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intracellular Ca2+ pool (unpublished).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 4th 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.