In silico Screening of Phytocompounds of Vitex negundo ...

PSGCAS Search: A Journal of Science and Technology Volume : 3 No. : 1, ISSN: 2349 – 5456 1

In silico Screening of Phytocompounds of Vitex negundo Linn. Leaves for Fungal Chitinase

Sharanya M.1 and *Sathishkumar R.2

1Department of Bioinformatics, Bharathiar University, Coimbatore, Tamil Nadu 2 Department of Botany, PSG College of Arts and Science, Coimbatore, Tamil Nadu * Corresponding author: [email protected]

ABSTRACT

Plants are serving human as everlasting source in different ways, most specifically in curing many human ailments. The plant sources are still acting as the reservoir for the novel structure. Recently more research work focuses on identifying novel plant compounds and plant based products. Most of the therapeutics is achieved from plant through various available techniques. In the present work, the phytocompounds of Vitex

negundo that have been isolated and reported in the literature were evaluated using bioinformatics approach for their potential fungal chitinase inhibition. The chitinase enzymes are involved in the synthesis and repair of cell wall component, chitin, which is unique for fungal cell walls. From high throughput virtual screening performed with schrodinger, it was found that the compounds agnuside (-5.8055), negundoside (-5.4376) and procatechuic acid (-5.0584) of V.negundo were showing least and significant docking scores when compared to others. In the present scernario bioinformatics is becoming a part in pharmaceutical companies which aids in the identification of lead compounds in a short period. Therefore, from the results obtained it can be further considered for QSAR studies for developing lead molecule specifically to inhibit fungal chitinase.

Keywords: Vitex negundo Linn., Chitinase, High throughput virtual screening, XP Docking, Agnuside.

INTRODUCTION

Fungi are the most common organisms

found in the environment, which are the causative

agents of infections either superficially,

subcutaneously or systemically. In the present

world, more number of factors is available for

acquiring fungal infections. Fungal infections are

common among the immunocompromised

patients rising as a life-threatening agents. In

addition, the population undergoing invasive

surgery, immunosuppressive therapy during

organ transplantation, treatment with broad-

spectrum antibiotics and glucocorticoids, receipt

of peritoneal dialysis or hemodialysis and

cancer are critically susceptible to fungal

infections [1]. Though, the available group

drugs, such as azoles, allylamines, polyenes,

etc., which are partially effective and the

longevity in course of intake lead to fungal

resistance [2]. Hence, the present era should

focus on searching the novel drug molecules to

treat fungal infections. From earlier days, plants

are serving as a reservoir for novel compounds

in treating various ailments. The plant products

like secondary metabolites, phenolic

compounds, essential oils and

Sharanya M. and Sathishkumar R


extracts were evaluated for their potential

antifungal activity. The treatment using

medicinal plants has historical basis of

therapeutic health care. Therefore, identifying

new and effective drugs from plant source would

be economically accessible and affordable in

developing countries [3, 4]. Terpenes, terpenoids,

saponins, alkaloids, phenolic compounds,

flavonoids, coumarins, xanthones, tannins,

lignans and other secondary metabolites of

plants are reported for promising antifungal

activity [3]. 1,2- dihydroxyxanthone is an

oxygenated xanthones showed activity on

clinical strains of Candida, Cryptococcus,

Aspergillus and Trichophyton mentagrophytes

and also had effect on sterol biosynthesis [5].

Similarly, terpenes including methyl chavicol

and linalool of Ocimum sanctum affected the

synthesis of ergosterol and caused membrane

cell damage in Candida species [6]. Furthermore,

the fungicidal effect of carvacrol and thymol are

originated from the inhibition of ergosterol

biosynthesis which correspondingly disrupts the

membrane integrity [7]. In this study, the

medicinal plant Vitex negundo Linn.

(Verbenaceae), also called nochi in Tamil was

chosen. Leaves are aromatic, bitter, acrid,

astringent, anodyne, anti-inflammatory,

antipyretic or febrifuge, tranquilizer, bronchial

smooth muscle relaxant, anti-arthritic,

anthelmintic and vermifuge [8]. The leaves

extracted with water-ethanol of 50:50 ratio

exhibited maximum antifungal activity against

Aspergillus niger and A. flavus showing MIC at

2.5 and 5mg/ml respectively [9]. The essential oil

obtained from seeds demonstrated promising

antifungal activity against Trichophyton rubrum

and Candida albicans [10].

Fungal cell wall in particular is a

unique and excellent target to be considered for

antifungal development. It is a complex

structure composed of chitin, glucans and other

polymers, cross-linked each other, where the

structure and biosynthesis are exclusive to the

fungi, where it is an essential objective to be

considered since both human and fungi are

under same eukaryote division [11]. In specific,

chitin is a polymer of β (1,4)-linked N-

acetylglucosamine which provide structural

rigidity and chemical/biological stability to the

fungus. Chitin has to be partially hydrolysed

during cell division and morphogenesis [12].

Chitinases are the enzymes that cleaves β-(1,4)

glycosidic bond of chitin[13]. During the chitin

disruption, the fungal cell lacks viability and/or

virulence [14]. Hence, the chitinases protein has

been selected as the antifungal target. In

bioinformatics, virtual screening is a

computational technique in drug discovery

which uses the computer programs to evaluate



the very large libraries of compounds [15]. The

two major virtual screening approaches are

target structure-based screening and screening

using active compounds as templates. The

structure based virtual screening involves the

docking analysis of small molecules into the

known molecular structure of a protein target.

Based on the scoring functions, the ligands

showing higher affinity are estimated to

possess inhibitory action[16]. The ligand based-

virtual searches otherwise known as

neighborhood behavior searches are made

against the in-house library of available

compounds to find the compounds with known

actives (similarity searching) or posses a

pharmacophore or substructure in common

with a known active pharmacophore

substructure searching [17, 18]. The target

structure-based screening for chitinase protein

has been studied in the present investigation.

In the present study, targeting chitinase

enzyme, the phytochemical constituents of Vitex

negundo L. leaves were screened through High

Throughput Virtual Screening (HTVS) and the

best scored compound has been docked to

analyze its possible mode of

interaction with the active site residues.

MATERIALS AND METHODS

Class III Chitinase (CHIA1) protein of

PDB ID: 2XVP was retrieved from Protein Data

Bank (PDB) (Figure 1&2). The phytocompounds

of V.negundo leaves (Table.1) were retrieved

from PubChem compound database. The protein

was prepared by removing the metal ions, water

molecules and cofactors. The grid was generated

around the active site residues Asp172, Glu174,

Tyr23, Gln230, Trp312, Tyr125, Ala124, Gln37,

Gln207, Asp170, Phe60 and Asn76 which are

chosen based on the study conducted by

Rush et al. (2010) [14]. The screening of

phytocompounds (Table 2) was carried out in

high throughput virtual screening (HTVS) of

Schrodinger software. Where the HTVS reduces

the number of intermediate conformation

throughout the docking funnel and reduces the

thoroughness of the final torsional refinement.

Hence, HTVS aids in identifying specific

molecules of interest from the rest of the

molecules. However, the XP docking was also

carried out only for the compounds showing

least G.score from virtual screening in order to

produce more sophisticated scoring function than

HTVS.

RESULTS AND DISCUSSION

Plants produce a variety of medicinal

components as secondary metabolites such as

phenolic compounds, essential oils, tannins,

terpenes, etc. that can inhibit pathogen growth

and are mostly evaluated for its sustainability

and affordability [3]. The compounds reported in



V.negundo leaves were virtually screened for the

target protein chitinase of 310 aminoacid length.

Among the 17 compounds, agnuside was found

showing least G.score of -5.805 Kcal/mol, which

was followed by negundoside of G.score -5.596

Kcal/mol. Agnuside is an iridoid glycosides

where the lipidated derivative and analogs are

specifically reported for its immune adjuvant

activity [19]. Until, the compound had not been

specifically evaluated for its antifungal ability,

the plant V.negundo possesses antimicrobial

activity.

The plant V.negundo is traditionally

reported for its use in the treatment of cough,

asthma, fever, eye disease, inflammation,

intestinal worms, skin diseases, nervous

disorder, leprosy and rheumatism[20]. The leaf

was evaluated for its efficient activity against

A.niger, A.flavus, A.fumigatus and the

dermatophyte Microsporum gypseum. The

growth inhibition was found higher for A.niger

and M.gypseum (43 and 56%, respectively) and

minimal for A.flavus and A.fumigatus (26 and

27%, respectively) [21]. The efficiency of the leaf

extracts (specifically benzene and water extracts)

to inhibit the growth of A.niger and A.flavus was

indicated by Aswar et al. (2009) [9].

The XP docking was carried out only for

agnuside and the knowledge on its mode of

interaction with chitinase protein was clearly

predicted. The G.score was -10.66Kcal/mol and

had 10 bond formations between the ligand

molecule and amino acid residues of protein.

Further the result was compared with the binding

efficiency of synthetic drug ketoconazole which

showed only -3.48Kcal/mol of G.score and 5

hydrogen bond interactions. The residues

Glu174, Gln230 and Asn233 were found

common in sharing bond formation in both the

molecules. Glu174 and Gln230 were the only

active residues participated in the interaction.

The negatively charged polar residue Glu174

shared two electrons with the agnuside, both of

1.5Å distance, whereas with ketoconazole only

one interaction of 2.0Å bond length was

observed.

The residue Glu174 (the catalytic acid)

was also reported to interact with the argifin, a

natural compound and the inhibitory action was

categorized [2]. In addition to Glu174, Asp172

and weak hydrogen bond with Tyr232 and

Trp312 was also observed. The compound

agnuside lacks interaction with the residues

Asp172, Tyr232 and Trp312. The groove of the

protein was observed to line up with residues

Trp312, Gln37 and Ala124, Tyr125 were the

backbone atoms. Phenyl moiety Phe60 served as

the floor for the active site. The active site

residues were found located in the loops

connecting the β-barrel and the hydrophobic

In silico Screening of Phytocompounds of

PSGCAS Search: A Journal of Science and Technology Volume

amino acid residue Phe60 alone was

situated in the β-barrel. So far, few of the

naturally occurring inhibitors (allosamid

styloguanidines,cyclo-L-Arg-D-Pro,psammaplin,

argadin, argifin) for family

(exochitinases) were reported [22, 2]

Gln230 was involved in single hydrogen

bond formation in both the ligands where the

bond length for agnuside and ketoconazole was

Table 1

Structure and

Agnuside

17750979

4-hydroxybenzoic acid

135

Screening of Phytocompounds of Vitex negundo Linn. Leaves for Fungal Chitinase

PSGCAS Search: A Journal of Science and Technology Volume : 3 No. : 1, ISSN: 2349 – 5456

amino acid residue Phe60 alone was found

barrel. So far, few of the

naturally occurring inhibitors (allosamidin,

Pro,psammaplin,

argadin, argifin) for family-18 chitinases [22, 2].

involved in single hydrogen

bond formation in both the ligands where the

bond length for agnuside and ketoconazole was

2.2Å and 2.0Å respectively. The other

interactions of both the molecules were tabulated

(Table 3) and the interactions were shown in

Figure 3&4. In the similar way, the numerous

loads of small molecules can be screened and

used further in QSAR studies.

pharmacophores of agnuside can further be

considered for developing lead

specific for inhibition of chitinase.

Phytocompounds of Vitex negundo leaves

Structure and Name of the Compound with PubChem ID

Negundoside

16655052

Procatechuic acid

Viridifloral

94174

γ-Terpinene

Linn. Leaves for Fungal Chitinase

5

2.2Å and 2.0Å respectively. The other

interactions of both the molecules were tabulated

interactions were shown in

. In the similar way, the numerous

loads of small molecules can be screened and

used further in QSAR studies. The

pharmacophores of agnuside can further be

developing lead drug molecule

chitinase.

leaves

PubChem ID

Procatechuic acid

72

Terpinene

7461


Globulol 101716

Caryophyllene oxide

14350

Oleanolic acid

10494

Sabinene

18818



Casticin 5315263

Vitamin C

Betulinic acid

64971

β-sitosterol222284

β-caryophyllene

5281515

4-terpineol

11230

Ursolic acid

64945

6

Vitamin C

5785

sitosterol 222284

terpineol

11230



Table 2 Virtual Screening of Phytoconstituents of Vitex negundo with 2XVP

Compound Name Glide score Compound Name Glide score Compound Name Glide score

Agnuside -5.805544 Negundoside -3.798527 Agnuside -3.118459

Agnuside -5.642732 Negundoside -3.758094 Caryophyllene oxide -3.111491

Negundoside -5.596881 Negundoside -3.757107 Oleanolic acid -2.890047



Agnuside -5.346562 Sabinene -3.650355 Negundoside -4.155089

Agnuside -5.313043 Negundoside -3.73272 β -sitosterol -2.943789

Agnuside -5.27015 Sabinene -3.64663 Negundoside -2.995692


Procatechuic acid -5.058439 4-terpineol -3.625607 Betulinic acid -2.88686

Agnuside -4.986446 Ursolic acid -3.632867 Negundoside -4.068281

4-hydroxybenzoic acid -4.953423 Agnuside -3.609137 globulol -2.849704

Agnuside -4.925231 Globulol -3.603247 Negundoside -2.922291

Negundoside -5.00465 Vitamin C -3.591461 Negundoside -2.921495

Agnuside -4.86693 β –sitosterol -3.575115 Betulinic acid -2.79587

Agnuside -4.85481 Ursolic acid -3.568113 Oleanolic acid -2.788313

Agnuside -4.789056 Agnuside -3.548539 Oleanolic acid -2.786311

Negundoside -4.782231 Oleanolic acid -3.533695 Caryophyllene oxide -2.774832


Agnuside -4.652641 Negundoside -4.304723 Ursolic acid -2.761505

Agnuside -4.509521 Betulinic acid -3.073707 Negundoside -2.826387

Negundoside -4.572242 β –sitosterol -3.047771 β -sitosterol -2.734605

Viridifloral -4.466438 Betulinic acid -3.047491 Negundoside -3.931117

Agnuside -4.322214 Negundoside -3.584428 Negundoside -2.807946

Agnuside -4.318595 Globulol -3.4947 Negundoside -3.927665

Gamma-Terpinene -4.296278 Ursolic acid -3.498548 Negundoside -2.800235

Agnuside -4.242411 Agnuside -3.483214 Negundoside -3.91084

Negundoside -4.264531 β –sitosterol -3.472244 Ursolic acid -2.711328

Negundoside -4.260427 Negundoside -3.551157 Negundoside -3.90499

Agnuside -4.17532 Negundoside -3.542928 Ursolic acid -2.70601

globulol -4.117585 Agnuside -3.445337 Negundoside -2.752477

Negundoside -4.200619 Viridifloral -3.432724 Betulinic acid -2.676288

Negundoside -5.28341 Viridifloral -3.409811 Negundoside -2.725571

Vitamin C -4.073547 β –sitosterol -3.403517 Betulinic acid -2.649623

Negundoside -4.128234 Caryophyllene oxide -3.393659 Negundoside -3.841447

Viridifloral -4.038 Negundoside -3.475597 β -sitosterol -2.631512


Casticin -4.038799 Negundoside -4.551403 β -sitosterol -2.599807



globulol -4.007818 Negundoside -3.42743 β -sitosterol -2.57152

Compound Name Glide score Compound Name Glide score Compound Name Glide score

Viridifloral -3.98237 Agnuside -3.336441 Oleanolic acid -2.578688

Viridifloral -3.976207 Globulol -3.333719 Betulinic acid -2.574339

Negundoside -4.050725 β -caryophyllene -3.32961 β -sitosterol -2.564132

Caryophyllene oxide -3.967512 β –sitosterol -3.315208 Betulinic acid -2.554102

Negundoside -5.128337 β –sitosterol -3.310557 Negundoside -3.745517

Caryophyllene oxide -3.920685 Globulol -3.271274 β -sitosterol -2.533876


Agnuside -3.867515 Negundoside -4.465426 Oleanolic acid -2.516963

Vitamin C -3.862849 Negundoside -4.434436 Betulinic acid -2.511944

Betulinic acid -3.847658 Globulol -3.220195 Ursolic acid -2.504947

Negundoside -5.040844 Betulinic acid -3.220191 β -sitosterol -2.490329

β -sitosterol -3.835479 Agnuside -3.206394 β -sitosterol -2.472868

Globulol -3.809153 β –sitosterol -3.197575 Betulinic acid -2.479468

Oleanolic acid -3.795189 Negundoside -4.397661 Ursolic acid -2.479461

β -caryophyllene -3.765089 Agnuside -3.188079 Oleanolic acid -2.4683

4-terpineol -3.748123 Ursolic acid -3.193443 Ursolic acid -2.449965

β -sitosterol -3.724172 β –sitosterol -3.142034 Betulinic acid -2.435094

Vitamin C -3.723751 β –sitosterol -3.12716

Table 3 XP Docking of Agnuside and Acetozolamide with Chitinase

S.No. Compound name G-Score (Kcal/mol)

No. of H- bond

Interacting residues

Bond Length(Å)

1 Agnuside -10.66 10

GLN37 (O-H) ALA124 (H-O) ALA124 (H-O) ASN174 (H-O) GLU174 (H-O) ASN233 (H-O) ASN233 (H-O) GLN230 (H-O) TYR232 (H-O) GLU174 (H-O)

2.4 2.0 2.5 2.6 1.5 2.3 2.2 2.2 2.0 1.5

2 Azetozolamide -3.48 5

GLN207 (H-O) GLN230 (H-O) ASN233 (H-O) ASN233 (H-N) GLU174 (H-O)

1.9 2.0 2.5 2.2 2.0

In silico Screening of Phytocompounds of


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Fig.3. Interaction of Agnuside chitinase protein

Fig.1. Fungal chitinase PDB ID: 2XVP

Screening of Phytocompounds of Vitex negundo Linn. Leaves for Fungal Chitinase


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