DBT-PU Interdisciplinary Programme in Life Science for Advanced Research and Education (DBT-PU IPLS)
Ref: BT/PR14554/INF/22/125/2010 and 20.10.2010
Final Report(2010-2017)
Dr.N.ArumugamCoordinator,
BUILDER Programme (Formerly IPLS)Pondicherry University
Pondicherry 6050140413-26545200413-26545449944338491
Progress Report for R&D Project (Year 2016 - 2017)
Section – A: Project Details
A1. Project Title DBT-PU Interdisciplinary Programme in Life Science for Advanced Research and Education (DBT-PU IPLS)
A2 DBT Sanction Order No. and Date
BT/PR14554/INF/22/125/2010 and 20.10.2010
A3 Name of the Coordinator
Previous Coordinator
First Coordinator
Dr. N. Arumugam (Oct 2016 – March 2017)
Prof. K. Srikumar
Prof. P. P. Mathur
A4 Institute Pondicherry University
A5 Address with Contact Nos. (Landline & Mobile) & Email
Department of BiotechnologySchool of Life SciencesPondicherry UniversityPondicherry 6050140413 2654 520 / [email protected]
A6 Total Cost 973.00Lakhs
A7 Duration 5Years
A8 Approved Objectives of the Project
1. To initiate research in interdisciplinary area in the identified areas (Bio-prospecting of natural products and analogues for therapeutic use)
2. To strengthen the teaching programme in the School of Life Sciences
3. To provide industrial and translational research exposure to students
4. To initiate intra and inter- departmental collaborative research programme
A9 Specific Recommendations made by the Taskforce :
Accorded extension of the project till March 2017 to facilitate JRF and SRF engaged in the project to complete their PhD.
Section-B: Scientific and Technical Progress
B1.Progress made against the Approved Objectives, Targets & Timelines during the Reporting Period:
Main Objective 1: To initiate research in interdisciplinary area in the identified areas.
Progress: 11 Ph.D. students have been trained and they are in the verge of submission of their thesis by March 2017. Our research involved interdisciplinary area as out lined in objective. This group has been done bio prospecting of biomolecules of natural and synthetic origin. About 12 lead molecules have been identified, tested on specific cell lines and animal models for diagnosis of leukemia, colon cancer, diabetic mellitus and male sterility. Considerable progress has been made. Licarin A, sesamin, curcumin and curcumin-indole analogues were identified to have anticancer properties, while homobrassinolides as antidiabetic, astaxanthins as antiaging, piperin as antifertility, acylhomoserine and novel bacteriocins as antimicrobial molecules. Results of these researches have been published in high impact factor peer review Journals. These molecules are ready to be taken forward as the potent molecules for developing into drugs.
Objective 2: To strengthen teaching programme in Life Sciences
Progress: PG students from other life science and physicochemical science departments were allowed to use BUILDER instrumentation facility for their project work. They were also given special training in the use of Animal cell culture facility, ITC, Rotavapour, HPTLC, Lyophilizer etc. A list of users of the facility is appended to this report. The facility in fact has been instrumental in facilitating of a large number research publications from our University in recent time. And most of them involve interdisciplinary areas of research (List of publication is enclosed).
Objective 3: To provide industrial and translational research exposure to students.
Progress: JRFs and SRFs under the programme as well as doctoral students from School of Life Sciences attended traning programme organized by IPLS on animal handling and cell culture work. Experts from Veterinary College, Chennai and NCCS, Pune, tutored the trainees.
Objective 4: To initiate intra and inter- departmental collaborative research programmes
Progress:Having viewed the PU-DBT BUILDER instrumentation facility that we have developed, UGC has identified our University as a potential place for funding under their Startup Centre scheme. In addition many interdisciplinary project proposals have been submitted and some of them are in pipeline for funding.
SUMMARY OF REPORT OF INDIVIDUAL FINDINGS
1. Isolation and characterization of Licarin A from Myristica fragrans
Licarin A (Fig 1.1) is a lignan obtained from the seeds of Myristica fragrans (nutmeg). It is proven to have anticancer property is suspected to follow autophagy mode for the same.
Fig 1.1. Molecular Structure of Licarin A
Results and DiscussionIsolation and characterization of Licarin A: Nutmeg seeds were purchased from Kureekunnel Agricultural Nursery and Gardens, kerala, India. The seed were ground, extracted with methanol and dried under reduced pressure to obtain crude extract. About 4g extract was obtained from 100g seed. The molecule was purified by column chromatography (silica gel 60-120 mesh size). HPTLC (60 F254, Merk) of Fraction-2 indicated that it contain single compound which was later could be crystallized in n-hexane environment. The Rf of the compound was 0.67 and was 88.24% pure. 1H NMR, C13 NMR and FT-IR confirmed the compound to be Licarin A. Molecular formula of the compound was determined to be C20H22O4 (m/z 327.160) from [M+ H] Mass spectrometry.
In vitro assay: The cell viability was evaluated by an MTT assay on A549 (Lung Cancer Cell lines) at 5×103 cells/well in a 96-well plate format and incubated at 37°C overnight. The cells were treated with concentration ranging from 10 µM to 100µM of compound 1 and the control cells were treated with the vehicle (DMSO). The cells were then treated with LicarinA (IC50) for 48hrs and the cell morphology was observed under inverted microscope. As shown in Fig.1.2, the treated cells showed elongated morphology. DAPI staining was done in a 6 well plate and incubated at 37°C Overnight. The cells were then treated with LicarinA (IC50) for 24hrs. Following incubation cells were washed with PBS, fixed using 4% para-formaldehyde and permeabilized with 70% ethanol. Then the cells were stained with DAPI(1mg/ml) and viewed under fluorescent microscope (UV filter). Cell Cycle analysis was carried out by trypsinization and staining with Propidium iodide and analyzed using Flow cytometer.
Acridine orange and Ethidium Bromide staining for Apoptosis/necrosis detection: The mode of cell death was assessed by Acridine orange (AO) and Ethidium Bromide test with necessary control (Vincristine at100nM) and negative control without drug for 24hours. AO/EB staining AO/EB staining is commonly used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. AO is a vital dye that can penetrate normal cell membrane
and exhibit green fluorescence in live cells. On the other hand, EB will stain only cells that have lost membrane integrity. Early apoptotic cells will appear bright green, while the late apoptotic cells will exhibit orange fluorescence as they incorporate EB. The cell that had undergone necrosis appears red. In the study the treated cells showed orange coloured bodies indicating late apoptosis as shown in Fig. 1.3.
10 20 30 40 50 60 70 80 1000
20
40
60
80
100Licarin A on a549
24 hrs48hrs72hrs
Concentration (µM)
cell
inhi
bitio
n %
Fig 1.2: Cell Viability assay (MTT). Licarin A showed dose and time dependent decrease in the cell viability in A549 cell line with a IC50 of 40µM.
Fig 1.3: Induction of Apoptosis by Licarin A (IC50). Positive control is Vincristine(100nM), negative control had no drug and treatment lasted for 24hours. After treatmemt cells were stained with Acridine orange and Ethidium Bromide staining for apoptosis identification. Treated cells showed orange coloured bodies indicating late apoptosis.
Acidic vacuoles staining by Acridine orange: DAPI staining is done to observe nuclear changes associated to apoptosis like nuclear condensation and fragmentation. The cells are made permeable and then stained with DAPI which binds to the DNA and fluoresces when viewed under UV filter. In the study treated cells showed more condensed DNA fluorescing bright compared to control cells indicating nuclear fragmentation and condensation, apoptotic sign. Acridine orange being a vital dye permeabilize into the cells easily and fluoresce red whrn bound
Vincristine 100nMLicarin A IC50Control
to acidic vacuoles like autophagic vacuoles. In the study cells were stained with Acridine orange for to observe any acidic vacuole formation. The treated cells exhibited autophagic vacuoles indicating that the treatment induces autophagy (Fig.5). Cell cycle analysis by Propidium iodide flow cytometry revealed that the percentage cells in Gi phase was increased when compared between the Control and treated cells indicating that the compound act by arresting G1 phase of the cell cycle (Fig.6).
Fig 4: DAPI Staining for nuclear fragmentation and condensation. Treated cells showed more condensed DNA fluorescing bright compared to control cells indicating nuclear fragmentation and condensation, an apoptotic sign.
Fig 5: Acridine orange staining for acidic vacuoles in autophagy. Acridine orange is a dye that stains acidic vacuoles (autophagosome and lysosome) in live cells and fluoresces orange. Treated cell showed more acidic vacuoles indicating that autophagy is activated.
Control Licarin A IC50 Vincristine 100nM
Sub G: 1.05%G1: 82.20%S: 6.76%G2-M: 4.34%
Licarin A IC50ControlSub G: 2.29%G1: 69.20%S: 11.07%G2-M: 18.82%
Control Licarin A IC50 Vincristine 100nM
Fig 6: Cell Cycle analysis by flow cytometry. Cells were treated with Licarin A (IC50), positive control Vincristine(100nM) and negative control with no drug. Treated cells showed G1 arrest as compared to control.
Conclusion: Licarin A showed cytotoxic effect against A549 cell lines, in a dose and time dependent manner. IC50 of the molecule for A549 was 40µM, 48hours. In 72 hours of incubation cells were found to be resisting the treatment in lower doses of 10, 20, 30, 40µM. Phase contrast microscope revealed treated cells were more elongated and stress compared to the control. Acridine orange and Ethidium Bromide staining showed orange coloured bodies indicating late apoptosis. These dyes stain acidic vacuoles (autophagosome and lysosome) in live cells and fluoresce orange. Treated cell showed more acidic vacuoles indicating that autophagy is activated. DAPI Staining revealed that treated cells showed more condensed DNA, brighter fluorescence as compared to control cells indicating nuclear fragmentation and condensation leading to apoptosis. Cell Cycle arrest as determined by Propidium iodide staining and flow cytometry revealed that the treated cells were arrested in G1 phase.
2. Bio-prospecting of the natural compound Astaxanthin for Drug discovery
Saccharomyces cerevisiae is potent model system for fundamental studies that can be directly translated in higher eukaryotic system including man. Deletion strain collection of this yeast system, constructed in the BY4741 (MATahis3∆1: leu2∆:met15∆:ura3∆) was obtained from Thermofischer Scientific USA, who works in (Thermo Fisher scientific in conjunction with Saccharomyces genome deletion project) in a 96-well format. Each deletion strain (total of4, 800 mutants strains are in the library) carries a defined deletion of a characterized or putative open reading frame, in which the open reading frame has been replaced with the kanMX4marker by PCR. Strains were routinely stored in the 96-well format at - 80°C in YPD medium.
Methods: The experiments conducted were: LC50 of Astaxanthin for screening using Wild type yeast, Sensitivity to hydrogen peroxide (H2O2) and Tertiary butyl Hydroperoxide (TBH) induced oxidative stress, Protection of oxidative stress by Astaxanthin in yeast mutant DNA repair mutant cells, Oxidative stress resistance by CFU, Analysis of intracellular ROS production, Acridine Orange and Ethidium Bromide Staining to measure the cell viability, DAPI Staining to measure DNA fragmentation, Molecular Docking of Astaxanthin with DNA and DNA Protection AssayResults:
Optimization of Astaxanthin Concentration: Treatment with Astaxanthin (10-40 µM) was found non toxic for the wild type strain BY4741 (Fig 2.1). Since the cell were not affected by Astaxanthin and continued to reach 100% growth, we have randomly chosen 30µM of Astaxanthin to study protection against oxidative stress mediated DNA damage.Astaxanthin protects oxidative stress mediated DNA repair mutants in yeast: Based on the growth inhibition protection by astaxanthin against oxidative stress caused by hydrogen peroxide in the liquid media, the mutants were categorized as high Protection (60-80 % above growth) and protection (40-60% growth) (Table 1). Among the 10 yeast mutants, the mutants such as rad1∆, rad51∆, ntg1∆, apn1∆ and apn2were protected more by Astaxanthin. The phenotype expression
analysis revealed that the mutants rad1∆, rad51∆, ntg1∆, apn1∆ and apn2 were sensitive to Hydrogen peroxide at 2.25mM and 2.5mM plates (Figure 2.2). Whereas Astaxanthin treated cells grew better than hydrogen peroxide induced stress indicating Astaxanthin rescued the DNA repair mutants from oxidative stress. The CFU studies by treating cells with Astaxanthin followed by treatment with1mM H2O2 showed that the number of cells and percentage survivability is increased by astaxanthin treatment in wild type and mutants in both H2O2
treated and untreated condition. The results implicates Astaxanthin scavenges the hydroxyl radical that are generated in the cells and increases the viability of cells.
Astaxanthin reduces intracellularlar ROS production: In this experiment cells were treated with the redox sensitive fluorochrome (H2DCF-DA) as described in method. The levels of intracellular oxidation was measured and observed in Fluorescent microscope. Direct exposure of H2O2to cells produced an increase of H2DCF fluorescence in the mutants compare to wild type strain. However after Astaxanthin treatment a reduction of H2DCF oxidation was observed in DNA repair mutants (WT, rad1∆, rad51∆, ntg1∆,apn1∆,apn2∆)indicating astaxanthin reduces oxidative stress mediated DNA damage. From the figure 4, it is clear that the number of cells that stained positive for H2DCFDA dye is more in H2O2treated cells compared to control indicating more accumulation of ROS in H2O2treated in mutants. Astxanthin pretreated cells were less stained indicating rescue against oxidative stress. The Relative fluorescence units of the dye indicated the reduction of ROS generation in mutants.
Acridine Orange and Ethidium bromide Staining for Cell viability: The results showed that the control untreated cells appeared green in color with intact nuclei (Figure 2.3). After Hydrogen peroxide treatment cells appeared orange to red color with condensed and fragmented nuclei indicated the cells undergone to apoptosis by oxidative stress whereas Astaxanthin treatment exposed to hydrogen peroxide significantly reduced the apoptosis caused by oxidative stress.
DNA fragmentation by DAPI Staining: DAPI associates with the minor groove of Double stranded DNA with a preference for adenine- thymine clusters. DAPI has a greater photostabilitystain preferentially to double stranded DNA and it has great photostability, Yeast can be DAPI stained to visualize the DNA fragmentation caused by the oxidative stress by observing the morophological change. Our experimental result showed that the mutant’s apn1∆ and rad51∆ showed high DNA fragmentation which was caused by Hydrogen peroxide whereas Astaxanthin treatment showed protection against H2O2 induced DNA fragmentation.
Molecular Docking of Astaxanthin and DNA: The docking results are given in Table 2 and the docked structures are presented in Fig 2.4 . The ranking of ligand (astaxanthin) was based on the scores generated by the docking server.
DNA Protection Assay: Plasmid DNA damage protection by the Astaxanthin is shown in Fig. 2.5. The control plasmid showed a band of linear, Relaxed and circular DNA on agarsoe gel electrophoresis. The Fenton reaction showed disappearance of circular DNA whereas the plasmid DNA with astaxathin treatment after fenton reaction displayed protective activity resulting in appearance of a circular band. Further densitometric analysis was carried out using
ImageJ software for the quantification of plasmid DNA relative to the untreated reaction. The quantification of plasmid DNA, shown in graph, revealed that the Astaxanthin was effective in protecting DNA by inhibiting the nicking caused by Fenton’s reagent.The results lead us to infer that the plasmid DNA with fenton reaction entirely destroyed the DNA while Astaxanthin protects against fenton reaction. Linear, and relaxed forms of images were visualized in gel Doc. By this result it confers Astaxanthin protects DNA against oxidative stress (Figure 8). Therefore it might be used to study in prevention of cancer.
Figure 2.1. To Optimize Astaxanthin concentration: Survival of S cerevisiae (BY4741) cells exposed to increased concentrations of Astaxanthin A) The results expressing the percentage of survival of wild type culture at different concentration of Astaxanthin. B) Phenotype expression of WT cells treated with different concentrations of Astaxanthin.
Table 2.1 DNA Repair Sensitivity and Protection of Astaxanthin against H2O2
Mutants H2O2 Sensitivity Astaxanthin Protection against H2O2 Sensitivity
rad27∆ ++ *rad1∆ + **rad51∆ ++ **mec3∆ ++ Not Protectedrad9∆ ++ Not Protectedrad17∆ + *ntg1∆ + **mag1∆ ++ Not Protectedapn1∆ ++ **apn2∆ ++ **
Figure 2.2:Astaxanthin protects DNA repair mutants under oxidative stress: Mid log phase grown DNA repair mutants rad1∆, rad51∆,ntg1∆,,apn1∆,apn2∆ and wild type were incubated with astaxanthin for 2 hour at 30 °C and 6time 10 fold serial dilutions were spotted on YPD and 2, 2.25 and 2.5mM H2O2 YPD Plate.
Figure 2.3. Acridine Orange and Ethidium Bromide Staining for Cell viability in yeast cells: Control cells are in green color indicating full viability. Hydrogen Peroxide treated cells fluoresce red-orange indicating the cells are dead due to oxidative damage caused by Hydrogen peroxide and Astaxanthin + Hydrogen Peroxide treated cells remain green indicating the rescue of cells from oxidative damage caused by H2O2 by Astaxanthin.
Table 2.2: Docking Properties of Astaxanthin interaction with DNA
B)
Fig 2.4 Docking posture of Astaxanthin with DNA
Figure 2.5: DNA protection of Astaxanthin: Plasmid treated with Astaxanthin (100 and 150µM in absence and presence of DNA nicking Fenton’s reagent followed by the densitometry analysis of relaxed (R), linear (L), and circular (C) forms of Plasmid DNA (underneath agarose gel). Lane1: Control (Deionisedwater + DNA), Lane 2: Negative Control (DNA + Fenton’s reagent), Lane 3: positive control (DNA + Astaxathin (100µM+ Fenton’s reagent), Lane 4: positive control (DNA + Astaxathin (150µM+ Fenton’s reagent)
Conclusion: Oxidative stress has been recognized as an important source of DNA damage and
mutation which leads to various cancer, neurological diseases and aging etc. The budding
yeast S. Cerevisiae is an excellent model system for identifying natural products targets for their
biological activity. In the present study, we carried out the protective effect ofAstaxanthinon
stress deficient DNA repair mutants. Our results showed that, Astaxanthin treatment increases
the oxidative stress resistance to DNA repair mutants rad1∆, apn1∆, apn2∆, rad51∆ and ogg1∆.
And also showed that Astaxatnhin will bind to DNA without causing any damage to DNA.
From this we can conclude that Astzaxanthinprotecs oxidative mediated DNA Damage by
scavenging, and oxidizing the free radicals which are responsible for DNA damage
3. Bioprospecting of inhibitors for Bcr-Abl for the treatment of CML
Bcr-Abl is a non-receptor tyrosine kinase that is deregulated in Chronic Myeloid Leukemia
(CML) and has become an attractive target for design of drugs for treatment of the disease.
Imatinib, Nilotinib, Dasatinib, Bosutinib and Ponatinib are the drugs currently approved for
CML treatment. In addition there are few other inhibitors in different stages of pre-clinical and
clinical trials. These drugs require long duration of treatment which results in non-hematological
toxicities. Hence, there is an urgent need to develop inhibitors preferably from natural products
to avoid side effects. By virtual screening we identified two natural inhibitors ZINC08764498
(hit1) and ZINC12891610 (hit2) that meet the said criteria from natural product database. In
silico analyses of the protein-inhibitor complex using docking and molecular dynamic
simulations, and the reactive nature of the inhibitors by DFT study showed that both have
potential for in vitro testing.
Experimental testing of the selected hits on Bcr-Abl positive K-562 and Bcr-Abl-negative HEK-
293 cell lines revealed that of the two, “hit 1” had an IC50 of 30 µM on K-562 cell line.
Subsequent experiments showed apoptosis induction in terms of both morphology and quantity.
This was in turn confirmed by increased caspase-3 protein expression levels. Both hit1 and hit2,
belongs to coumarin class of naturally occurring oxygen heterocyclic compounds which are
known for their potentialanti-cancer properties. The coumarin derivative ZINC08764498,
reported here for the first time to have anti-CML activity, needs further investigation to delineate
the mode of anti-cancer action.
4. Bio-prospecting of Sesamin as am anticancer drug
Sesamin and its analogues have been reported recently to have anticancer property against colon
cancer. Sesamin is a natural lignan obtained from the Sesamum indicum. Sesamin isolated from
the commercially available gingelly oil yielded a mixture of 88% sesamin and 12% Sesamolin.
This mixture was tested and compared with commercially available seasmalin for their biological
effect on human cell lines. In silio analysis was carried out on β-catenin as potential target.
PDB structure of sesamin and some of its analouges were subjected to docking with β-catenin of
βcatenin/Tcf-4 protein complex formed in the colon cells. It was observed that Sesamin was the
most interactive with the pocket residues the β-catenin followed by the sesangolin. The potency
of the sesamin, sesamin isolate and Sesamol were subjected to wet lab analysis. They were tested
on the colon cancer cell lines SW480 and the HT-29 for various time period. It was observed that
isolated sesamin which in fact is a mixture with 12% sesamolin showed a higher potency. It was
followed by the pure sesamin obtained commercially. The response was comparable with the
currently used standard 5-fluorouracil bu without any side-effects. The observed IC50 for
sesamin isolate was 20 µM for 72 hrs period of incubation. Sesamol did not show inhibitory
activity on SW480 but was acting as a potent molecule on the HT-29 cells with the IC50 of
20µM at 72 hrs period of incubation and the effect was similar to that of the sesamin isolate and
pure sesamin. The underlying mechanisms for this differential activity of the one of the sesamin
analogue, sesamol has to be understood and explored. The anti-proliferative activity of these test
lignans were studied using the MTT anti-proliferative assay. This was followed by the nuclear
morphological observation of the treated cells with the one of the nuclear stain DAPI (4',6-
diamidino-2-phenylindole). The changes in the nuclear morphology were observed in 24, 48 &
72hrs period of incubation. All the tested compounds resulted in the formation of various
morphological structures like crescent shaped nuclei, apoptotic bodies, and the fragmented and
sheered nuclei. All above described nuclear morphological changes led us to infer that sesamin,
sesamin isolate and the sesamol along induce apoptotic cell death of the colon cancer cell. The
experiments to find out other molecular events involved in the regulation at translational and
transcriptional levels require further study.
5. Antimicrobial and Anticancer properties of signalling molecules produced by nosocomial pathogen Acinetobacter baumannii
Secretory N-acyl homoserine lactones (AHL) mediate quorum sensing (QS) in bacteria. AHLs
are known for inhibiting unrelated group of bacteria and for mimicking host signaling
mechanism thereby subverting the regulatory events in host cells. This study investigated
isolation and characterization of AHL produced by Acinetobacter baumannii and its effect on
other pathogenic bacteria and mammalian cells. The AHL isolated had an m/z of 325 with a
molecular formula C18H31NO4. It showed inhibitory potential against Staphylococcus aureus.
In the case of Gram-positive bacteria peptidoglycan hydrolases (LysM) are required for
separation of daughter cells during cell division where YSIRK-G/S signal peptide delivers the
enzyme to the cross-wall compartment. Perturbed regulation of LysM and YSIRK-G/S signal
peptide results in associated defective cell shape and increased autolysis. TrxA (thioredoxin)
a redox/oxidative system that interact with the RNA polymerase RpoA subunit C-terminal
domain leads to changes in gene expression to enable sustenance of viability in stressed
conditions. σB-Down regulation (Fold change 60.13) of general stress protein 3-hexulose-6-
phosphate synthase was noted in this study. NFκB (nuclear factor kappa-light-chain-enhancer of
activated B cells) is a protein complex that controls cell proliferation and cell survival. Some
tumor cells often secrete factors that cause NFκB to become active. Inhibition of NFkB activity
increases the sensitivity to apoptosis in cancerous cells. These results suggest that agents which
inhibit the NF-kappaB would increase the PARP cleavage. In-vitro experiments indicate the
bacteriostatic AHL pose a time-dependent activity and induce apoptosis in cancer cell lines.
Therefore this compound can serve as a potential structural backbone for constructing new AHL
analogues for inhibiting bacteria such as S. aureus. The findings also emphasizes on
reevaluating all the previously characterized AHLs for added new biological functions other
than QS.
6. Identification of P-gp inhibitors to overcome drug resistance in cancer
Development of multi-drug resistance is common in chemotherapy failure. P-glycoprotein (P-
gp) has been a well known a drug transporter that has been implicated in efflux
chemotherapeutic drugs thus playing a vital role in the development of drug resistance in cancer.
To date, a number of compounds have been screened in the clinical trials to identify potent P-gp
inhibitors. However, none have been approved so far. This is solely due to the unpredictable
toxicity and cross reactivity of those molecules with other proteins. Compounds of natural origin
were thought to be the best resource for P-gp inhibitory activities due to their less toxic effects.
Therefore this study done to find the P-gp inhibitors from natural origin and to design and
synthesis their analogues by computer aided drug design approach to get the potent small
molecule P-gp inhibitors. Therefore it was intended to Design, synthesize and evaluate drug like
properties of piperine analogs as P-gp inhibitors to overcome drug resistance to cancer mediated
by this marker. The experiments conducted include Cell viability assay by MTT, Clonogenic
assay, Western blot analysis of P-gp, Immunofluorescence detection of P-glycoprotein
localization, Rhodamine 123 accumulation assay and fluorescence microscopic analysis of cell
death by acridine orange/ethidium bromide dual staining.
Results and discussion: Two piperine analogs (Pip1, Pip2) were designed and synthesised
through computer aided drug design approach (Fig 6.1). In silico docking studies and molecular
dynamic simulation (50 ns) studies were carried out using the homology modelled human P-gp
protein to explore binding efficiency of these analogues with the P-gp protein. In vitro testing
was carried out in P-gp over expressing cancer cells. The in silico docking study showed that the
synthesised analogues were able to bind effectively than piperine and molecular dynamic
simulation results showed that both the analogues maintained hydrophobic interaction
throughout the simulation period (50ns).
Fig 6.1. Synthesis of piperine analogs. (a) 6,7-Dimethoxy-1,2,3,4-tetrahydro-isoquinoline, isobutyl chloroformate, N-methylmorpholine, THF, 0 °C to rt, 25%. (b) 2-(3,4-Dimethoxy-phenyl)-ethylamine, isobutyl chloroformate, N-methylmorpholine, THF, 0 °C to rt, 51%.
The in vitro cell viability assay results showed that both Pip1 and Pip2 were able to reverse
vincristine resistance in P-gp over expressing cancer cells by 8.8- and 2.9-folds, respectively at 2
micro molar concentrations. Moreover, Pip1 which has a 6, 7-dimethoxytetrahydroisoquinoline
moiety was as potent as a standard inhibitor verapamil that exerted reversal of vincristine
resistance by 11-folds. Moreover, the rho 123 accumulation assay confirmed the increased
accumulation of rho 123 in P-gp over expressing cancer cells in the presence of these analogues
compared to normal cells (please refer to publication). Therefore, these piperine analogues may
serve as a lead molecule to synthesise more potent P-gp inhibitors. We have recently published
these results in the Scientific Reports (Nature) and the copy is appended herewith.
7: Characterization of Novel Bacteriocins from probiotic bacteria from fermented foods
Probiotic bacteria were isolated from fermented food products, strains were screened for
antimicrobial activity against pathogens related to dental biofilm, food borne and aquaculture
pathogens. Among 115 strains, two strains were selected to study which revealed better
antimicrobial activity. Strains were identified as Lactobacillus fermentum and Lactobacillus
mucosae and characterized by biochemical and molecular methods. The bacteriocins were
isolated and purified from culture free supernatant of the strains by ammonium sulfate
precipitation and fractionated by gel permeation chromatography. Bacteriocins of L.fermentum
and L. mucosae had inhibited pathogens such as Streptococcus sp, Streptococcus mutans,
Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus, Salmonella typhi and
Escherichia coli. Crude bacteriocins were subjected to Sephadex column chromatography and
active fractions were collected, further purified using RP- HPLC, the molecular weights of the
peptides were determined by SDS-PAGE and masses were confirmed by Matrix Assisted Laser
Desorption Ionization- time of flight Mass Spectroscopy, isolated probiotic bacterial peptide
masses were confirmed and subjected to stability parameters such as different temperatures, pH,
and enteric enzymes. Present study concluded that bioactive peptides and viable probiotic
bacteria could be useful for probiotic therapy in dental caries and useful in Agri-food industry as
preservatives.
B3. Details of New leads obtained, if any:
Lead Molecule Identified Biological Activity Test Model Target Protein
Homobrassinolide Anti cholesterogenic and anti carcinogenic
K562, A549, Hep3B Liver X Receptor
ZINC08764498 Anticancer K562 BCR-ABL
ZINC04654813 Anticancer MDA MB-231 AKT1
Piperine Antifertility Wistar Rat Androgenic receptors
Piperine analog P-gp inhibition A549 MDR cell lines developed for testing
Astaxanthin Antiaging Yeast cells Antioxidant proteins
Licarin A Anticancer A549 Autophagy
Curcumin Anticancer A549, K562, SW480 Gene expression analysis
Sesamin Anticancer SW480 β-catenin/TCF4 complex
Acylhomoserine Lactone Antimicrobial A.baumannii
and S. aureusD-alanine - D alanine synthetase
Novel Bacterocins Antimicrobial Lactobacillus fermentum
Probiotic in treatment of dental caries & as preservative in Agro-food industry
B4. Summary and Conclusion:
Inter disciplinary programme in Life Sciences (IPLS) is an interdepartmental project sanctioned by DBT, GOI in October, 2010 (File no. BT/PR14554/INF/22/125/2010, dated 28/09/2010) to facilitate Infrastructure Development of our University and to train Man Power in the area of Biotechnology. The sanctioned equipment were procured, staffs and research scholars appointed by October 2012 and cater to the needs of both internal as well as all students School of Life Sciences of the University.
We had successfully met and completed all the four objectives set for this project. Our research involved isolation and identification of bioactive molecules or metabolites from microbes,plants and animal as well as some synthetic ones and assess their efficacy as antimicrobial and anticancer agents. So far 11 (Eleven) molecules including Sesamin, Licarin A, Astaxanthin, Piperin and its analogue have been isolated and characterized by HPLC, HPTLC, FTIR and NMR. The molecules were tested positive for antimicrobial and / or anticancer properties. Some of these have also been tested in animal models as well. In the process we had also trained a dozen of Ph.D. students in the area of inter disciplinary research. The faculties from all four Departments of the University worked in a coordinated manner in the capacity of co-guidance of researcher or in the research investigation. The high impact publication that we have made is the testimony to this effect. From the project we have published nearly 30 publications and many more to come. Apart from these there are publications from faculties and Research Scholars outside IPLS who have acknowledged the usage of the IPLS facility.
The sanction for the year 2016-17 was received on March 2017.We had prepared to successfully complete the project by due date of September 2017as is quoted in e-Promise page of DBT website. With the hope of receiving an extension letter till September 2017 as mentioned in e-Promise we had committed some expenditure by that date. Since we received the letter of extension of the Project up to March 2017, only in September 2017, it is requested that the over spent expenditure till September, 2017 may kindly be permitted.
B5. Details of Publications and Patents, if any:
1. Safiulla Basha Syed, Hemant Arya, I-Hsuan Fu, Teng-Kuang Yeh, Latha Periyasamy, Hsing-Pang Hsieh and Mohane Selvaraj Coumar (2017). Targeting P-glycoprotein: Investigation of piperine analogs for overcoming drug resistance in cancer. Sci Rep (Nature) 7: 7972. Epub 2017 Aug 11.
2. Gopichand Chinta, Mohane Selvaraj Coumar and Latha Periyasamy (2017). Reversible Testicular Toxicity of Piperine on Male Albino Rats. Pharmacogn Mag 2017 Oct 26;13(Suppl 3):S525-S532. Epub 2017 JuSyed, S. B.; Coumar, M. S., P-Glycoprotein Mediated Multidrug Resistance Reversal by Phytochemicals: A Review of SAR & Future Perspective for Drug Design. Curr Top Med Chem 16 (22): 2484-508.
3. John J and Prashanth K. (2016). Quorum sensing molecule N-Acylhomoserine lactone produced by Acinetobacter baumannii Displays Antibacterial and anticancer properties. Biofouling 32 (9): 1029–1047 (Impact factor 3.7)
4. John J and Prashanth K. Fatal infection in adults by pneumolysin and autolysin producing, non- vaccine serotype Streptococcus pneumoniae: Re-think on current strategy of adult pneumococcal vaccination. Indian J Med Res. 2016; 143(4): 514–517. (Impact Factor 1.6)
5. Gopichand Chinta,Safiulla B. Syed,Mohane S. Coumarand LathaPeriyasamy(2015) Piperine: A Comprehensive Review of Pre-clinical and Clinical Investigations. Current Bioactive Compounds 11:156-169.
6. Safiulla Basha Syed and Mohane Selvaraj Coumar (2016) P – Glycoprotein Mediated Multidrug Resistance Reversal by Phytochemicals : A review of SAR & Future Perspective for Drug Design. Current Topics in Medicinal Chemistry 16:1-25(I.F 3.402)
7. Madhu Dyavaiah, Phaniendra A and Sudharshan SJ (2016)Microbial Keratitis in Contact Lens Wearers,JSM Ophthalmol 3(3):1036.
8. Aejaz Ahmad Dar ,Nitish Kumar Verma, Neelakantan Arumugan (2014) An updated method for isolation, purification and characterization of clinically important antioxidant lignans – Sesamin and sesamolin, from sesame oil Industrial Crops and Products Volume: 64 201-208.(I.F 3.20)
9. Athithan, V., Premalatha R, Srikumar. K. ed., (2014) Down Regulation of Plasma and Tissue Biomarkers by Homocastasterone. International Journal of Drug Delivery 6.(I.F 1.02)
10. Chinta, G., RamyaChandar Charles, M., Klopčič, I., SollnerDolenc, M., Periyasamy, L., and SelvarajCoumar, M. (2015) In Silico and In Vitro Investigation of the Piperine's Male Contraceptive Effect: Docking and Molecular Dynamics Simulation Studies in Androgen-Binding Protein and Androgen Receptor. Planta Med. (I.F 2.152)
11. Chinta, G. C., Janarthanan, R., Jesthadi, D., Shanmuganathan, B., and Periyasamy, L. (2014) Effect of Piperine On Goat Epididymal Spermatozoa: An In Vitro Study. Asian Journal of Pharmaceutical and Clinical Research 7.(I.F 0.74)
12. Cincin, Z. B., Unlu, M., Kiran, B., Bireller, E. S., Baran, Y., and Cakmakoglu, B. (2015) Anti-proliferative, apoptotic and signal transduction effects of hesperidin in non-small cell lung cancer cells. Cellular Oncology 38, 195-204 (I.F.3.032)
13. Durairaj, V., Hoda, M., Shakya, G., Babu, S. P., and Rajagopalan, R. (2014) Phytochemical screening and analysis of antioxidant properties of aqueous extract of wheatgrass. Asian Pac J Trop Med 7S1, S398-404.(I.F 1.062)
14. Durairaj, V., Shakya, G., Pajaniradje, S., and Rajagopalan, R. (2014) Effect of wheatgrass on membrane fatty acid composition during hepatotoxicity induced by alcohol and heated PUFA. J Membr Biol 247, 515-521.(I.F 2.457)
15. Durairaj, V., Shakya, G., and Rajagopalan, R. (2014) Anti-hyperlipidemic effect of wheatgrass on alcohol and▵pufa induced liver toxicity in male albino wistar rats. International Journal of Pharmacy & Pharmaceutical SciencesVol6suppl 2,.(I.F 0.55)
16. Shakya, G, VaralakshmiDurairaj, M. H. a. Rajagopalan, R., (2015) RP-HPLC Analysis and Oxidative Stress Mediated Cytotoxic Effect of Methanol extract of Wheatgrass by Modulating Nrf2 Level in Hep-2 Cells. International Journel of Pharma Sciences Volume 5897-903(I.F. 1.04)
17. Muthanna, H. S. P. R. a. N. (2015) Variations in the Blaise Reaction: Conceptually New Synthesis of 3-Amino Enones and 1,3Diketones. European Journal of Organic Chemistry, 1525-1532.(I.F 3.065)
18. Kennedy, R. K., Naik, P. R., Veena, V., Lakshmi, B. S., Lakshmi, P., Krishna, R., and Sakthivel, N. (2015) 5-Methyl phenazine-1-carboxylic acid: a novel bioactive metabolite by a
251 rhizosphere soil bacterium that exhibits potent antimicrobial and anticancer activities. ChemBiol Interact 231, 71-82. (I.F 2.577)
19. Kennedy, R. K., Veena, V., Naik, P. R., Lakshmi, P., Krishna, R., Sudharani, S., and Sakthivel, N. (2015) Phenazine-1-carboxamide (PCN) from Pseudomonas sp. strain PUP6 selectively induced apoptosis in lung (A549) and breast (MDA MB-231) cancer cells by inhibition of antiapoptotic Bcl-2 family proteins. Apoptosis 20, 858-868. ( I.F. 3.685)
20. Mishra, S. K., Ferreira, J. M., and Kannan, S. (2015) Mechanically stable antimicrobial chitosan- PVA-silver nanocomposite coatings deposited on titanium implants. CarbohydrPolym 121, 37-48.(I.F 4.074)
21. Mohankumar, K., Pajaniradje, S., Sridharan, S., Singh, V. K., Ronsard, L., Banerjea, A. C., Benson, C. S., Coumar, M. S., and Rajagopalan, R. (2014) Mechanism of apoptotic induction in human breast cancer cell, MCF-7, by an analog of curcumin in comparison with curcumin--an in vitro and in silico approach. ChemBiol Interact 210, 51-63.(I.F 2.577)
22. Mohankumar, K., Pajaniradje, S., Sridharan, S., Singh, V. K., Ronsard, L., Banerjea, A. C., Selvanesan, B. C., Coumar, M. S., Periyasamy, L., and Rajagopalan, R. (2014) Apoptosis induction by an analog of curcumin (BDMC-A) in human laryngeal carcinoma cells through intrinsic and extrinsic pathways. Cell Oncol (Dordr) 37, 439-454.(I.F 3.032)
23. Muthu, K., Panneerselvam, M., Topno, N. S., Jayaraman, M., and Ramadas, K. (2015) Structural perspective of ARHI mediated inhibition of STAT3 signaling: an insight into the inactive to active transition of ARHI and its interaction with STAT3 and importinβ. Cell Signal 27, 739-755 .(I.F 4.315)
24. Nakkala, J. R., Mata, R., Gupta, A. K., and Sadras, S. R. (2014) Biological activities of green silver nanoparticles synthesized with Acorouscalamus rhizome extract. Eur J Med Chem 85, 784-794.(I.F 3.447)
25. Pajaniradje, S., Mohankumar, K., Pamidimukkala, R., Subramanian, S., and Rajagopalan, R. (2014) Antiproliferative and apoptotic effects of Sesbaniagrandiflora leaves in human cancer cells. Biomed Res Int 2014, 474953(I.F 1.579)
26. Panneerselvam, M., Muthu, K., Jayaraman, M., Sridharan, U., Jenardhanan, P., and Ramadas, K. (2013) Molecular dynamic simulations of the tubulin-human gamma synuclein complex: structural insight into the regulatory mechanism involved in inducing resistance against Taxol. MolBiosyst 9, 1470-1488.(I.F 3.183)
27. Parthiban, A., Kumaravel, M., Muthukumaran, J., Rukkumani, R., Krishna, R., and Rao, H. S. P. Design, synthesis, in vitro and in silico anti-cancer activity of 4H-chromenes with C4-active methine groups. Medicinal Chemistry Research, 1-15.(I.F 1.402) 252
28. Periyasamy, L., Kumar, Y., and babujestadi, D. (2014) “Preliminary Phytochemical Screening, DNA Protection, Antioxidant and Anti-proliferative Effect of Seed Extracts of Bixaorellana L.”. American Journal of Phytomedicine and Clinical Therapeutics 2014 2, 8. (I.F 0.69)
29. Premalatha, R., Srikumar, K., Vijayalaksmi, D., Kumar, G. N., and Mathur, P. P. (2014) 28-Homobrassinolide: a novel oxysteroltransactivating LXR gene expression. MolBiol Rep 41, 7447-7461.(I.F 2.024)
30. Shakya, G., Pajaniradje, S., Hoda, M., Durairaj, V., and Rajagopalan, R. (2014) GC-MS Analysis, In Vitro Antioxidant and Cytotoxic Studies of Wheatgrass Extract. American Journal of Phytomedicine and Clinical Therapeutics 2, 877-893.(I.F 0.69)
B6. Snap Shots of some recent publication:
Section-C: Details of Grant Utilization:
C1. Equipment Acquired with Actual Cost:
Sl No. Items Name Total Cost (Rs. In Lacs)
1 Carbon Dioxide (CO2) Incubator (2) 102 Isotheral titration calorimeter 663 Lyophilizer with vacuum concentrator 144 Rota Evaporator (2) 185 Fluorescence Inverted Microscope 156 Thermocycler (4nos) 157 -800C Freezer 78 Autosampler for Liquid Chromatography –Mass
Spectrometer and accessories33
11 High Performance Thin Layer Chromatography 3212 Fermentor 23 Total 0
1.
C2. Manpower Staffing and Expenditure Details:
SCHOLARS BEING TRAINED UNDER BUILDER PROGRAMME
Mr. GopichandChinta - SRF
Mr. Sudharshan S.J - SRF
Mr. James John - SRF
Ms. BindumadhuriCavuturu - JRF
Mr. ShamimAkhtar Sufi - JRF
Mrs. Uma Maheshwari - JRF
Ms. Pragnalakshmi.T - JRF
Mr. Victor Mukherjee - JRF
Mr. PhanikrishnaParcha - JRF
Mr. SafiullaBasha Syed - JRF
Mr. RepallyAyyanna - JRF
STAFF TRAINED UNDER BUILDER PROGRAMME
Dr.Anshul Nigam - Asst. Professor, currently working in RusanPharma
Ms.G.V.Jayachitra - Technical Officer
Mr. Thamilarasan - Technical Officer
Mrs. NishammaBeevi - Technical Assistant
Ms. Shanthi - Technical Assistant
C3. Details of Recurring Expenditure: Please see PDF files of UC and SoE sent separately
C4. Financial Requirement for the Next year with Justifications: Nil. However, we would like to convey that with the hope of receiving an extension letter till September 2017 as mentioned in e-Promise we had committed some expenditure by that date. Since we received the letter of extension of the Project up to March 2017, only in September 2017, we request that the over spent expenditure till September, 2017 may kindly be permitted.
