK, where screening of 1million compounds was carried out, (2) fluorescent substrate preparation for whole HTS campaign (Bhat et. al. 2006), (6) setting up of a coupled assay of MraY and MurG (E.coli) for secondary screening, (3) purification of two proteins and a plasmid in large scale for secondary screening, (4) setting up of bacterial respiration studies using oxygraph instrument for secondary screening, and (5) membrane preparation for another HTS assay (bacterial peptidoglycan biosynthesis) required for screening campaign of 1million compounds, (6)  setting up of a coupled assay of MraY and MurG (E.coli) for secondary screening, etc.

 

In  NCL ( 2002- present)

                  Since my joining at National Chemical Laboratory, CSIR, Pune on 4^th March, 2002, apart from taking the responsibility of setting up of High Throughput Screening Facility, I started basic research on analyzing the importance of nitrogen metabolic pathway for identifying novel compounds as inhibitor of tuberculosis bacilli (Khan et. al. 2008, Khan et.al 2008). Controlling tuberculosis is one of the major concerns India is trying to solve. We have already developed few target based and whole cell based high throughput screening protocols for identifying novel inhibitors (Singh et al. 2005; Khan et.al 2008; Singh and Sarkar 2006; Singh et. al. 2010). Availing the advantage of being in NCL, my group paid attention in screening different libraries in collaboration with the synthetic organic chemists and already identified few novel anti-tubercular scaffolds (Complete specification filed at PCT, 2011; Provisional Patents filed in 2010). Our results clearly showed that there is an active nitrate/ammonia metabolic pathway present in the mycobacterium bacilli that helps in survival of the pathogen in host (Akhtar et. Al. 2013). Recently our studies on 1,2,4-trialzolethiols has clearly established that chaperonin 2 as an anti-tubercular target (Provisional Patent filed 2011). As part of NRM activity, I have already developed a screening facility with a library of 3000 natural products extracts/fractions as well as 3000 synthetic compounds. Biochemical characterization has revealed a mechanism based enzyme inactivation occurs as a result of interaction between glutamine synthetase and its inhibitor L-methionine S-sulfoximine (Unpublished observation). A detailed study from our group on host-pathogen interaction has revealed the development of hypoxic environment within infected macrophages when glutamine synthetase as well as nitrate reductase plays important role in the survival of the bacilli before necrosis of the host cell to start another round of infection (Provisional Patent 2011).

                          Along with nitrogen metabolic pathway, we are also involved in identifying the role of cellular redox components in development and maintenance of latent stage of the bacilli. Our unpublished observation suggests that hydroxyl radical as a signal for activation of tubercular bacilli from its latent stage. Currently, we are involved in identifying the proteins involved in this signaling cascade leading to activation of the pathogen which is an important physiological event for sudden development of disease in human. It has also been observed that Identified scaffolds from our facility are being tested for their in vivo studies on different models. I have also standardized a) Antiproliferation assay using MTT dye, b) Antiapoptotic assay using Caspase 3/7 kit and c) Cytotoxicity assay using LDH release detection kit on 12 different cell lines as part of my plan to enter into anti-cancer development program. Apart from screening programs, we are currently involved in biochemical evaluation of potential targets, their interaction with the known inhibitors and identification of targets for our lead molecules by applying proteomic approach as well as understanding the molecular basis of reactivation of dormant bacilli into the active stage as part of our research interest.