1

PROFORMA – I

PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH

AND

DEVELOPMENT, PROGRAMME SUPPORT

PART I: GENERAL INFORMATION

1. Name of the Institute/University/Organisation submitting the Project Proposal:

Vivekananda Institute of Tropical Mycology (VINSTROM), Ramakrishna Mission

Vidyapith, Chennai-600004.

2. State: Tamil Nadu

3. Status of the Institute: Non-profit organization of the Ramakrishna Mission

4. Name and designation of the Executive Authority of the Institute/University

forwarding the application : Swami Shukadevananda, Chairman, Vivekananda

Institute of Tropical Mycology, Ramakrishna Mission Vidyapith, 45, Oliver Road,

Mylapore, Chennai 600 004.

5. Project Title : A study of the biodiversity and bioactive natural products of non-

sporulating fungi associated with mangroves and sponges of

Andaman Islands 6. Category of the Project : R&D/ Programme Support

7. Specific Area : 6.2 Marine Biology

8. Duration : 3 Years

9. Total Cost (Rs.) : Rs. 92,49,960-

10. Is the project Single Institutional or Multiple-Institutional (S/M) ? : M

11. If the project is multi-institutional, please furnish the following:

Name of Project Coordinator : Dr. T.S. Suryanarayanan

Affiliation : Director, VINSTROM

Address : Ramakrishna Mission Vidyapith

45, Oliver Road

Mylapore, Chennai 600 004.

12. Scope of application indicating anticipated product and processes

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For the first time, non-sporulating endophytes of marine habitat would be

explored for their diversity and production of novel antibiotics, antialgal, antifungal, ,

antioxidant and antimalarial compounds.

13. Project Summary

Non-sporulating endophytic fungi associated with mangrove trees (leaf endophytes,

bark phellophytes and root endophytes) and sponges of the Andaman Islands (Rifleman,

Manjeri, Wandoor, and Pluto Islands) will be isolated using suitable growth media and

culture conditions. Emphasis will be laid on isolation and culturing of non-sporulating

fungi since these have not received enough attention for their diversity and metabolic

potential. The fungi will be identified based on DNA sequences and maintained in our

culture collection. The species diversity, tissue and host specificity of mangrove

endophytes and sponge fungi will be worked out. The extracts of conditioned media from

these fungi will be screened for production of antibiotic, antialgal, antifungal,

antioxidant and antimalarial metabolites. Those extracts which appear to be promising

will be purified and chemically characterized. A collection of rare and bioactive

metabolites producing fungi will be maintained. Likewise a library of extracts of

candidate fungi will be maintained. Voucher specimens of fungi which produce novel

and interesting metabolites will be deposited in a culture collection centre.

The secondary metabolites extracted from the culture filtrates of the fungi would

be tested for antialgal, antibacterial and antifungal activities by bio-autogram and agar

diffusion methods. The antioxidants will be assayed by DPPH radical scavenging assay,

hydroxyl radical scavenging assay and total reducing power assay.

Antibiotic metabolites active against Gram positive and negative microorganism

will be assayed by Muellar-Hinton agar plates. Metabolites active against Plasmodium

will be assayed by SYBR Green fluorescence based assay of P. falciparum growth in

microtiter plate wells.

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Those extracts that appear promising would be subjected to activity guided

RPHPLC purification in fraction collection mode and their structure determined using

FTIR, MS, and NMR

PART II: PARTICULARS OF INVESTIGATORS

Principal Investigator:

14. Name : Dr. T.S. SURYANARAYANAN

Date of Birth : 25.07.1951 Sex: Male.

Designation : Director

Department : -

Institute/University : Vivekananda Institute of Tropical Mycology

Address : Ramakrishna Mission Vidyapith

45, Oliver Road, Mylapore,

Chennai PIN: 600 004.

Telephone: 9791186036 E-mail: t_sury2002@yahoo.com

Number of research projects being handled at present: 2

Co-Investigator – 1

15. Name : Dr. MUKESH DOBLE

Date of Birth : 16.09.1950 Sex: Male

Designation : Professor

Department : Department of Biotechnology

Institute/University : Indian Institute of Technology Madras

Address : Indian Institute of Technology Madras, Chennai

PIN : 600 036

Telephone : 91-44- 22574107 Fax 91-44-22574102 E-mail : mukeshd@iitm.ac.in

Number of Research projects being handled at present: 2

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Co-Investigator – 2

Name : Dr. DINKAR SAHAL

Date of Birth : 18TH

JANUARY 1956 Sex: Male

Designation : STAFF RESEARCH SCIENTIST

Department : MALARIA

Institute/University : International Centre For Genetic Engineering And

Biotechnology,

Address : Aruna Asaf Ali Road, New Delhi 110067

Telephone : : 9871980190

E-mail : : dinkar@icgeb.res.in, dsahal@gmail.com

Number of Research projects being handled at present: 1

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PART III: TECHNICAL DETAILS OF PROJECT

16. Introduction

16.1 Origin of the proposal

Fungi associated with sponges are the single most prolific producers of novel

metabolites (Bugni and Ireland, 2004; Rateb and Ebel, 2011). Similarly, endophytic fungi

of mangroves are also recognized as abundant producers of bioactive compounds (Bugni

and Ireland, 2004; Raghukumar, 2008; Rateb and Ebel, 2011). Furthermore, sterile fungi

isolated as endophytes also produce novel bioactive compounds but have not received as

much attention as the sporulating endophytes (Hussain et al., 2009). The islands of

Andaman have several exclusive mangrove communities in isolated patches represented

by different mangrove and mangrove associates species. Marine sponges of different

species are also found here. It would be worthwhile to study the biodiversity and

metabolites profile of mangrove endophytes and sponge fungi of Andaman islands since:

(1) sponge-associate fungi are prolific producers of bioactive metabolites but have not

been studied adequately for this trait (Suryanarayanan, 2012).

(2) these two classes of fungi (the mangrove endophytes and sponge-associate fungi) are

the least studied in India for bioactive metabolites.

(3) non-sporulating, sterile fungi associated with sponges and mangroves have not been

studied for their species diversity and bioactive metabolites.

16.2 (a) Rationale of the study supported by cited literature

Most of the currently used antibiotics are derived from restricted molecular

frameworks whose effective lives have been stretched by synthetic modifications of the

molecules (Fischbach and Walsh, 2009). Prolonged and indiscriminate use of such

antibiotics with limited structural diversity has resulted in bacteria developing antibiotic

resistance genes (resistomes), culminating in the appearance and spread of multiple drug

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resistant (MDR) pathogens (Wright, 2007; Kumarasamy, 2010). To control MDR

pathogens, it is essential to look for new molecular structures with antibiotic properties.

Here, natural products research is imperative because combinatorial chemistry has fallen

short of our hyped expectations of it (Ortholand and Ganesan, 2004). Indeed, marine

invertebrates harbor in their tissues a series of microorganisms such as bacteria, and fungi

and, in some cases, associated micro-organisms may constitute up to 40% of the biomass,

this bacterial concentration exceeding that of the surrounding sea water by two or three

orders of magnitude. Although the real contribution of the microorganisms to the

secondary metabolism of marine invertebrates has not yet been fully evaluated,

essentially because of the difficulties encountered in culturing sponge-associated

microbes, it is generally accepted that these harbored microorganisms play a significant

role in the biosynthesis of the natural products isolated from the invertebrate (Fattorusso

(Fattorusso and Scafati 2009).

Sorbicillactone A was isolated from a strain of Penicillium chrysogenum cultured

from a sample of the Mediterranean sponge Ircinia fasciculata; it possesses a unique

bicyclic lactone structure, seemingly derived from sorbicillin. The compound exhibits

promising activities in several mammalian and viral test systems, in particular a highly

selective cytostatic activity against murine leukemic lymphoblasts (L5178y) and the

ability to protect human T cells against the cytopathic effects of HIV-1(Bringmann and

Muller 2003) .

Cyclosporin A (CsA) (a hydrophobic cyclic peptide produced by a fungus) is widely used

as an immunosuppressive agent to inhibit the rejection of transplanted organs Azouzi et

al. 2010.

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In this context, screening microbes from unexplored and little-studied ecological

niches for novel metabolites appears to be a promising exercise (Fischbach and Walsh,

2009). Among such organisms marine fungi associated with sponges, corals, seaweeds,

mangroves, marine detritus, tunicates, fish, seagrasses, marine sediments, etc. appear to

be prolific producers of molecules of unprecedented structures with potential

pharmaceutical importance (Bugni and Ireland, 2004; Schulz et al., 2008; Raghukumar,

2008; Kjer et al., 2009; Suryanarayanan, 2010b). In addition, the marine derived fungi,

especially from mangroves and sponges, produce novel metabolites with unusual

bioactivites thus making them choice candidates for bioprospecting (Rateb and Ebel ,

2011).

Marine fungi represent a source of fascinating and significant chemical diversity.

So far, more than 1000 novel compounds have been described from marine fungi and

current studies show that they are still a relatively unexplored group of organisms for

novel biochemicals (Rateb and Ebel, 2011) (Fig. 1).

Fig. 1. Types of novel metabolites so far obtained from marine-derived fungi.

In the recent years, marine derived fungi associated with mangrove trees and

sponges have been the subject of intense study as they produce bioactive metabolites of

M. E. Rateb and R. Ebel . 2011.

Secondary metabolites of fungi

from marine habitats

Nat. Prod. Rep., , 28, 290–344

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novel and even unprecedented molecular architectures (Proksch et al., 2008; Höller et al.,

2000). In a broad-based assay of fungi from sponges, Höller et al. (2000) isolated several

novel compounds with unique bioactivities; that were found to inhibit malarial parasite,

tuberculosis bacterium and HIV-1 reverse transcriptase. They therefore concluded that

sponge-associated fungi are a good source of novel bioactive metabolites. This study was

followed by those of Edrada et al. (2002); Lin et al (2003) which confirmed that the

biosynthetic potential of sponge associated fungi is higher than that of their terrestrial

conspecifics. In the recent years, these findings were bolstered by the studies of Proksch

et al (2008) and Liu et al (2011).

Similarly, endophytic fungi which reside inside the living tissues of mangrove

trees without expressing any disease symptoms are also well known for their heightened

synthetic activity (Kumar and Hyde, 2004; U‟Ren et al., 2012). Kjer et al (2009) isolated

14 different natural products from an Alternaria sp. that was endophytic in the mangrove

tree Sonnretia alba. Aly et al (2008) identified a protein kinase inhibitor from a

mangrove endophyte. Lopanick et al (2004) and Mitchell et al (2008) identified several

novel metabolites from endophytes. Three unique metabolites, namely, phomopsis-H76

A (1), B (2), and C (3), were produced by Phomopsis sp. which is a mangrove endophyte

(Yang et al., 2010). Bugni and Ireland (2004) posit that mangrove endophytes are an

untapped source of novel bioactive chemicals. In this context, sterile endophytic fungi

(those that do not sporulate in culture) are also an unexplored source for novel chemicals

(Vinale et al., 2010). For instance Vinale et al (2010) extracted veratryl alcohol (VA) and

4-(hydroxymethyl)-quinoline which showed antibiotic and antifungal effects from a

sterile endophytic fungus. Similarly, Maria et al (2005) showed that a sterile endophyte

from a mangrove produced novel antibiotics that was active against several bacteria.

However, there are very few studies in the tropics on non-sporulating marine derived

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fungi of sponges and endophytes of mangroves with regard to their diversity and

synthetic ability. There is hardly any study on the bioactive metabolite of these ecological

groups of fungi from India.

(b) Hypothesis:

Fungi associated with marine sponges and endophytic fungi of mangrove trees are

metabolically more innovative than their terrestrial counterparts. Together, these two

sources of fungi account for most of the novel metabolites described from marine habitat

Fig. 2; Rateb and Ebel, 2011).

Fig. 2. Contribution by different ecological groups of marine fungi to the tally of novel

metabolites.

This is perhaps an outcome of their constant need to interact with their host milieu.

Endophyte–plant host interactions are different from pathogen–plant host interactions

since neither associate really „wins‟; neither disease symptoms develop on the plant host

nor is the fungus eliminated by the plant host (Pinto et al., 2000; Schulz et al., 2002;

Stone et al., 2004; Sieber, 2007; Saikkonen, 2007). This situation entails sustained and

prolonged reactions against the defense mechanisms of the host by the endophyte and this

M. E. Rateb and R. Ebel . 2011.

Secondary metabolites of fungi

from marine habitats

Nat. Prod. Rep., , 28, 290–344

10

could act as selection pressure for developing novel metabolic pathways – a potentially

beneficial situation for bioprospectors (Weber et al., 2007; Suryanarayanan et al., 2010a).

We feel that many novel bioactive compounds can be isolated from sponge fungi and

mangrove endophytes of Andaman Islands since they support rare and unique organisms

which evolved thorough millions of years owing to the physical separation between the

islands and from the main lands.

(c) Key questions:

What non-sporulating species of fungi occur as endophytes in the mangroves and

what fungi are associated with the marine sponges of the Andaman Islands ?

What is the chemical diversity of these endophytes and sponge fungi?

What antibiotic, antialgal, antifungal, insecticidal, antioxidant and antimalarial

metabolites are produced by these fungi?

16.5 Current status of research and development in the subject

Filamentous fungi are revealed only by actinomycetes and plants in producing

diverse types of secondary metabolites showing varied biological activities. Among the

fungi, those occurring inside the tissues of sponges which are either symbionts or casual

residents (acquired by the sponge in the process of ingestion - Baker et al., 2009) are

unique in producing novel metabolites (Hirot et al., 2004; Suryanarayanan 2012).

Interestingly, unlike the bacteria associated with marine sponges, the fungi produce

metabolites different from those of their sponge hosts (König et al., 2006). In the recent

years, there has been a considerable increase in the studies on microbes associated with

sponges (Fig 3). Ding et al (2010) isolated strong antimicrobial compounds from

Nigrospora sp. associated with the sponge Clathrina luteoculcitella occurring in the

South China Sea. Aspergillus versicolor, another fungus isolated from South China Sea

sponge elaborates a peptide cyclo (L-Trp-L-Phe) having both agricultural and

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pharmaceutical potential (Chu et al., 2011). Another A. versicolor isolated from a marine

sponge produces a novel aromatic polyketide derivative, two xanthones, and five

anthraquinones (Lee et al. 2010). Rateb et al (2010) reported three hitherto undescribed

dibenzofurans from an ascomycete isolated from a Fijian marine sponge; these

compounds inhibited the growth of many bacterial species and the activity of tyrosine

kinase. Proksch et al (2008) isolated 81 fungal strains belonging to 20 different genera

from the sponge Suberites domuncula; from these a total of 19 different fungal

metabolites, including three new natural products were characterized. Li et al (2011)

reported the production of new hexylitaconic acid derivatives by a marine sponge-derived

Penicillium sp. A sponge associated Acremonium sp. elaborates new sesquiterpenoids

with anti inflammatory properties (Zhang et al., 2009). Hao et al (2008) obtained several

new polyketide-originated metabolites from a mycelia sterilia fungus isolated from

sponge. Aspergillus aculeatus¸asssociated with the sponge Xestospongia testudinaria,

elaborates Aspergillusol A, an r-Glucosidase Inhibitor (Ingavat et al., 2009).

Trichopyrone showing antioxidant activity was isolated from Trichoderma viride

associated with the Caribbean sponge Agelas dispar (Abdel-Lateff et al., 2009). Liu et al

(2009) extracted seven new drimane sesquiterpenoids along with the known compounds

deoxyuvidin B, strobilactone from cultures of the fungus Aspergillus ustus, which was

isolated from the marine sponge Suberites domuncula. The compounds showed cytotoxic

activity against a panel of tumor cell lines, including L5178Y, HeLa, and PC12 cells.

Suryanarayanan (2012) in his recent review states that our knowledge of the diversity and

metabolites of sponge-associated fungi is inchoate.

With reference to mangrove endophytes, although many have been isolated

(Kumaresan et al., 2002), their synthetic potential is being recognized only recently

(Raghukumar, 2008). Huang et al (2010) isolated three new dimeric naphtho-γ-pyrones,

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named rubasperone A, rubasperone B and rubasperone C from the mangrove

endophytic fungus Aspergillus tubingensis; these were active against tyrosinase and α-

glucosidase. Maria et al (2005) showed that the culture extracts of mangrove endophytes

inhibit several Gram positive and Gram negative bacteria. Chaeprasert et al. (2010)

isolated several metabolites from endophytes of 10 mangrove species in Thailand and

reported the following: The antimicrobial potential of 71 endophytic fungi isolated from

mangrove plants towards selected bacteria (Bacillus subtilis, Pseudomonas aeruginosa,

Escherichia coli and Staphylococcus aureus) was tested using ethyl acetate extracts of

fungi cultivated under static conditions. All test bacteria were inhibited by a

Cladosporium sp. isolated from the leaves of T. populneoides and an endophytic Xylaria

sp. 1 isolated from A. ilicifolius leaves caused considerable inhibition to Gram-positive

and Gram-negative bacteria. Additionally, the crude extracts of 84 endophytic fungi were

tested for anticancer activities by the MTT assay against A375 (human malignant

melanoma), SW620 (human colorectal adenocarcinoma), Kato III (human gastric

carcinoma), HepG2 (human liver hepatoblastoma) and Jurkat (human acute T cell

leukemia). Most extracts had cytotoxicity against some cancer cell lines.

Iwatsuki and Omura (2011) isolated five tropolone compounds from the culture broth of

Penicillium sp. FKI-4410. Among them, puberulic acid exhibited potent antimalarial

inhibition, with IC50 values of 0.01μg/ml against chloroquine-sensitive and resistant

Plasmodium falciparum strains with low cytotoxicity (IC50 : 57.2μg/ml) in vitro.

Butyrolactones isolated from the fungus Aspergillus terreus BCC 4651 showed

antiplasmodial and antimycobacterial activity ( Haritakun and Isaka 2010). Kasettrathat

and Kittakoop (2008) showed that tetronic acid and nodulisporacid A isolated from a

marine-derived fungus Nodulisporium sp. CRIF1 from a Thai soft coral, was moderately

antiplasmodial (IC50:1–10 μM) against chloroquine-resistant P. falciparum strain 94.

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Pontius and Konig (2008) isolated a heterocyclic substituted xanthone, chaetoxanthone B

from cultures of a marine derived fungus Chaetomium sp. that showed selective activity

towards P. falciparum K1 strain (IC50: 0.5 μg/mL). Wright and Lang-Unnasch (2005)

reported that pycnidione isolated from the marine fungus Phoma sp., had significant

antiplasmodial activity against three strains of Plasmodium falciparum (IC50: 0.15–0.4

μM)

Increasing research interest in marine sponge-microorganism associations. Number of publications retrieved from the

ISI Web of Science database..

16.6 The relevance and expected outcome of the proposed study

Though marine fungi from different substrates are known to be a good source of

bioactive compounds (Bugni and Ireland,2004; Raghukumar 2008), the symptomless

endophytic fungi associated with mangroves and marine sponges have not been studied

in detail for such chemicals in our country. Hence, this proposal is relevant and

pioneering in nature.

Taylor, M.W., Radax, R., Steger, D. &

Wagner, M. 2007. Sponge-Associated

Microorganisms: Evolution, Ecology,

and Biotechnological Potential.

MICROBIOLOGY AND

MOLECULAR BIOLOGY REVIEWS,

June 2007, p. 295–347.

Fig. 3.

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Expected outcome

Novel metabolites of pharmaceutical importance

Identification of potential producers of antibacterial, antifungal antialgal,

antiinsect, antioxidants and antimalarial chemicals.

Biodiversity and ecology of endophytes of mangroves and fungi of sponges of the

Andaman Islands – this study will be the first of its kind for Andaman Islands.

A collection of cultures of non-sporulating fungi associated with marine sponges

and mangroves of Andaman Islands

16.7 Preliminary work done so far

The Principal Investigator‟s lab is the first to have studied endophytic fungi of mangrove

plants (Suryanarayanan et al., 1998). This paper has been cited by many workers. Hyde

and Soytong (2008) in their review state that our lab has made significicant contribution

to knowledge of tropical endophytes. The principal investigator‟s lab is also the first to

isolate endophytes from seagrasses (Devarajan et al., 2002). The principal investigator‟s

lab is specializing in techniques that help isolating, purifying and identifying fungal

endophytes (Suryanarayanan et al., 2002 – cited in Ganley RJ, Brunsfeld SJ and

Newcombe G (2004) A community of unknown, endophytic fungi in western white pine.

Proceedings of the National Academy of Sciences USA 101: 10107-10112;

Suryanaryanan et al., 2003 – cited in Clay K (2004) Fungi and the food of the Gods.

Nature 427: 401-402). It is also engaged in screening of endophytes for antibacterial,

antialgal and antifungal as well as antimitotic properties (under two Indo-German

projects sanctioned by the DBT). We have isolated several bioactive compounds in the

DBT-funded Indo-German project (Suryanarayanan et al., 2009); these include

cytochalasins, enniatins, apicidin, aphidicolin, etc (Suryanarayanan et al., 2009). In a

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preliminary study, we found that the culture extract of a marine fungus isolated from a

marine sponge off Mandapam coast had metabolites that inhibited cancer cell lines

(Suryanarayanan et al., 2010a).

In a collaborative research by the PI and Co-PI 1 that is funded by the DBT, it has

been shown that the fungi associated with seaweeds produce several novel antibiotics and

antioxidants (Suryanarayanan et al., 2010b). We also reported the production of novel

macrolides by Curvularia lunata, a fungus isolated from a seaweed (Geetha et al., 2011).

In a collaborative research funded by the DBT project “Pharmaceuticals from

marine and marine derived fungi associated with seagrasses and seaweeds of Tamil Nadu

coast”, the PI and co PI have studied the fungal associates of marine sponges occurring

along the coast of Rameswaram Southern India. A paper describing the results of this

study (including the diversity and distribution of sponge fungi and their bioactive

compounds) has been published in Fungal Diversity (Thirunavukkarasu et al., 2012).

The PI has published a paper ("L-asparaginase from marine derived fungal endophytes of

seaweeds", 2011, Mycosphere, Online Journal of Fungal Biology - ISSN 2077 7019)

on the anti neoplastic enzyme of fungi associated with marine algae. Currently, about

100 culture extracts obtained from these fungi by the PI are being screened for anti

malarial activity in Co-PI 2's lab.

Contibution by Co- Principle Investigator – 1

Currently completed a DBT sponsored 3 year project titled “Anti-TB compounds from

marine actinomycetes” in collaboration with Tuberculosis research centre, Chennai. A

novel metabolite was identified and its structure was elucidated using analytical tools. A

patent has been filed for the novel compound which shows anti-TB and anti –HIV

activity (New Antituberculosis antibiotic from marine actinomycetes strain R2,

247/DEL/2011, February 2, 2011). So the co-PI 1 has extensive experience in isolating

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metabolites from natural sources using chromatographic and determining the structures

using several analytical tools.

Contribution by Co- Principle Investigator – 2

The CO-PI 2‟s laboratory at the ICGEB, New Delhi has deep interest and wide

experience in activity guided purification of molecules and methods of screening crude

extracts and purified molecules against Plasmodium falciparum strains in human red

blood cell culture. The screening methods include SYBR Green fluorescence dependent

microtiter plate HTP assay and Giemsa stain based microscopy based assays. The former

is useful for HTP screening and the latter is vital for firm validation and mechanistic

studies (using fluorescent dyes like Hoechst 33342 and JC-1). Both assays are in frequent

use in the laboratory. His laboratory is also equipped with MTT based assays for

quantitative estimation of mammalian cell cytotoxicity. Selectivity of molecules is thus

assessed through Selectivity index which is the ratio of IC50

(P.falciparum)/IC50(Mammalian cells). .

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17. Specific objectives

Determination of the distribution, diversity, host and tissue restriction of non

sporulating fungal endophytes and of non sporulating sponge fungi of Andaman

islands

Creation of a genetic resource of these unique fungi for use in bioprospecting by

other labs

Identification of fungal metabolites which are anti algal, antifungal, antibacterial,

antimalarial and antioxidant in nature

Chemical characterization of bioactive molecule isolated from endophytes and

sponge fungi

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18. Work Plan:

PI’s Lab:

1. Isolation of endophytes from mangroves (various tissues and various species)

2. Isolation of sponge – associated fungi.

3. 3. Identification of the fungi

4. Maintaining pure cultures of the isolated fungi

5. Extraction of metabolites and testing for anti algal and antifungal

metabolites

6. Extracts sent to CoPI 1 and CoPI 2 labs

CoPI 1’s Lab:

1. Test extracts for anti oxidants

2. Test extracts for antibiotics

CoPI 2’s Lab:

1. Test extracts for anti plasmodial activity

2. Antiplasmodial Molecule Isolation

Extracts which show promising activities will be subjected to the following detailed

study. Fungi which produce promising metabolites would be grown in large

quantities (PI 1’s lab) and extracts will be given to CO PI 1 and CO PI 2’s Labs for

separation and isolation. These isolated compounds would be tested again for their

activity and finally chemically catheterized by Co PI 1 and CO PI 2’s labs.

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Identification( by culture criteria &

PCR and gene sequencing, followed

by BLAST search) &

Fig. 2. Bioassays for detecting secondary metabolites from fungi

Antialgal assay

Antifungal assay

Culture filtrate extracted with

hexane & butanolConcentrated

(30 ml in to 1.5 ml fungal extracts)

Sponge/Mangrove

tissue

Isolation &

purification on

PDA

Cultivation in seawater

medium, 21 days

PI’s Lab

Antibiotics Assay

Antioxidants assay

Co PI 1’s Lab

Antimalarial assay

Co PI 2’s Lab

AntialgalAntifungal

Identification( by culture criteria &

PCR and gene sequencing, followed

by BLAST search)

Fig. 2. Bioassays for detecting secondary metabolites from fungi

Antialgal assay

Antifungal assay

Culture filtrate extracted with

hexane & butanolMetabolites will be eluted using methanol,

methanol evaporated in preweighed tubes

and residue subjected to quantitative

potency determination assays listed below

Sponge/Mangrove

tissue

Isolation &

purification on

PDA

Cultivation in seawater

medium (containing the resin

XAD for adsorption of

secondary metabolites), 21

days

PI’s Lab

Antibiotics Assay

Antioxidants assay

Co PI 1’s Lab

Antimalarial assay

Co PI 2’s Lab

XAD samples will be sent by Dr.Suryanarayanan

To Dr. Dhoble and Dr. Sahal

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Promising fungi will be cultivated Broth will be subjected to

on large scale in fermentor water butanol partition

XAD

NO

Antifungal Antibiotic Antioxidant Antialgal Antiplasmodial XAD 1 XAD 2 XAD 3 XAD 4 XAD 5 XAD 6

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Large-scale fermentation of

promising strains on rice medium

(Based on chemical

screening/bioactivity) PI’s lab

Solvent extraction, purification

confirmation of activity by bioassay

(PI’s lab and Co-PI 1 & 2’s lab)

Fractionation and structure elucidation

(CoPI 1 and 2’s labs). The fungal extracts

which show many bioactivities will be further

fractionated in PI 1 and PI 2 labs and tested in

all the 3 labs to identify the active fractions

and metabolites.

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18.1 Work plan

Figure 4 gives the summary of the work plan to be carried out in the PI and CoPI

1 and 2‟s labs.

Work in PI’s Lab:

A. Leaf, bark and root tissues of mangroves and sponge samples occurring along

Rifle man, Manjeri, Wandoor and Plueto Islands of Andaman will be collected

and sampled for isolation of associated endophytes and fungi.

B. The samples will be processed within 48 hours of collection from mangroves will

be isolated using appropriate surface sterilization method (depending on the

texture of the tissue). Sponge samples would be comminuted in sterile sea water

and spread over suitable agar media to isolate associated fungi.

C. Fungi will be isolated, purified and identified based on culture and DNA

sequences. Cultures will be maintained in VINSTROM.

D. Using the data collected, ecological parameters such as species diversity,

colonization frequency, similarity co-efficient, etc. will be completed.

E. This study will throw light on endophyte assemblages of Andaman mangroves

and fungal assemblages of Andaman sponges.

F. Fungi would be grown in sea water amended liquid media and the culture filtrates

will be extracted using suitable solvents. The solvents will be assayed for anti

algal, anti fungal, and anti insect compounds by auto-biogram and maggot assay.

G. A library of extracts of candidate fungi will be maintained. Voucher specimens of

fungi which produce novel and interesting metabolites will be deposited in a

culture collection centre.

Co-PI’1 lab –IIT

Antioxidants assay including (1) DPPH radical scavenging assay, (2) hydroxyl

radical scavenging assay and (3) total reducing power assay will be performed.

Antibiotic activity against Gram positive and negative microorganism will be

performed using Serial dilution MIC method.

The promising compound (s) will be purified using prep TLC, and column

chromatography. Then the structures will be determined with FTIR, GC-MS, 1H

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NMR, and 13C NMR. The novelty of the structures will be ascertained with

SciFinder data base.

ikCo-PI’2 lab - ICGEB

Following work will be done at Malaria Research Lab, ICGEB New Delhi

1) Primary antimalarial screening : Fungal extract provided by PI lab will be screened

for antiplasmodial activity using SYBR Green fluorescence based assay of P. falciparum

growth in microtiter plate wells in vitro. In order to check parasite growth inhibition by

the above extracts, malaria parasite will be grown in vitro (Trager and Jensen1976).

Chloroquine sensitive (3D7) and resistant (INDO) strains of P. falciparum will be used in

culture. The parasite would be cultured in RPMI 1640 supplemented with HEPES,

sodium bicarbonate, gentamycin, 10% human serum or albumax and human erythrocytes.

The parasite will be grown in plates incubated at 370C in carbon dioxide incubator. The

parasitemia would be monitored by microscopic examination of blood smears after

staining with Giemsa stain. For various studies the parasite cultures would be prepared

after Sorbitol mediated synchronization to ring stage.

In vitro drug susceptibility testing in P. falciparum will be determined by a SYBR

Green I-based fluorescence method described previously by Smilkstein and Riscoe

(2004). Stock solutions of each test drug will be prepared in DMSO/ sterile distilled

water. The 50% inhibitory concentration (IC50) will be determined by analysis of dose-

response curves.

2) PURIFICATION OF ANTI-PLASMODIAL MOLECULES

Purification of molecules from active fungal extracts will be done by solvent

extraction followed by reverse phase HPLC using UV dual wavelength and ELSD

24

detection. Antiplasmodial molecules will be identified in the backdrop of bioassay guided

fractionation.

3) Determination of Therapeutic Potential of Purified Antiplasmodial Molecules:

Therapeutic index is a ratio of toxic concentration (TC50) for host to inhibitory

concentration (IC50) for the pathogen. Therapeutic indices will be determined by

measuring the cytotoxicity of test molecules on mammalian cells. Animal cell lines

(HeLa and fibroblast) will be used to determine drug toxicity by using MTT assay for

mammalian cell viability as described by Mosmann (1983).

4) STRUCTURAL ANALYSIS OF ACTIVE MOLECULES:

Structure determination will help in identification and chemical synthesis of nature

derived antiplasmodial molecules. This way we can use nature`s molecules without the

need to perturb ecological equilibrium when the drugs are required in huge amount.

Structural characterization of active molecules will be done using Spectroscopic tools

including Mass Spectrometry, UV-Visible Spectrometry, FT-Infrared Spectrometry, CD

Spectrometry and Nuclear Magnetic Resonance. Our laboratories are well equipped with

these tools that will facilitate structure determination.

5) STUDYING DRUG IN ACTION:

i) In vitro stage dependence of action: Stage of parasite life cycle on which

molecules are acting will be determined by microscopic analysis of drug effect on

different stages (e.g. ring, trophozoite, Schizont) of parasite. Cultures at the ring/troph/

schizont stages will be seeded in 96-well plates. The test compounds will be added to

25

parasite cultures at different stages and drugs will be removed after 6h (Schizonts), 24h

(Trophozoites) , and 48 h (rings) by spinning and washing with complete medium. The

washed cells will be further incubated for 48h under standard drug free culture condition.

Slides will be made and analyzed microscopically. Stage specificity of action will be

assessed by observing the stage transitions in drug treated samples against control.

Parasite invasion inhibition will be examined by action on schizont and formation of ring

as index of invasion in fresh red cells.

ii) Drug Action In-vivo

Plasmodium species that cause human disease are essentially unable to infect non-

primate animal models. So, in vivo evaluation of anti-malarial compounds typically

begins with the use of rodent malaria parasites. In-vitro purified potent antimalarial

molecules will be used to analyze their potential in-vivo using rodent malaria parasite P.

berghei / P. yoelii (Peters and Warhurst, 1977). Briefly, groups of four to five mice will

be intravenously inoculated with 5 x106 P. berghei / P. yoelii infected erythrocytes and

treatment will be initiated when parasitemia has reached 1-3% (Day 0). Drugs will be

administered once-daily on days 0, 1, and 2. Blood drop will be collected 24 h after each

treatment and the smears will be analyzed microscopically. Results will be expressed as

the dose reducing parasitemia to 50% (ED50), 75% (ED75), and 90% (ED90). The

animals will be considered cured if they survived 30 days after the infection without any

detectable parasite.

i) Studying Synergy in Drug Action.

To determine the potential of drugs in combination therapy, action in synergy with

known drugs like chloroquine and artemisinin will be assessed by isobologram analysis

26

using fixed-ratio combination. Briefly, drugs will be diluted in fixed ratios of sub

inhibitory starting concentrations to generate well-defined concentration response

curves. The FICs (Fractional Inhibitory Concentrations) will be calculated by the

following formula: FIC (A) = IC50 of drug A in combination/ IC50 of drug A alone; FIC

(B) = IC50 of drug B in combination/ IC50 of drug B alone; FIC index = FIC (A) +FIC

(B). The isobolograms will be constructed by plotting a pair of FICs for each

combination of drug and the selected compound. An interpretation of a straight diagonal

line (FIC index = 1) on the isobologram indicates an additive effect between the two

drugs. A concave curve below the diagonal line (FIC index < 1) indicates synergy of the

combination, while a convex curve above the diagonal line (FIC index > 1) indicates

antagonism (Kelly and Rescoe, 2009).

18.2 Connectivity of the participating institutions and investigators

The PI and Co-PI ‟1 (IIT, Chennai) have already collaborated in a previous DBT

funded marine biotechnology project to study pharmaceuticals from marine fungi

(BT/PR10169/AAQ/03/376/2007). The PI is already collaborating with Co PI ‟2 (Dr.

Dinkar Sahal, ICGEB, New Delhi) in a preliminary study that screens marine fungal

metabolites for antimicrobial compounds.

18.3 Alternate strategies

Preliminary investigations in the PI‟s lab have shown that sponge fungi and

mangrove endophytes are good sources of novel metabolites. Biodiversity of marine

fungi associated with sponges and mangroves of Andaman Island will be worked out. A

culture collection of this rare fungi will be available for further work. A library of

bioactive metabolites will be available for further development on the path to their

transformations into drugs for clinical use..

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33

19. Timelines:

Period of study Achievable targets

6 months Procuring Instruments, appointing JRF. 2 collection trips, isolation of

fungi; 2 collection trips to Rifle man Islands of Andaman, isolation of

fungi, characterization of fungi using r-DNA sequences

12 months 3 collection trips to Rifle man, Manjeri Islands of Andaman, isolation

of fungi, characterization of fungi, screening for antialgal, antifungal,

antibacterial, insecticidal, antioxidant and antimalarial activities.

Isolation, purification of interesting compounds.

18 months 2 collection trips to Andaman islands (Wandoor Islands). Isolation of

fungi, characterization of fungi, screening for antialgal, antifungal,

antibacterial, insecticidal and antioxidant activities. More isolation

and continuation of search for the above metabolites. Isolation,

purification of interesting compounds. Computation of ecological

parameters of mangrove endophytes. Chemical characterization of

metabolites. Primary Bioassay Guided fractionation of active extracts.

24 months 2 collection trips to Andaman islands (Wandoor and Plueto Islands).

Isolation of fungi, characterization of fungi, screening for antialgal,

antifungal, antibacterial, insecticidal antioxidant and antimalarial

activities. A culture collection as a result of these collections.

Identification of the chemicals that inhibit microbes. Computation of

ecological parameters of sponge associated fungi. Large Scale

purification of identified metabolites

34

30 months 2 collection trips to Andaman islands (Wandoor and Plueto Islands).

Isolation of fungi, characterization of fungi, screening for antialgal,

antifungal, antibacterial, insecticidal antioxidant and antimalarial

activities. Cultures of interesting fungi, and extracted metabolites to

be placed in VINSTROM, IIT and ICGEB. Voucher specimens to be

deposited in a National Culture Collection centre. Large Scale

purification of identified metabolites, Cytotoxicity, In vivo drug

efficacy study of purified molecules, Structural characterization of

active molecules.

36 months Preparation of final report, papers for publication and filing of patents.

Large Scale purification of identified metabolites, Cytotoxicity, In

vivo drug efficacy study of purified molecules, Structural

characterization of active molecules.

20. Name and address of 5 experts in the field:

Sl.

No.

Name Designation Address

1 Prof. V. K. Bhasin Professor Department of Zoology, Delhi

University, India

2 Prof . Santosh K. Kar Professor Centre for Biotechnology,

Jawaharlal Nehru University, New

Delhi, India

3 Prof. Aparna Dixit Professor Centre for Biotechnology,

Jawaharlal Nehru University, New

Delhi, India

4 Dr. Peter Proksch Professor Institute for Pharmaceutical

Biology & Biotechnology,

Heinrich-Heine-Universität

Duesseldorf, Duesseldorf,

Germany

5 Dr. Florenz Sasse Professor Helmholtz Centre for Infection

Research, Department of Chemical

Biology, Braunschweig, Germany

35

Product Development:

Products in the form of drugs or drug leads against Malaria, fungal infections, anti

insects, anti algal and antioxidants are likely to be discovered in the project.

36

PART IV: BUDGET PARTICULARS (Principal Investigator - VINSTROM)

Budget (In Rupees)

A. Non-Recurring

Sl. No. Item Year 1 Year 2 Year 3 Total

1.

2.

Horizontal

autoclave (50 L

capacity)

ELISA Reader

40,000

8,00,000

- - 8,40,000

Sub-Total (A) =8,40,000

B. Recurring

B.1 Manpower

Sl.

No.

Position No. Consolidated

Emolument

Year 1 Year 2 Year 3 Total

1. JRF Rs 12000 pm

+30%

HRA(Rs.

14000 pm

+30% HRA-

third year

only)

1,87,200 1,87,200 2,18,400 5,92,800

Sub-Total (B.1) = 5,92,800

B.2 Consumables

S.

No.

Item

Quantity Year 1 Year 2 Year 3 Total

1. Glassware,

Chemicals for

media, HPLC,

TLC plates, etc.

1,50,000 1,50,000 1,50,000 4,50,000

Sub-Total (B.2) = 4,50,000

Other items Consolidated

Emolument

Year 1 Year 2 Year 3 Total

B.3 Travel

35,000 25,000 35,000 95,000

B.4 Contingency

50,000 50,000 50,000 1,50,000

B.5 Overhead

(20%)

84,440 82,440 90,680 2,57,560

37

Sub-total of B

(B.1+B.2+B.3+B.4+B.5)

15,45,360

Grand Total (A + B) 23,85,360

1. A JRF is needed for collecting samples, isolating endophytes and to culture

them as well as to screen them for metabolites.

2. An ELISA Reader is essential for rapid screening of the hundreds of isolates

that we expect and to identify the candidate fungi for further study.

3. Chemicals (high grade) are needed to obtain pure culture of the fungi;

solvents are needed to extract secondary metabolites from mycelia and

culture extracts.

38

PART IV: BUDGET PARTICULARS (Co-Investigator - 1 & 2 from IIT

Madras and ICGEB)

Budget : IIT

A. Non-Recurring

Sl. No. Item Year 1 Year 2 Year 3 Total

1. - - - - -

2. - - - - -

Sub-Total (A) =

Recurring

B.1 Manpower

Sl.

No.

Position

No.

Consolidated

Emolument

Year 1 Year 2 Year 3 Total

1. JRF Rs 12000 pm

+30% HRA

1,87,200 1,87,200 1,87,200 5,61,600

Sub-Total (B.1) = 5,61,600

B.2 Consumables

Sl. No. Year 1 Year 2 Year 3 Total

Chemicals/solvents/glass

ware/plastic ware

2,00,000 2,00,000 2,00,000 6,00,000

Sub-Total (B.2) = 6,00,000

Other items. Year 1 Year 2 Year 3 Total

B. 3 Travel 30,000 30,000 30,000 90,000

B.4 Contingency 75,000 75,000 75,000 2,25,000

B.5Overhead (20%) 98,440 98,440 98,440 2,95,320

Sub-total of B

39

(B.1+B.2+B.3+B.4+B.5)

Grand Total (A+B) 5,90,640 5,90,640 5,90,640 17,71,920

1. A JRF (with Biochemistry background)is needed to do the activity studies and

structure elucidation

2. We require plenty of solvents for column purification hence the consumable

budget will be high

3. We have to carry out LC-MS and CHN analysis on payment basis, hence require

contingency

Budget : ICGEB

B. Non-Recurring

Sl. No. Item Year 1 Year 2 Year 3 Total

1. SPD-M20A-

PDA Detector

To aid in

purification of

coeluting

molecules that

are spectrally

distinct.

~ 600000 ~ 600000

2. Multiple sample Parallel Evaporation device: Syncore Syncore comprises two instruments and a broad range of accessories suitable for all aspects of multiple sample processing.Its design concept allows one unit to perform as a parallel evaporator (Polyvap) which can elegantly be converted to a parallel concentrator (Analyst). Thus its applications include fast, parallel evaporation of multiple samples, the smooth concentration to predefined residual volumes and sample preparation and purification with SPE.

2200000 2200000

40

Sub-Total (A) = ~ 2800000 (Subject to quotation)

Recurring

B.1 Manpower

Sl.

No.

Position

No.

Consolidated

Emolument

Year 1 Year 2 Year 3 Total

1. JRF/SRF 16000/18000

+ 30% HRA

280800 280800 280800 842400

Sub-Total (B1) = 842400

B.2 Consumables

Sl. No. Item Quantity Year 1 Year 2 Year 3 Total

Biological

reagents,

media,

Fluorescent

dyes,

Solvents,

Glass ware

Purchase

and

maintenance

of mice for

in vivo

antimalarial

testing of

lead

compounds

400000 400000 400000 1200000

Sub-Total (B2) = 1200000

Other items. Consolidated

Emolument

Year 1 Year 2 Year 3 Total

B. 3 Travel 0 40000 40000 80000

B.4 Contingency 100000 100000 100000 300000

B.5Overhead (20%) 628480

Sub-total of B

(B.1+B.2+B.3+B.4+B.5)

2292680

Grand Total (A+B) 5092680

41

PART V: EXISTING FACILITIES

Resources and additional information

1. Laboratory: Separate Mycology lab 1300 Sqft. Area.

a. Manpower

b. Equipment

Principal Investigator’s lab:

Air Conditioners, Autoclave, Automatic Shaker, Electrophoresis Unit,

UV Transilluminator, Freeze Dryer (Lyophilizer), Hot air Ovens, Laminar air flow

chambers, Microscopes, NUV lamps, Digital SLR Camera, Zoom lens, Pentax 80-

200 Zoom lens, Computer with Laser Jet printer, Refrigerated Centrifuge, Rotary

Shaker, Rotavapor, Electronic Balance, Vortex Mixer, Magnetic Stirrer, Vacuum

Pump, Growth Chamber, UV-Vis Spectrophotometer, Glass Distillation Unit, Deep

freezer and HPLC.

In Co-PI’1 Lab: IIT

Bacteriological Incubator, Spectrophotometer, Protein Purification System,

Gel Electrophoresis Units, Micro plate Spectrometer, Bench top Freeze Dryer,

Gel Documentation System, High Pressure Liquid Chromatography, Fast

Protein Liquid Chromatography, Membrane ultra-filtration unit , Lyophilizer

Vacuum flash evaporators, UV-Vis spectrophotometer with accessories

UV trans illuminator , Fourier transform Infra Red Spectroscopy,

Ultracentrifuge, pH stat, rotavap, Millipore distillation system, Gel permeation

Chromatography, Sonicator , MALDI-TOF, SEM

Other resources such as clinical material:

Cell culture lab, General Biochemistry Lab

In Co-PI’2 Lab: ICGEB

42

EXISTING FACILITIES

S.

No.

Name of equipment/

accessories

Make Funding

Agency

Year of

Procurement

1 PREP HPLC Gilson DBT 2006

2 Microscope Nikon DBT 2006

3 Coolant circulator Julabo DBT 2006

4 NMR 500 MHz Bruker ICGEB 2009

5 FT-IR Perkin Elmer ICGEB 2006

6 Multiwell florescence

Reader

Perkin Elmer DBT 2007

Other resources such as clinical material, animal house facility, glass house.

Experimental garden, pilot plant facility etc.

Animal House facility on a payment basis is available at the ICGEB, New Delhi

43

PART VI: DECLARATION/CERTIFICATION

It is certified that

a) the research work proposed in the scheme/project does not in any way duplicate the work

already done or being carried out elsewhere on the subject.

b) the same project proposal has not been submitted to any other agency for financial support.

c) the emoluments for the manpower proposed are those admissible to persons of corresponding

status employed in the institute/university or as per the Ministry of Science & Technology

guidelines (Annexure-III)

d) necessary provision for the scheme/project will be made in the Institute/University/State

budget in anticipation of the sanction of the scheme/project.

e) if the project involves the utilisation of genetically engineered organisms, we agree to submit

an application through our Institutional Biosafety Committee. We also declare that while

conducting experiments, the Biosafety Guidelines of the Department of Biotechnology would

be followed in toto.

f) if the project involves field trials/experiments/exchange of specimens, etc. we will ensure that

ethical clearances would be taken from concerned ethical Committees/Competent authorities

and the same would be conveyed to the Department of Biotechnology before implementing

the project.

g) it is agreed that any research outcome or intellectual property right(s) on the invention(s)

arising out of the project shall be taken in accordance with the instructions issued with the

approval of the Ministry of Finance, Department of Expenditure, as contained in Annexure-

V.

h) we agree to accept the terms and conditions as enclosed in Annexure-IV. The same is signed

and enclosed.

44

45

PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF

INVESTIGATORS

Name : Dr. T.S. Suryanarayanan

Designation : Director

Department/Institute/University : Vivekananda Institute of Tropical Mycology

(VINSTROM)

Ramakrishna Mission Vidyapith

Chennai 600 004

Date of Birth : 25.07.1951 Sex: Male

Education

Sl. No. Institution Place Degree Awarded Year Field of Study

1. University of Madras M.Sc 1973 Botany

2. University of Madras Post M.Sc.,

Diploma

1974 Mycology and

Plant

Pathology

3. University of Madras Ph.D 1979 Mycology

A. Position and Honors

Position and Employment

Sl. No. Institution Place Position From To

1. Dept. of Botany

RKM Vivekananda

College

Reader and

Head of the

Department

Dec. 1978 July 2009

2. Vivekananda Institute

of Tropical Mycology

(VINSTROM)

RKM Vidyapith

Director August 2009 Till date

Honors/Awards

Fulbright Nehru Senior Fellowship, The Ohio State University, USA, September

2011 – March 2012.

Fulbright visiting faculty, Arizona State University, Tempe, Arizona, USA. May

2004.

Honorary research Associate, University of New Brunswick, St. John, Canada.

46

Invited Lectures

Some strategies to better-utilize fungal endophytes – Intergovernmental Framwork for

European Cooperation in Science & Technology – Symposium on Endophytes in

Biotechnology & Agriculture – Trento, Italy November 2012.

Fungal Endophytes: Ecology and Economic Potential. – Helmholtz Centre for

Infection Research, Braunschweig, Germany. May 2007.

Ecology of fungal endophytes: contribution from VINSTROM. – Plant Pathology

Seminar, University of Arizona, Tucson, Arizona, USA, November 2006.

Tropical Fungal Endophytes: ecological insights gained from forests of Southern

India – 25th

Genetics of Microorganisms Congress, São Paulo, Brazil, March 2006.

Tropical Endophytes: An Ecologist’s Perspective – Technical University of

Braunschweig, Germany, November 2005

Biotechnological Potential of Fungal Endophytes: An Overview – Pipe line Seminar

at the German Research Centre for Biotechnology, Braunschweig, Germany,

November, 2005.

Endophytes of Sonoran Cacti - School of Life Sciences, Arizona State University,

USA. 2004

Fungal endophytes of the mangrove Rhizophora apiculata – Asian Mycological

Congress, Hong Kong, 2000

Diversity of endophytes in tropical forests: the Western Ghats experience – 3rd Asia-

Pacific Mycological Congress on Biodiversity and Biotechnology, 2002, Kunming,

China.

B. Publications

Book chapters 12 Research Papers 88

Selected peer-reviewed publications

1. Suryanarayanan, TS and Hawksworth, DL (2005) Fungi from little-explored and

extreme habitats. In Bio-diversity of fungi: Their role in human life (SK

Deshmukh and MK Rai, eds.): 33-48. Oxford & IBH Publishing, New Delhi.

2. Suryanarayanan, T.S., Ravishankar, J.P., and Muruganandam V. (2010). Drug

discovery: going with the tide. Current Science 99:1308.

3. Kumaresan, V and Suryanarayanan, TS (2001) Occurrence and distribution of

endophytic fungi in a mangrove community. Mycological Research 105: 1388-

1391.

4. Suryanarayanan, TS and Kumaresan, V (2000) Endophytic fungi of some

halophytes from an estuarine mangrove forest. Mycological Research 104: 1465-

1467.

5. Suryanarayanan, TS, Kumaresan, V and Johnson, JA (1998) Foliar fungal

endophytes from two species of the mangrove Rhizophora. Canadian Journal of

Microbiology 44: 1003-1006.

47

6. Suryanarayanan, T.S., Thirunavukkarasu, N., Govindarajulu, M.B., Sasse, F.,

Jansen, R. and Murali, T.S. 2009. Fungal Endophytes and Bioprospecting. Fungal

Biology Reviews 23: 9-19.

7. Ravishankar, JP, Muruganandam, V and Suryanarayanan, TS (1996) Effect of

salinity on amino acid composition of the marine fungus Cirrenalia pygmea.

Current Science 70: 1087-1089.

8. Ravishankar, JP, Muruganandam, V and Suryanarayanan, TS (1995) Isolation and

characterization of melanin from a marine fungus. Botanica Marina 38: 413-416.

9. Ravishankar, JP, Muruganandam, V and Suryanarayanan, TS (1994) Effect of

salinity on fatty acid composition of Cirrenalia pygmea, an obligate marine

fungus. Botanica Marina 37: 479-481.

10. Suryanarayanan, T. S., Thirumalai, E., Prakash, C.P., Govinda rajulu, M.B. and

Thirunavukkarasu, N. 2009. Fungi from two forests of southern India: a

comparative study of endophytes, phellophytes and leaf litter fungi. Canadian

Journal of Microbiology 55: 419-426.

List maximum of five recent publications relevant to the proposed area of work.

1. Suryanarayanan, T.S., Murali, T.S., Thirunavukkarasu, N., Govinda Rajulu, M.B.,

Venkatesan, G., Sukumar, R. (2011). Endophytic fungal communities in woody

perennials of three tropical forest types of the Western Ghats, southern India.

Biodiversity and Conservation 20: 913-928.

2. Govinda Rajulu, M.B., Thirunavukkarasu, N., Suryanarayanan, T.S.,

Ravishankar, J.P., El Gueddari, N.E. and Moerschbacher, B.M. (2011).

Chitinolytic enzymes from endophytic fungi. Fungal Diversity 47:43-53.

3. Thirunavukkarasu, N., Suryanarayanan, T.S., Murali, T.S., Ravishankar, J.P.,

Gummadi, S.N. (2011). L-asparaginase from marine derived fungal endophytes of

seaweeds. Mycosphere 2:147–155.

4. Suryanarayanan, T.S., Ravishankar, J.P., Muruganandam V. (2010). Drug

discovery: going with the tide. Current Science 99:1308.

5. Suryanarayanan, T.S., Venkatachalam, A., Thirunavukkarasu, N., Ravishankar,

J.P., Doble, M. and Geetha, V. (2010) Internal mycobiota of marine macroalgae

from the Tamilnadu coast: distribution, diversity and biotechnological potential.

Botanica Marina 53:456-468.

B. Research Support

Ongoing research Projects

Sl.

No.

Title of Project Funding

Agency

Amount Date of

sanction and

Duration

1. The potential of fungal

endophytes as biocontrol

organisms

DBT 36,59,000/- February

2011

(3 Years)

48

2. Endophytic fungi of endemic

plants of Arunachal Pradesh:

Diversity and novel metabolites

DBT 14,80,000 May 2012

(3 years)

Completed research Projects

Sl.

No.

Title of Project Funding

Agency

Amount Date of

completion

1. Screening of tropical fungal

endophytes for novel secondary

metabolites

DBT 24,95,000/- 31.08.2008

2. Diversity of microfungi in leaf

litter of the different forests of

Western Ghats

MoEF 12,60,000/- 15.02.2009

3. Enzymes of pharmaceutical

importance from tropical

microfungi

DBT 25,30,000/- 22.05.2010

Place : Chennai

Date : Signature of Investigator

49

PART VII : PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS

Name : Dr. Mukesh Doble

Designation : Professor

Department/Institute/University : Dept. of Biotechnology, Indian Institute of

Technology Madras, Chennai 600 036.

Date of Birth : 16.09.1950 Sex: Male

Education

Sl. No. Institution Place Degree Awarded Year Award

1. IIT, Madras B.Tech 1972 I class

2. IIT, Madras M.Tech 1974 I class

3. University of Aston,

Birmingham

Ph.D 1980

Position and Honors

Position and Employment

Sl. No. Institution Place Position From To

1. Dept. of

Biotechnology, IIT

Chennai.

Professor April 2004 Till Date

Research Experience in various institutions

Professor, IIT Madras, Department of Biotechnology, IIT Madras, (April 2004-

Faculty, Centre for Biotechnology, Anna University, Chennai, (July 2003, April

2004)

Program Leader, Chemical engineering modelling group, GE India Technology

centre, Bangalore (2000 - 2003 )

Process modelling of monomer and polymer reactions

Setting up of chemical engineering lab and polymer reaction lab

Group Manager, ICI India R&T centre , Bombay (1983-88, 1989-2000)

Process development/scale-up, process engineering and technology

development modelling & simulation, reaction kinetics and mechanisms

molecular modelling and QSAR studies

Research Fellow, Texas A&M University, USA (1988-89)

Fischer-Tropsch catalyst design

Research Fellow, University of Cambridge, UK (1980-83)

Car exhaust catalysis design and modelling

Asst Engineer , KCP Ltd, Madras (1974-77)

Pneumatic conveying

sugar processing

50

B. Publications

Books 7 Research Papers 109

Selected peer-reviewed publication

1. Sivakumar, PK and Mukesh Doble, COX-2 Enzyme and its Inhibitors, Current

Bioactive Compounds, 2006 2 (2), 161-179.

2. Mukesh Doble, Biological Treatment of VOCs, Chemical Engineering, 2006, 35-

41.

3. Mukesh Doble and Anil Kumar K, Experimental and modelling studies on

antifungal compounds, Central European Journal of Chemistry 4 (3), 2006, 428-

439.

4. Hemalswarya, Mukesh Doble, Potential synergism of natural products in the

treatment of cancer, Phytotherapy Research, 20, (4)2006.

5. Devarajan, Padma V, Sonavane, Ganeshchandra S and Mukesh Doble, Computer

Aided Molecular Modeling: A Predictive Approach in the Design of

Nanoparticulate Drug delivery System, Journal of Biomedical Nanotechnology,

1(4),2005, 375-383.

6. Bhagwatr, SS, Bevinakatti, Mukesh Doble, Transesterification of substituted

ethanols with ethyl acetate-modelling studies, j. biochem. Engg., 2005, 22(3),

253-360.

7. Mukesh Doble, Putting Six-Sigma processes to work, Chemical Engineering,

2003, 62-67.

8. Mukesh Doble, scale up of chemical reaction, 2002, 48-56.

9. Bhaduri, S, Lahari GK, Munish P and Mukesh Doble (2000) Catalysis Letters,

65,1.

List maximum of five recent publications relevant to the proposed area of work.

1. K. Anil Kumar, Sanjay Kumar Singh, B. Siva Kumar and Mukesh Doble,

Synthesis, anti-fungal activity evaluation and QSAR, studies on podophyllotoxin

derivatives, Central European Journal of Chemistry 5(3) 880-897 (2007).

2. P M Sivakumar, Geetha Babu and Mukesh Doble, QSAR studies on Chalcones

and Flavonoids as Anti-Tuberculosis Agents using genetic function

approximation (GFA) method, Chem. Pharm. Bull. 55(1) 44−49 (2007).

51

3. M. Sudhakar, Priyadarshini, Mukesh Doble,P.Sriyutha Murthy and R.

Venkatesan, Marine Bacteria Mediated Degradation of nylon 66 and 6,

International Biodeterioration and Biodegradation

doi:10.1016/j.ibiod.2007.02.002

4. P. M. Sivakumar, Mukesh Doble, S. Prabu Seenivasan and Vanaja Kumar,

Synthesis, Antimycobacterial activity evaluation and QSAR studies of chalcone

derivatives, Bioorganic & Medicinal Chemistry Letters , 17, (6), 1695-1700

(2007)

5. Puratchikody, Mukesh Doble, Antinociceptive and antiinflammatory activities

and QSAR studies on 2-substituted-4, 5-diphenyl-1H-imidazoles, Bioorganic and

Medicinal Chemistry, Bioorg Med Chem. Jan 15; 15(2):1083-90 (2007).

C. Research Support

Ongoing research Projects

Sl.

No.

Title of Project Funding

Agency

Date of sanction and Duration

1. Enhanced biosurfactant production

for improving biodegradation of

poly propylene

DST May 2007

(3 Years)

2. Synthesis and characterization of

curdlan

DBT August 2006

(3 Years)

Place :

Date : Signature of Investigator

52

PART VII : PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS

Name : DR. DINKAR SAHAL

Designation : SENIOR RESEARCH SCIENTIST

Department/Institute/University : Malaria Research Group

I.C.G.E.B., P.O.Box 10504, Aruna Asaf Ali

Marg,

New Delhi-110 067.

Date of Birth : 18 JAN 1956

Sex (M/F) : Male

Education (Post-Graduation onwards & Professional Career)

SI No. University/Institution Degree awarded Year

1. M.S. university,

Baroda, India

M.Sc (Biochemistry)

1977

2. All India Institute of

Medical Sciences

New Delhi.

Ph.D (Biochemistry) 1984

RESEARCH EXPERIENCE IN VARIOUS INSTITUTIONS:

Professional Experience:

1977 - 1984 : Senior Research Fellow,Department of Biochemistry,All india

institute of Medical sciences, New Delhi, India .

1984 -1986 : Research Associate, Molecular Biophysics Unit, Indian Institute of

Science, Bangalore 560012.

1986 -1988 : Research Fellow, Department of Molecular Genetics, Beckman

Research Institute of the City of Hope, Duarte, California.

1988 -1992 : Lecturer, Department of Biophysics, University of Delhi, South

Campus New Delhi . I was engaged in teaching physical biochemistry to Post graduate students and

conducting research on (a) Peptide models of Protein phosphorylation and

(b)Chromogenic peptides as substrates in testing pyrogens by the amoebocyte lysate

assay from the horse shoe crab system,

1992- till now: Senior Research Scientist, ICGEB, New Delhi I was actively engaged in developing laboratory technologies for production of gene

products both by recombinant and chemical synthesis routes. I have synthesized human

53

Oxytocin by solution phase synthesis in a final yield of 70 grams and human

Adrenocorticotropic hormone by solid phase synthesis in a final yield of 4 grams. By the

recombinant approach, human Insulin was synthesized in milligrams scale following

expression of insulin chains as fusion protein inclusion bodies in E.coli, cyanogens

bromide cleavage , sulfitolysis and A+B chain assembly into chromatographically pure

and biologically active human insulin. At the ICGEB, New Delhi, Sahal made

significant contributions to the study of (a) anomalous mobility of sulfitolysed proteins in

SDS-PAGE and improvising this into a simple method to monitor the kinetics of

sulfitolysis, (b) the invention of a charcoal based method for removal of Iodine used in

the formation of disulfide bonds as exemplified by the synthesis of Oxytocin (c) down

stream processing of recombinantly expressed insulin precursors and (d) peptide cocktails

for application in ELISA based diagnosis of hepatitis C. In more recent times, Sahal has

made a mark by playing a key role in the de novo design and chemical syntheses of

helical hairpin peptides. The first such helical hairpin reported in PNAS (2001) was

designed for proximal orientation of a left handed helix with a right handed helix. Soon

thereafter, Sahal and companions reported in STRUCTURE (2004) the crystal structure

and circular dichroism studies of an analogous homochiral hairpin in which both helices

were right-handed. Most recently, Sahal‟s lab has reported the design, synthesis and

spectroscopic characterization of Trpzip- a de novo designed water soluble mini protein

displaying a motif. Sahal‟s investigations have traversed a large canvas from

structure to function. Thus, one of the major current interests of Sahal‟s laboratory is the

design of potent antibiotic peptides. Mechanistic studies of these peptides are revealing

important clues on the design of the much needed novel antibiotics acting at new targets.

Sahal is currently a Principal Investigator of the DBT sponsored project on “Potentiation

of de novo designed conformationally constrained helical peptides for antibiotic action”.

His talk on antibiotic peptides at the Satellite meeting of the joint 15th

IUPAB & 5th

EBSA International Biophysics Congress on “Cell Penetrating Peptides” (Aug 2005)

held at Montpellier, France was well received. Sahal has made important contributions in

proposing new models for our understanding of the antimalarial action of Artemisinin.

He has played a key role in developing validated high through put methods for the

discovery of novel blood stage antimalarials. These methods have been patented,

presented at the World conference on Magic Bullets-to celebrate Paul Ehrlich‟s 150th

birthday, Nurnberg 2004 and published. Using these methods, Sahal‟s laboratory has

identified promising antimalarial hits from chemical combinatorial libraries on the one

hand and marine organisms on the other. The exploration of hits from chemical

combinatorial libraries is a programme in collaboration with CDRI, Lucknow. Sahal is a

Principal Investigator in the DBT sponsored multi institutional project (ICGEB,New

Delhi- Malaria Research Centre, Delhi and Marine Research Lab,University of

Trivandrum) entitled “Discovering antimalarials from marine Organisms”.

RESEARCH SEMINARS PRESENTED

(a)Self-nonself discrimination : a model

(b)An excursion with Peptides

(c)How do Information and Energy play the game of Biology?

(d)Human Chorionic Gonadotrpin: Structure, Function and Immunobilogy

(e)Cryptobiosis

(g) Some significant properties of the peptide bond

(h)Theory of Chromatography

(i) Thermodynamics of Evolution

54

(j) From Ellman‟s reagent to disulfide biochemistry

(k) The chirophilic Glycine Crystal

(j) Origins of biomolecular handedness

(k) In vivo Crystallization and Biomineralization

(l) Protein Electrostatics

(m) Insulin and IGF-I Receptors: a comparative study of isoreceptor Protein

Tyrosine Kinases using exogenous substrates, inhibitors and stimulator

(n) Innovations in Protein Kinase assay

(o) Joys and Travails of Drug discovery

(p) Quality control in Protein Pharmaceuticals

(g) Artemisinin : a colorless drug targets heme a colored cofactor to bring out its

antimlarial action

(h) Making way through the haze of drugs and bugs

(i) Bacterial knock at the SIMIR club

(j) Sensing the color of heme towards discovery of novel antimlarials

REVIEWS

Deciphering ionic interactions in Peptides and Proteins.

Dinkar Sahal in Biophysical Processes in Living Systems. 2001,Editor: P.Pardha

Saradhi, Oxford & IBH Publishing Co.Pvt. Ltd. New Delhi, pp 55-74.

MONOGRAPHS:

1. Production of Human Oxytocin by Solution Phase Chemical Synthesis (1997)

D.SAHAL, ICGEB, New Delhi.

2. Production of Human Adrenocorticotropic Hormone by Solid Phase Chemical

Synthesis (1996) D.SAHAL, ICGEB, New Delhi.

Sahal’s Commentary on the 1992 Nobel Prize in Physiology and Medicine:Key to Life’s

Dynamism- The work of Krebs and Fischer has given a new perspective on controls in

Biological processes. The Economic Times 24th

October 1992, p.11.

Reviewer/Referee/Critic : I have been reviewing manuscripts written by friends, and

papers or proposal received from grant giving agencies like the DBT/DST/CSIR. Also I

am a reviewer of papers sent by various journals like Biochemie, Analytical

Biochemistry, Blood, Current Science, J Photochem. PhotoBiol. etc. I am the guest editor

of the Journal of Amino acids for its special issue entitled “Peptides: Applications from

Drug Discoveries to Materials and Molecular Electronics"

Book review by Sahal

Biophysics reviewed by Dinkar Sahal Current Science 83,(10) 25 Nov 2002, 1267-1269

List of important publications

1. Sahal D, Ramakrishnan,S; Iyer,K.S.N.; Das,C;and Talwar,G.P (1982) Immunobiological properties of a

carboxyterminal 53 aminoacid peptide of the beta subunit of human chorionic gonadotropin. J.

Reproductive Immunology 4,145-156

55

2. Talwar,G.P; Gupta S.K ; Singh,V; Sahal,D; Iyer,K.S.N; and Singh,O. (1985) Bioeffective monoclonal antibody

against the decapeptide gonadotropin release hormone: reacting determinants and action on ovulation and

estrus suppression. Proc. Natl.Acad.Sci.U.S.A 82,1228-1231

3. Iyer,K.S.N.; Sahal,D; and Talwar,G.P. (1986) Chemical synthesis and immunobiological properties of a cyclic

eicosapeptide(Gly )82-101 of the beta subunit of human chorionic gonadotropin. Int.J.Peptide Protein Res.

27,604-612.

4. Sahal,D; and Balaram,P. (1986) Peptide models of electrostatic interactions in proteins:nmr studies on two

beta turn tetra peptides containing Asp---His and Asp---Lys salt bridges. Biochemistry 25, 6004-6013.

5. Sahal,D; and Fujita-Yamaguchi,Y. (1987) Protein kinase assay by paper trichloroacetic acid method:high

performance using phosphocellulose paper and washing an ensemble of samples on flat sheets.

Anal.Biochem 167, 23-30

6. Sahal,D;Ramachandran,J; and Fujita-Yamaguchi.Y. (1988) Specificity of tyrosine protein kinases of the

structurally related receptors for insulin and insulin like growth factor-1:tyr-containing synthetic polymers

as specific inhibitors or substrates. Archives of Biochem. Biophys. 260 , 416-426.

7. Yoko Fujita-Yamaguchi, David B.Sacks, Jay M.Mcdonald, Dinkar Sahal and Satish kathuria. (1989) Effect of

basic polycations and proteins on purified insulin receptor:insulin- independent activation of the receptor

tyrosine-specific protein kinase by poly(l-lysine). Biochem.J. 263, 813-822.

8. Dinkar Sahal and Yoko Fujita-Yamaguchi. (1989) Solid-phase tyrosine specific protein kinase assay in

multiwell substrate-immobilized polyacrylamide gel. Anal.Biochem 182, 37-43.

9. Dinkar Sahal,Shu-Lian Li and Yoko Fujita-Yamaguchi. (1991) Solid-phase protein kinase assay. Methods in

Enzymology 200 , 90-98.

10. Jyoti verma and Dinkar Sahal. ,(1992) Chromatographic and proton nmr study of ion pair interaction

between dodecyl histidine methyl ester and dodecyl hydrogen phosphate in chloroform. Bull.Chem.Soc.Jpn

65, 1719-1721

11. N.Sankar and Dinkar Sahal. (1993) A phosphate staining reagent revisited. Bull.Chem.Soc.Jpn. 66, 2579-

2581.

12. Malhotra,M. and Sahal, D. (1996) Anomalous mobility of sulfitolyzed proteins in SDS- PAGE:Analysis and

Applications. Int.J.Peptide Protein Res 48, 240-248

13. Vijay Kumar , N.Jayasuryan , Honey Reddi , Dinkar Sahal & S.K.Panda. (1998) A monoclonal antibody against

the X protein of hepatitis B virus.Fine mapping of its epitope and application in a quantitative ELISA of the

X protein in sera of Hepatitis B patients. Hybridoma 17 (2) , 157-164.

14. Dinkar Sahal. (1998) Physiology and overcrowding. Current Science 74 (12) 1033.

15. D.Sahal. (1999) Removal of iodine by solid phase adsorption to charcoal following oxidation of

acetamidomethyl-protected peptide precursors to their disulfide bonded products: oxytocin and a Pre-

S1 peptide of hepatitis B virus illustrate the method. J. Peptide Research 53, 91-97.

16. Dinkar Sahal. Deciphering ionic interactions in peptides and proteins. In "Biophysical Processes in Living

Systems", Ed. P.Pardha Saradhi. Oxford & IBH Publishers

17. Udupi A. Ramagopal , Suryanarayanrao Ramakumar, Dinkar Sahal and Virander S.Chauhan. (2001) De novo

design and characterization of an apolar helical hairpin peptide at atomic resolution: Compaction

mediated by weak interactions. Proc.Natl.Acad.Sci (USA) 98 (3), 870 - 874.

56

18. Sahal D, Kannan R, Sinha A, Babbarwal V, Gnana Prakash B, Singh G, Chauhan VS. (2002) Specific and

instantaneous one-step chemodetection of histidine-rich proteins by Pauly's stain. Anal Biochem. Sep

15;308 (2):405-8.

19. Kannan R, Sahal,D, Chauhan V.S. (2002) Heme-artemisinin adducts are crucial mediators of the ability of

artemisinin to inhibit heme polymerization. Chem Biol. Mar; 9 (3): 321-32

20. Hasan Korkaya, Shahid Jameel, Dinesh Gupta, Shweta Tyagi, Ravinder Kumar, Mohammad Zafrullah, Manjari

Mazumdar, Sunil Kumar Lal, Li Xiaofang, Deepak Sehgal, Suman Ranjan Das, and Dinkar Sahal (2001) The

ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK. J Biol Chem. Nov

9;276(45):42389-400.

21. Sahal,D , Kannan R, , Chauhan V.S. (2003) Applying malaria parasite's heme detoxification system for

screening potential antimalarial drugs. Anal Biochem. Jan 15;312(2):258-60.

22. Dinkar Sahal. (2002) Book Review of Biophysics (Vasantha Pattabhi and N.Gautham,Narosa Publishing

House,New Delhi Current Science 83, 1267

23. Sahal,D. (2003) Absence of condensing agent. Current Science 84,978.

24. Rudresh; Ramagopal, U.A.; Inai, Y.; Goel, S.; Sahal, D.; Chauhan, V.S. (2004) De novo design and

characterization of a helical hairpin eicosapeptide: Emergence of an anion receptor in the linker region.

Structure 12, 389-396.

25. Padmashri, R; Chakrabarti, K.S.; Sahal, D; Mahalakshmi,R.; Sarma, S.P.; and Sikdar. S.K. (2004) Functional

characterization of the pentapeptide QYNAD on rNa(v)1.2 channels and its NMR structure. Pflugers Arch.

447, 895-907.

26. Chetal, P.; Chauhan,V.S.; and Sahal, D. (2005) A Meccano set approach of joining trpzip a water soluble β-

hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide. J. Peptide Res. 65,

475-484.

27. Kannan, R, Kumar, K, Sahal, D, Kukreti, S, Chauhan, V.S. (2005) Reaction of artemisinin with haemoglobin:

Implications for antimalarial activity. Biochemical Journal 385(2) 409-418

28. Jagdish Rai, S. Raghothama and Sahal D. (2007) De novo design of F containing peptides. Chemical Biology

and Drug Design 69 (2):119-123.

29. Jagdish Rai, S. Raghothama and Sahal D. (2007)Tyrosine-heme ligation in Heme- peptide complex: design

based on conserved motif of catalase. J Pept Sci. 13 (6):406-412.

30. Ipsita Pal-Bhaumick, Ramendra Pati Pandey, Gotam K.Jarori, Santosh Kar, Dinkar Sahal (2007). Structural

and functional studies on Ribonuclease S, Retro S and Retro Inverso S peptides. Biochem Biophys Res

Commun. Oct 18; 17963728

31. Sahal, D. (2009) “Synthetic Life and Artificial Organisms”, chapter in the volume “Life and Organicism”:

Project on History of Indian Science, Philosophy and Culture (PHISPC) Rangaswamy, N.S. (ed)

32. Pooja C.Dewan, Aparna Anantharaman, Virander S Chauhan and Dinkar Sahal (2009) Antimicrobial Action of

Prototypic Amphipathic Cationic Decapeptides and Their Branched Dimers Biochemistry, 48, 5642-5657

33. Anantharaman, Meryam Sardar and Dinkar Sahal (2010) Synergy with rifampicin and kanamycin enhances

potency, kill kinetics and selectivity of de novo designed antimicrobial peptides. Aparna Antimicrobial

Agents and Chemotherapy, 54 ,1693-1699

34. Aparna Anantharaman and Dinkar Sahal (2010) Reverse Engineering Truncations of an antimicrobial peptide

dimer to identify the origins of potency and broad spectrum of action. J Med Chem, 53 (16), 6079–6088

57

35. Wanare G, Aher R, Kawathekar N, Ranjan R, Kaushik NK, Sahal D. (2010) Synthesis of novel alpha-

pyranochalcones and pyrazoline derivatives as Plasmodium falciparum growth inhibitors. Bioorg Med Chem

Lett. 20(15): 4675-8

36. Kumar R, Mohanakrishnan D, Sharma A, Kaushik NK, Kalia K, Sinha AK, Sahal D (2010) Reinvestigation of

structure activity relationship of methoxylated chalcones as antimalarials: Green Synthesis and evaluation

of 2,4,5-trimethoxy substituted patterns as lead candidates derived from abundantly available natural β-

asarone. Eur J Med Chem. 45(11):5292-301

37. Bagavan A, Rahuman AA, Kaushik NK, Sahal D (2011) In vitro antimalarial activity of medicinal plant

extracts against Plasmodium falciparum. Parasitol Res. Jan;108(1):15-22

38. Rao JL, Reddy PS, Mishra RN, Gupta D, Sahal D, Tuteja N, Sopory SK, Reddy MK (2010) Thermo and p H

stable ATP- independent chaperone activity of heat-inducible Hsp 70 from Pennisetum glaucum. Plant

Signal Behav. Feb;5(2):11-121.

39. Bagavan A, Rahuman AA, Kamaraj C, Kaushik NK, Mohanakrishnan D, Sahal D. (2011)Antiplasmodial activity

of botanical extracts against Plasmodium falciparum. Parasitol Res. 108(5):1099-109

40. Aher RB, Wanare G, Kawathekar N, Kumar RR, Kaushik NK, Sahal D, Chauhan VS (2011)

Dibenzylideneacetone analogues as novel Plasmodium falciparum inhibitors. Bioorg Med Chem Lett.

15;21(10):3034-6

41. Tanwar, P.a , Yadav, G.C.a , Jaitley, U.K.b , Kaushik, N.c , Sahal, D (2011) Synthesis and antimalarial activity

of novel N-{2-[2-(2-aminoethoxy) ethoxy] ethyl}-7-chloroquinolin-4-amine and its derivatives Indian Journal

of Chemistry - Section B Organic and Medicinal Chemistry, vol. 50, no. 2, pp. 233–241

42. Sayani Dasgupta, Sharmishtha Samantaray, Dinkar Sahal

and Rajendra P Roy (2011)

Isopeptide Ligation Catalyzed by Quintessential Sortase A: Mechanistic Cues from Cyclic and Branched

Oligomers of Indolicidin. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 27, pp. 23996–24006, July 8,

2011.43.

43. Tandem allylic oxidation–condensation/esterification catalyzed by silica gel:an expeditious approach

towards antimalarial diaryldienones and enones from natural methoxylated phenylpropenes. Abhishek

Sharma, Naina Sharma, Amit Shard, Rakesh Kumar, Dinesh Mohanakrishnan, Saima, Arun K. Sinha and Dinkar

Sahal (2011) Organic & Biomolecular Chemistry 9(14):5211-9.

44. Stilbene-Chalcone Hybrids: Design, Synthesis and Evaluation as a New Class of Antimalarial Scaffolds that

Trigger Cell Death through Stage Specific Apoptosis. Sharma, N, Mohanakrishnan D, Shard A, Sharma A,

Malik S, Sinha, A, Sahal D. (2012) J Med Chem 55,297-311.

45. Antimalarial activities of medicinal plants traditionally used in the villages of Dharmapuri regions of South India Chinnaperumal Kamaraj, Naveen Kumar Kaushik, Abdul Abdul Rahuman

a, Dinesh Mohanakrishnan, Asokan

Bagavan, Gandhi Elango, Abdul Abduz Zahir, Thirunavukkarasu Santhoshkumar, Sampath Marimuthu, Chidambaram Jayaseelan, Arivarasan Vishnu Kirthi, Govindasamy Rajakumar

a, Kanayairam Velayutham

a, Dinkar

Sahal (2012) J Ethnopharmacology

46. Anti-plasmodial action of de novo-designed, cationic, lysine-branched, amphipathic, helical peptides,

Naveen K Kaushik, Jyotsna Sharma and Dinkar Sahal. Malaria Journal (In Press)

PATENTS:

(a) Solid Phase Protein Kinase Assay

58

Dinkar Sahal, U.S.Patent Application Serial No. 321,298

(1989),Date of Filing March 10,1989.

Canadian Patent Application Serial No. 2011768-1, Date of

filing: March 8,1990.

(b) An Improved Process For the Preparation of Alkyl Phosphate.

Dinkar Sahal and Arun Kumar Sinha. Indian Patent Application

: An Improved Process For the Preparation of Alkyl Phosphate.

Dinkar Sahal and Arun Kumar Sinha. Indian Patent Application

Serial Number: 744/Del/91, Date of Filing: 13th

August 1991.

(c) An improved process for the preparation of Chromogenic

Peptides Containing Arginine para Nitroanilide useful for

testing of bacterial pyrogens in parenteral fluids, drugs, food

products, recombinant products, Vaccines, Medical devices,

radiopharmaceutical products, Water supplies etc intended for

human use.

Dinkar Sahal and Subita Srimal

Indian Patent Application Serial Number:317/Del/93/457,

Date of Filing: Dec.1992.

(d) Method of screening a candidate substance as a

potential drug for treatment of malaria. Sahal,D; Kannan,R;

Chauhan,V.S. Indian Patent Application No. 705/DEL/2002.

(e) Antibiotic potential of de novo designed

Didehydrophenylalanine containing

conformationallyconstrained cationic amphipathic peptides and

peptidomimetics

Dinkar Sahal, Pooja Chetal, Aparna Anantharaman and

Virander S.Chauhan.

Indian Patent Application 1343/DEL/2005 filed May 2005.