5

2.0 REVIEW OF LITERATURE

edicinal plants are of great interest in the field of biotechnology and are

considered as rich sources of ingredients in drug development. The

increase in demand for plant based therapeutics both in developed and

developing countries is largely due to the growing credits conferred to natural

products as being non-norcotic, causing minimal side effects and easily available at

affordable prices. The withania plants are tremendously known for their roots rich in

steroids and alkaloids and are a valuable constitute of traditional Ayurvedic drug

preparations against many diseases (Kiritikar and Basu, 1975; Williamson, 2002). The

major biochemical constituents of withania plants are a class of secondary metabolites

known as withanolides. These withanolides are known to possess diverse therapeutic

properties and has been used for centuries to treat various disorders. Several

withanolides have been isolated and characterized until date, but withaferin A is

considered to be multifunctional with strong anticancer and anti-inflammatory

properties (Berghe et al., 2012; Szic et al., 2014).

The ruthless collection of these plants by local herbalists and Ayurvedic drug

companies has lead this genus to the verge of extinction. To meet with the growing

demand for pharmaceutical industries, it is found to necessitate in vitro propagation of

such endangered plants. The present investigation was made to in vitro propagate

W. coagulans by optimizing the potential growth regulators and efforts were made to

explore the potent withanolides for their anti-inflammatory properties against LPS

activated Bone Marrow Derived Macrophages (BMDM’s) and the under lying

mechanism.

The literature relevant to the present study entitled “Computational

bioactivity analysis of major withanolides and their experimental validation in

in vitro cultures of Withania species” is reviewed in this chapter under the following

headings:

2.1 Withania as a medicinal plant

2.1.1 Withania somnifera

2.1.2 Withania coagulans

2.1.3 Withanolides of withania species

M

6

2.2 Pharmacological properties of withanolides

2.2.1 Anti-microbial

2.2.2 Anti-inflammatory

2.2.3 Anti-cancer

2.2.4 Hypolipidemic and hypocholesterolemic activities

2.2.5 Hepatoprotective

2.2.6 Immunomodulatory

2.2.7 Antihyperglycaemic activity

2.2.8 CNS activity

2.2.9 Cardiovascular effect

2.3 Analytical tools employed to study plant secondary metabolites

2.3.1 Qualitative and quantitative analysis

2.3.2 High performance thin layer chromatography

2.4 In silico analysis

2.4.1 ADME predictions

2.4.2 Molecular docking

2.4.3 Scoring function

2.4.4 Target proteins

2.5 Toll-like receptors (TLRs) in inflammation

2.5.1 Toll-like receptor (TLR) 4 signalling

2.5.2 NF-kB and Mitogen-activated protein kinases activation

2.5.3 Chronic Inflammation

7

2.1 Withania as a medicinal plant

Since the beginning of human civilization, medicinal plants have been used

for their therapeutic values by mankind. Nature has been a source of therapeutic

agents for centuries and an impressive number of modern drugs have been developed

from natural sources. As source of medicines, plants continue providing us with new

remedies and around 25% of today’s medical prescriptions are based on plant derived

substances or analogues. The efficacy and safety of herbal medicines have turned the

major pharmaceutical population towards medicinal plants research (Sara et al.,

2009).

The family Solanaceae comprises of 84 genera that includes 3,000 species

scattered throughout the world. The sixty five known withania species are densely

distributed in the drier parts of tropical and subtropical zones (Schonbeck-Temesy,

1972; Hepper, 1991; Warrier, 1996; Hunziker, 2001). In Ayurveda, withania is

claimed to have potent aphrodisiac, rejuvenative, sedative and life prolonging

properties. The plant has been traditionally used to promote youthful strength,

endurance and health, nurturing the timely elements of the body and increasing the

production of body fluids such as muscle fat, lymph, blood, cells and semen. It also

helps to counteract chronic fatigue syndrome, bone and body weakness associated

with dehydration, impotency, emaciation, premature ageing and muscle tension.

Bruised leaves and fruits are locally applied to glands, ulcers and tumours

(Williamson, 2002). Among the genera withania, Withania somnifera and

Withania coagulans are the two most esteemed species having high economical and

medicinal significance, being used and cultivated in several regions such as Pakistan,

Afghanistan, Egypt, Iran, Palestine, Spain, Jordan, Morocco, Canary Island, Eastern

Africa, South Africa, Congo, Madagascar and India (Dymock et al., 1981; Javanshir,

2000; Sharma, 2004; Panwar and Tarafdar, 2006). The botanical description and the

illustration of these two prevalent species of Withania are described in Table 2.1 and

Figure 2.1.

2.1.1 Withania somnifera

Withania somnifera (L) Dunal, (Solanaceae) popularly known as

Ashwagandha is the most commonly used herb in Ayurvedic and indigenous medical

system for more than 3000 years. Various parts of the plant have been used for

8

centuries to treat a variety of ailments. Many pharmacological studies have been

carried out to illustrate multiple biological properties of W. somnifera (Mishra et al.,

2000). The similarity of therapeutic properties with those of Asian ginseng has led

Ashwagandha being called as Indian ginseng (Singh and Kumar, 1998). The species is

widely distributed in Africa, the Indian sub-continent and the Mediterranean. In India,

it grows well in the drier regions of tropical and sub-tropical areas of Punjab,

Haryana, Uttar Pradesh, Madhya Pradesh, Bihar, Jharkhand, Uttarkhand, Rajasthan

and Maharashtra and some parts of Jammu and Kashmir ascending upto 1,650 m in

the Himalayas (Kapoor, 2005).

Classification:

Kingdom : Plantae

Division : Angiosperm

Class : Dicotyledoneae

Order : Tubiflorae

Family : Solanaceae

Genus : Withania

Species : somnifera Dunal

(Singh et al.,2011)

Vernacular names:

Sanskrit : Asvagandha

Hindi : Asgandh

Kannada : Viremaddinagaddi

Malayalam : Amukkuram

Tamil : Amukkira

Telugu : Vajigandha

English : Winter cherry

2.1.2 Withania coagulans

Withania coagulans (L.) Dunal (Solanaceae) is commonly known as Indian

cheese maker, is well known for its ethnopharmacological activities. The fruit and

berries are used commercially for milk coagulation (Sanjay et al., 2007). Surveys of

existing literatures have shown that the plant is used in various traditional systems of

medicine like Ayurveda and Unani, and has been recommended for treating various

disorders including ulcers, rheumatism, bronchitis, and degenerative diseases (Maurya

et al., 2010; Khodaei et al., 2012). W. coagulans is a small ever green shrub that is

reputedly used for treating dyspepsia, flatulent colic and other intestinal disorders.

The plant is a native of the Asia-temperate (Western Asia- Afghanistan) and Asia-

tropical (Indian Subcontinent - India, Nepal) regions. This plant species is sparsely

distributed in the eastern Mediterranean region and extends to South Asia.

9

Table 2.1: Botanical description of W. somnifera and W. coagulans(Jain et al., 2012)

S.No. Description Withania somnifera (L.)Dunal

Withania coagulans (stocks)Dunal

1 Habit Undershrub Herb

2 Leaves Alternate, broadly ovate,subacute, entire margins

Alternate, elliptic lanceolate-coriaceous, obtuse, entiremargins, glabrous, coated withminute stellate hairs on boththe surfaces

3 InflorescenceAxillary, umbellatecymes

Axillary

4 Flowers Monoecious Dioecious

5 CalyxAccrescent,gamosepalous with5sepals

Campanulate, gamosepalouswith 5 sepals clothed with finestellate grey tomentum

6 Corolla Campanulate, greenish-yellow with 5 petals

Campanulate, greenish-yellowwith 5 petals

7 Androecium Anthers 1.2 mm long,broadlyovate

Anthers long and filamentousin male flowers, smaller infemale flowers

8 GynoeciumOvary ovoid/globose,glabrous

Ovary ovoid/globose, withoutstyle or stigma

9 Style Filiform Glabrous

10 StigmaMushroom-shaped, 2-lamellate

Mushroom-shaped, 2-lamellate

11 Fruit (Berry)Globose, enclosed inthepersistent calyx, seedsyellow,reniform

Globose, smooth, closely girtby the enlarged membranouspersistent calyx

12 SeedsGlobose, enclosed in thepersistent calyx, yellow,reniform

Globose, ear shaped, glabrous,enclosed in the persistentcalyx yellow, reniform

13 Flowering Throughout the year November-March*

10

Figure 2.1: Botanical descriptions of W. coagulans and W. somnifera (plantillustrations.org)

11

Classification:

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnolipsida

Order : Solanales

Family : Solanaceae

Genus : Withania

Species : coagulans

(Gupta, 2012)

Vernacular names:

Hindi : Puni-ke-bij, Akri

Persian : Tukhme- Kaknaje-hindi

Afgan : Spicebajja

Punjabi : Khamjira

Sindhi : Punir band, Punir-ja-fota

(Mathur et al., 2011).

2.1.3 Withanolides of withania species

The phytoconstituent profiles of withania have been of great interest to the

scientific and research community. Chemical characterization of withania started off

with Power and Salway (1911) who identified and isolated amorphous alkaloid

(C12H16N2) from South African strains of W. somnifera. Later in 1933, Majumdar and

Guha investigated W. somnifera plant from Bengal and confirmed the alkaloid

presence. Laboratory analysis till date has revealed over 35 chemical constituents in

the roots of W. somnifera (Rastogi and Mehrotra, 1998). Among these, the

biologically active constituents are steroidal lactones (withaferin A, withanolides

A-Y, withasomniferin A, withasomidienone, withasomniferols A-C, withanone, etc),

alkaloids (isopellertierine. Anferine), and saponins with additional acyl group

(sitoindoside) (Gupta and Rana, 2007; Maurya et al., 2010). Withanolides are

traditionally believed to account for the plants medicinal properties and they bear

resemblance to Ginsenosides both in their appearance and action. Withanolides from

the withania plants have been researched in a variety of clinical examinations for their

numerous therapeutic activities including cancer and immune functioning (Grandhi

et al., 1994).

Withanolides are a group of naturally occurring C28 steroidal lactones with an

intact or modified ergostane skeleton. They are mainly produced by the solanaceae

family, and in particular to the genera Withania, Physalis, Dunalia, Datura,

Tubocapsicum, Nicandra and Jaborosa (Glotter, 1991). Among these, plants,

Withania, Physalis and Datura have been widely distributed in the southern

peninsular regions of Tamil Nadu with rich content of withanolides (Gupta and Ray,

12

1991; Ravikumar et al., 2010). Nearly 400 withanolides or closely related congeners

have been discovered in 58 Solanaceae species under 22 genera (Eich, 2008). Some of

the withanolides have been discovered in certain Ajuga species like Parviflora Benth

and Tacca species such as Taccaceae (Huang et al., 2002), as well as in certain

marine organisms. Nevertheless, their occurrence is by far predominant in Solanaceae

(Eich, 2008). Different withanolides such as withacoagin and coagulan from the roots

and fruits of W. coagulans and withanolide A and withaferin A from the roots and

leaves of W. somnifera has been reported (Khare, 2007). The basic skeleton of

withanolides is shown in the Figure: 2.2.

Figure 2.2 Basic structure of withanolides. C28 steroidal lactones with anintact ergostane skeleton

Withanolides are synthesized via mevalonate pathway during terpenoids

formation and arise during initial cyclization of 3S-squalene-2,3-epoxide (Kreis and

Muller-Uri, 2010). Synthesised withanolides generally contain polyoxygenated

ergostane skeleton. The most characteristic feature of withanolides is the ability to

introduce oxygen functions in almost every functional site of the carbocyclic skeleton

and compounds side chain (Naz, 2002). These withanolides were initially classified

based on the chemotypes of withania species and the regions of the collected plant.

Chemically, these compounds may be classified as ergostane derivatives of their

structural pattern which are broadly divided into seven groups represented in Figure

2.3 (Glotter, 1991)

1. 5β, 6β –epoxides

2. 6α, 7α –epoxides

3. 5-enes

4. Intermediary compounds

13

5. 5α,6α –epoxides

6. 6β,7β –epoxides

7. Phenolic withanolides

Figure 2.3 Structural classifications of withanolides

The diverse structural analogues of withanolides provide a great opportunity

to study structure activity relationship, lead optimization and target identification.

Therefore, withanolides represent a promising lead compounds in development of

new drugs. The isolated withanolides from W. somnifera and W. coagulans are listed

in Table 2.2 and 2.3.

Table 2.2: Withanolides identified in W. somnifera

S.No. Chemical constituent References

1 Withaferin A

Lavie et al. (1965), Kirson et al.(1970), Devi (1996), Gupta et al.(1996), Anjaneyulu and Rao(1997), Ali et al. (1997), Mohanet al. (2004), Oh et al. (2008a)

2 Withanolide D, 2, 24-dienolide Lavie et al. (1968), Kirson et al.(1970)

3 27-deoxywithaferin A Kirson et al. (1970)

4 Withanone, Trienolide Kirson et al. (1971)

5 5, 20α ®-dihydroxy-6α, 7α-epoxy-1- Menben-Von and Stapel (1973)

14

oxo-(5α) - With a-2, 24-dienolide(steroidallactone) 2, 3-dihydrowithaferinA-3beta-O-sulfate

6Withanolide - WS 1 (aliphatic ketone),Withanolide - WS 2 (aliphatic ester)

Kundu et al. (1976), Khan et al.(1993)

7 Withanolide E Glotter et al. (1977)

8 Sitoindosides VII, VIII Bhattacharya et al. (1987)

9 Sitoindosides IX, X Ghosal et al. (1988)

10

Withanosides I, II, III, IV, V, VI, VII,5a,20aF(R)-dihydroxy-6a,7a-epoxy-1-oxowitha-2,24-dienolide, coagulin Qandphysagulin D

Matsuda et al. (2001)

11 withanolide A, withanoside VIII, IX, XI Zhao et al. (2002)

12Withanolide A,withanoside IV and VI

Kuboyama et al. (2002),Tohda et al. (2005)

13

Physagulin D (1,6)β-D-glycopyranosyl-(1-4)-β-D-glycopyranoside, 27-O-β-Dglycopyranosylphysagulin D, 27-O-β-Dglycopyranosylviscosalactone B, 4, 16dihydroxy-5β, 6β epoxyphysagulin D, 4-(1-hydroxy-2,3-dihydrowithaferin A,viscosalactone B, 27-desoxy-24,25-dihydrowithaferin A

Jayaprakasam and Nair (2003)

14Withanone, 27-hydroxy withanolide A,iso-withanoneand 6α, 7β-epoxy-1β,3β,5α-trihydroxywitha-24-enolide

Lal et al. (2006)

15 Ashwagandhanolide Subaraju et al. (2006), Mirjaliliet al. (2009a)

16 Withanolide B and Z, 7- hydroxylwithanolide Pramanick et al. (2008)

17 Withanoside IV, VI, physagulin D andwithastraronolide Ahuja et al. (2009)

18 Withanolidesulfoxide Mulabagal et al. (2009)

15

Table 2.3: Withanolides identified in W. coagulans

S.No. Chemical constituent References

1 Withaferin A Subramanian andSethi, 1969

2 Withanolide G, I, J, K Gottlieb andKirson, 1981

3 3β-hydroxy- 2,3-dihydrowithanolide F, Ergosta-5,25-diene-3β,24 ε -diol

Budhiraja et al.,1983

4 Withanolide D, Δ3 isowithanolide F Velde et al., 1983

5Withanolide H: 14α, 20αF, 27-trihydroxy-1-oxo-20R,22R-with a-2,5,24- trienolide, 3β,14α,20αF,27-tetrahydroxy-1-oxo-20R,22R-witha-5,24-dienolide

Ramaiah et al.,1984

6 (20S, 22R) 6α, 7α- epoxy- 5α-hydroxy- 1- oxo- witha-2 ,24- dienolide, Withacoagin Neogi et al., 1988

7 17β, 27 dihydroxy-14, 20- epoxy -1- oxo- 22R- witha-3,5, 24- trienolide

Rahman et al.,1993

8

14, 15β- epoxywithanolide I: [(20S, 22R) 17β, 20β-dihyroxy -14β, 15β- epoxy- 1- oxo- witha-3,5,24-trienolide], 17β- hydroxywithanolide K: [(20S, 22R) 14α,17β, 20β-trihydroxy 1- oxo- with a-2, 5, 24- trienolide],17β,20β- dihydroxy- 1- oxo- witha- 2,5,24- trienolide

Choudhary et al.,1995

9 Coagulin B, C, D, E, F, G, H, I, J, K, L, M, N, O Rahman et al.,1998(a, b, c, d)

10

Withahejarin: [ 20 β-hydroxy-1-oxo-(22R) – witha-2,5,24 trienolide, Withapakistanin: [ 17β, 20 β-dihydroxy- 14, 15β- epoxy-1-oxo-(22R)- with a-3,5,24trienolide], Withasomniferine-A: [ 17β, hydroxyl- 6α, 7α-epoxide-1-oxo-(22R)-witha-4,24-dienolide], Coagulin A

Shahwar, 1999

11 Coagulin P, Q, R Rahman et al.,1999

12(22R), 20β-hydroxy- 1-oxowitha- 2,5,24- trienolide,(22R)-14,20-epoxy-17ß-hydroxy-1-oxowitha-3,5,25-trienolide, Coagulin U

Naz, 2002

13

17β-hydroxy-14α,20α-epoxy-1-oxo-(22R)-witha-3,5,24-trienolide, 20β, hydroxy -1- oxo- (22R) – witha – 2, 5.24- trienolide, Withacoagulin: 20β,27-Dihydroxy-1-oxo-(22R)-witha-2,5,24-tetraenolide

Rahman et al.,2003

14 Coagulanolide: (17S,20S,22R)-14α,15α,17β,20β-tetrahydroxy-1-oxowitha-2,5,24-trienolide Maurya et al., 2008

16

15

(20R,22R)-14,20a,27-trihydroxy-1-oxowitha-3,5,24-trienolide, (22R)-14a,15a,17b,20b-tetrahydroxy-1-oxowitha-2,5,24-trien-26,22-olide, Withacoagulin A, B,C, D, E, F, Withanolide F, L

Huang et al., 2009

16 Coagulansins A, Coagulansins B Jahan et al., 2010

17 Withacoagulin D, Withanolide K and J Kuroyanagi et al.,2012

2.2 Pharmacological properties of withanolides

The integral constituents of withania genera have always been a great interest

to the research community. The bioactive components are alkaloids, steroidal

lactones with ergostane skeletons such as withanolide A-Y, withaferin A,

withasomniferols A-C, withanone, withasomniferin A, etc (Gupta and Rana, 2007;

Maurya et al.,, 2010). Withanolide A (5α,20α-dihydroxy- 6α,7α -epoxy-1-oxowitha-

2,24-dienolide) and withaferin A (4β,27-dihydroxy-5β,6β-epoxy-1-oxowitha-2,24-

dienolide) are the key active withanolidal principles responsible for a diverse array of

pharmacological activities. They have chemically similar back bone but differ in their

side chain constituents (Sanghwan et al., 2007; Hemalatha et al., 2008).

2.2.1 Antimicrobial activity

Antibacterial and anti-fungal properties have been demonstrated in isolated

withanolides from the extracts of various parts of withania (Khan et al., 1993;

Choudhary et al., 1995). The methanolic extract of W. somnifera possessed maximum

inhibitory activity against a wide range of bacteria. Oral administration of fruit

extracts of W. somnifera successfully obliterated Salmonella infection in mice

subjects as revealed by increased survival rate as well as less bacterial load in vital

organs of the treated animals (Owais et al., 2005). The methanol, diethyl ether and

hexane extracts from leaves and roots of W. somnifera were evaluated for their

synergistic antibacterial activity by agar disc diffusion assay against Escherichia coli

and Salmonella typhimurium (Arora et al., 2004; Jain et al., 2012). Lalsare et al.

(2010) demonstrated antimicrobial and antioxidant activities from various extracts of

W. coagulans fruits. The methanolic, dichloromethane and pertroleum extract of

W. coagulans were treated against a wide array of fungal infections caused by

Trichoderma viridis, Aspergillus flavus, Fusarium laterifum, Aspergillus fumigatus,

Trichophyton mentogrophytes, Microsporum canis and Candida albicans (Maurya

17

et al., 2010; Mughal et al., 2011). Also volatile oil from the fruits of W. coagulans has

antibacterial activity against Vibrio cholera and Staphylococcus aureus (Khare,

2007).

These anti-fungal and anti-bacterial properties have been demonstrated in

isolated withanolides from extracts of both W. somnifera and W. coagulans

respectively. Two withanolides (14, 15β-epoxywithanolideI[(20S,22R) 17β,20β-

dihydroxy-14β, 15β-epoxy-1-oxo-witha-3,5,24-trienolide] and 17β-hydroxy

withanolide K (20S,22R) 14α,17β,20β-trihydroxy- 1-oxo-witha-2,5,24-trien-olide])

were isolated from W. coagulans. The withanolides 17β- hydroxywithanolide K

(20S,22R) 14α,17β,20β-trihydroxy- 1-oxo-witha-2,5,24-trien-olide was found to be

active against a number of potentially pathogenic fungi Nigrospora oryzae,

Aspergillus niger, Curvularia lunata, Stachybotry satra, Allescheria boydii,

Drechsleraro strata, Microsporum canis and Epidermo phytonfloccosum and plant

pathogen Pleurotus ostreatus (Choudhary et al., 1995). The compound also showed

activity against gram positive bacteria (S. aureus) (Rahman and Choudhary, 1998a).

Withaferin A potentially exhibited significant antibacterial activity against Gram-

positive bacteria’s but were inactive against Gram-negative microorganisms and non-

filamentous fungi. Also another significant compound, withanolide D are proved with

antifungal cytotoxic activity against thirteen fungal species responsible for various

human infections (Roumy et al., 2010).

2.2.2 Anti-inflammatory activities

The anti-inflammatory potential of W. somnifera and W. coagulans has been

studied by several workers. Anbalagan and Sadique (1981) started off with

preliminary experiments and reported W. somnifera to possess efficient anti-

inflammatory activity compared to a common anti-inflammatory drug,

hydrocortisone. Budhiraja et al. (1984 and 1986) showed aqueous extracts of

W. coagulans fruits had significant anti-inflammatory activities in subacute models of

formalin-induced arthritis in rats. 3-β-Hydroxy-2, 3-dihydrowithanolide F was

isolated from W. coagulans extracts and produced the same pattern of anti-

inflammatory activity in formalin induced rat arthritis (Maurya et al., 2010). The

effect of W. somnifera on synthesis of glycosaminoglycans in the tissue granulation of

carrageenan-induced air pouch granuloma was studied by Begum and Sadique (1987).

18

It was found out that W. somnifera root powder decreased the glycosaminoglycans

content by 92%, much higher than the drugs phynylbutazone and hydrocortisone. The

same team again proved the efficiency of root powders of W. somnifera in comparison

to hydrocortisone succinate in rheumatoid rats (Begum and Sadique, 1988).

Granuloma tissue formation was inhibited when subcutaneous cotton-pellet implanted

rats were treated with extracts of aerial parts of W. somnifera and produced a high

anti-inflammatory activity compared to the drug hydrocortisone sodium succinate

(Al-Hindawi et al., 1992; Singh et al., 2010). Bhattacharya et al. (2000) reported the

synthesis of a live plasma protein, alpha-2-macroglobulin increased greatly upon

inflammation and this was decreased effectively upon treatment with W. somnifera

compared to other standard anti-inflammatory drugs. The hydroalcoholic extracts of

W. coagulans fruits showed significant anti-inflammatory activity against carrageenan

induced paw oedema rat models (Rajurkar et al., 2001; Gupta et al., 2013) followed

by, Lalsare and Chutervedi (2010) proved it with various solvent extracts of W.

coagulans fruits.

These activities are mainly attributed due to their high bioactive steroids,

especially the major withaferin A which is shown to have similar structure and

function to glucocorticoids and has a complex influence on inflammatory system

(Davis and Kuttan, 2000). Withaferin A is known to play as potent inhibitors of pro-

inflammatory mediators and a promising treatment against inflammatory cascades of

various disorders (Kaileh et al., 2007). Preliminary studies on withanolides anti-

inflammatory activity started with Subramanian and Sethi (1972) who assessed the

activity of withaferin A, withanone and other new withanolides on acute and subacute

models of inflammation and found significant variation in biological activities of

withanolides. Withanolide fraction from aerial parts of W. somnifera from Iraq had

antigranuloma activity and reduced the weight of inflammation induced adrenal

glands. Withaferin A was also found to have adrenal and granuloma inhibiting

activity without affecting the spleen and body weight (Al-Hindawi et al., 1992; Patel

et al., 2013). Other reports have also indicated withanolides as an inhibitor of NfkB

mediated inflammations (Bargagna-Mohan et al., 2006; Oh et al., 2008b; Kour et al.,

2009; Oh and Kwon, 2009; Maitra et al., 2009).

2.2.3 Anti-cancer activity

19

The anticancer property of withania species has been extensively studied by

several researchers and was identified as an effective agent in preventing cancer

through reduction in tumor size (Davis and Kuttan, 2000; Prakash et al., 2002;

Winters, 2006; Senthilnathan et al., 2006; Widodo et al., 2007; Singh et al., 2011;

Khazal et al., 2013; Szic et al., 2014). Initial studies started with oral administration

of W. somnifera extracts simultaneously with urethane for seven months and reduced

tumor incidence was found (Singh et al., 1986). Treatment with W. somnifera root

extracts on mice with induced skin cancer exhibited significant decrease in the

number of skin lesions compared to control group (Prakash et al., 2002). Also a

number of studies have reported the anti-cancer activities in the leaf extracts of

W. somnifera (Diwanay et al., 2004; Christina et al., 2004; Leyon and Kuttan, 2004;

Malik et al., 2007; Aalinkeel et al., 2010; Malik et al., 2009). Ichikawa et al. (2006)

demonstrated that the anti-carcinogenic effect of W. somniferais mainly mediated

through withanolides activity of anti-proliferation, antiangiogenic, antimetastatic,

anti-invasive and proapoptosis which then results in suppression of NF-kB regulated

gene products. Additionally, the extracts of W. coagulans were demonstrated to

inhibit the incorporation of thymidine and hence proliferation of carcinomas.

Withaferin A was identified as the responsible component in extracts of

withania with potent tumor inhibiting activity by inhibiting more than 50% of RNA

synthesis and acting as mitotic poison, resulting in the cell cycle arrest in various

human derived carcinomas (Jayaprakasam et al., 2003; Choudhary et al., 2010;

Maurya et al., 2010; Chen et al., 2014). Earlier studies on withaferin A proved their

radio-sensitising and growth inhibitory effects on experimental mouse tumors and

increase the tumor free survival in a dose dependent manner (Devi et al., 1995;

Sharada et al., 1996; Ganasoundary et al., 1997). Similarly withaferin A in tumor

cultures decreased the expression of nuclear factor-kappa β and tumor necrosis factor

resulting in the arresting of apoptotic signalling (Choudhary et al., 2010). Apart from

this, studies on withanolide A also showed growth inhibitory and radio sensitizing

effects on mouse carcinomas and resulted in the mitotic arrest of the chicken

fibroblast cells (Gupta and Keshari, 2013). These withanolides are also said to induce

apoptosis via mitochondria by cytochrome C release and caspase activation (Senthil

et al., 2007). The presence of unsaturated lactone as a side chain upon which allelic

primary alcohol groups are attached and highly oxygenated rings on the other end of

20

the molecule has been suggested as the chemical system with carcinostatic properties

(Rastogi et al., 1998; Khare, 2007).

2.2.4 Hypolipidemic and hypocholesterolemic activities

The aqueous extract of W. somnifera root and W. coagulans fruit have been

reported to decrease total amount of lipid, cholesterol and triglycerides in

cholesterolemic animals (Andallu and Radhika, 2000; Hemalatha et al., 2006).

W. somnifera root powder was effective in decreasing lipid profiles of normal

subjects. Visavadiya and Narasimhacharya (2007) investigated hypocholesterolemic

activity of W. somnifera in male swiss albino rats and suggested that the activity is

mediated through increased the HMG-CoA reductase activity and bile content of

liver. In another study, hypoglycemic and hypocholesterolemic effect of W. somnifera

roots were assessed on human subjects and suitable parameters were analysed in

blood and urine samples of the subjects after 30 days treatment. Significant increase

in urine sodium, urine volume and decrease in serum cholesterol levels and decreased

LDL and VLDL were observed indicating the roots of withania to be a potential

source of hypocholesterolemic agents (Andallu and Radhika, 2000; Gupta and Rana,

2007; Singh et al., 2010). Further, a significant decrease in lipid-peroxidation was

found after administration of W. somnifera extracts to hypercholesteremic animals

when compared to the control groups.

The hypolipidemic and hypocholesterolemic activities of W. coagulans were

also reported by Hemalatha et al. (2006) and Datta et al. (2013). Administration of

aqueous extracts of W. coagulans fruits to high fat diet-induced rats for 7 weeks

significantly reduced serum cholesterol, lipoprotein and triglyceride levels. The

extract also showed hypolipidemic property in triton induced hypercholesterolemia

(Jain et al., 2012). The histopathological examination of liver tissues of withania

extract treated rats showed comparatively lesser degenerative changes compared to

hyperlipidemic controls. The hypolipidemic effect of

W. coagulans fruits was much comparable to Ayurvedic product containing

“Commiphoramukkul” to treat high cholesterol levels (Hemalatha et al., 2006). The

hydroalcoholic extract of W. coagulans fruits were also effective and comparable to

drug “atorvastatin” in controlling lipid levels in high cholesterol diet induced rats.

Hoda et al. (2010) showed aqueous and chloroform extracts of W. coagulans fruits to

21

effectively decrease total cholesterol, triglyceride, LDL and VLDL levels. The

extracted coagulin L from the fruits of W. coagulans was found to be the responsible

element with effective anti-dyslipidemic effect on mice (Maurya et al., 2008).

2.2.5 Hepatoprotective activity

The W. somnifera root powder influenced the levels of lipid peroxidation

thereby provided hepatoprotection (Mohanty et al., 2008). In an in depth study

conducted to examine the effect of these extracts on hepatic cells of

Clarias batrachus reported flavonoids as the responsible molecules in the

W. somnifera extract, stimulating the neuroendocrine system resulting in hyper-

activity of the liver cells endomembrane and exit of molecules through surface

exocytosis (Verma et al., 2009). Studies on pharmacological properties of

W. coagulans by Budhiraja et al. (1986) and Rajurkar et al. (2001) reported the

hepatoprotective activity of extracts of W. coagulans fruits. 3β-hydroxy-2,3-dihydro

withanolide F isolated from W. coagulans was screened for its hepatoprotective effect

against CCl4 induced hepatotoxicity, and the compound was found capable with

marked protective effect after histopathological examinations (Maurya et al., 2010;

Gupta, 2012). Similarly, withaferin A at dose 10 mg/kg, significantly protected CCl4

induced hepatotoxicity as effective as hydrocortisone in rat models (Rastogi

et al., 1998; Khare, 2007).

2.2.6 Immunomodulatory activity:

Administration of W. somnifera extract was found to significantly reduce

leucopenia induced by cyclophosphamide (CTX) and sub-lethal dose of gamma

radiation by means of its effective immune regulation and chemoprotection activity

(Kuttan, 1996; Davis and Kuttan, 1998). W. somnifera treatment significantly

increased RBC count, platelet count and Hb concentration (Ziauddin et al., 1996). In

another study, administering powdered root extract from W. somnifera inhibited

cyclophosphamide–induced delayed type hypersensitivity (DTH) reactions and

enhanced the phagocytic activity of macrophages compared to control group

(Agarwal et al., 1999; Davis and Kuttan, 2000). Extracts of W. somnifera root are also

capable of inhibiting the mitogen induced DTH reactions and lymphocyte

proliferation in rats (Rasool and Varalakshmi, 2006). The extracts of withania are also

known to enhance total white blood cell count, haemoglobin concentration, red blood

22

cell count, platelet count and enhanced phagocytic activity of macrophages (Davis

and Kuttan, 2002).

Withacoagulins A-F along with ten other withanolides isolated from aerial

parts of W. coagulans exhibited strong inhibitory activity on excess proliferation of T

and B-cells and helped in immune modulation (Huang et al., 2009). Coagulin H

significantly inhibited IL-2 production by 80% and docking study predicted coagulin

H bound to IL-2 receptor binding site more effectively than the drug prednisolone.

Based on these computational and experimental results, coagulin H was identified as a

potent immunosuppressive candidate (Mesaik et al., 2006). Similarly withaferin A has

been reported to possess both immune suppressive and immune activating properties

and implement specific and selective effects on human B and T lymphocytes via

antigen recognition (Bahr and Hansel, 1982; Rastogi et al., 1998; Aggarwal et al.,

1999; Davis and Kuttan, 2000; Gautam et al., 2004; Rasool and Varalakshmi, 2006;

Narinderpal et al., 2013).

2.2.7 Antihyperglycaemic activity

W. coagulans has been used since time immemorial in Ayurvedic medicines

to treat diabetes (Gurson and Saner, 1971; Budhiraja et al., 1977; Huang et al., 2009;

Ojha et al., 2014). Administration of aqueous fruit extracts of W. coagulans was

found to significantly lower the blood glucose level (Hemalatha et al., 2004; Saxena,

2010). Long term study on diabetic rats showed a reduction of 54.1% and 52.9% in

post prandial glucose levels and fasting blood glucose levels after 30 days treatment

(Hoda et al., 2010). The ethanol extracts of W. coagulans fruits also reduced blood

glucose level by 52.6% and decrease of 75% sugar level in urine (Jaiswal et al.,

2010). Even W. somnifera has been evaluated for its hypoglycemic effects in human

subjects during clinical studies. Six type 2 diabetes subjects were treated with extract

powder for 30 days and a significant decrease in blood glucose levels were identified

upon comparison with control (Singh et al., 2010). Significant improvement in signs

and symptoms were observed and attained euglycemis in type 2 diabetes mellitus by

Lopez-Ridaura et al. (2004) and Jaiswal et al. (2009). Alam et al. (2009) reported the

combined effect of W. coagulans and Trigonellafoenum graecum in controlling Type

2 diabetes.

23

This hypoglycemic activity of withania is reported mainly due to their high

content of Ca and Mg which plays significant role in diabetes management (Giugliano

et al., 2000; Rai et al., 2007; Kumar et al., 2009). Jaiswal et al. (2010) reported

antidiabetic and hypoglycemic activity of aqueous fruit extracts of W. coagulans by

Laser Induced Breakdown Spectroscopy for detection of glycaemic elements present

in the extract and reported the trace elements like Ca and Mg responsible for

antidiabetic potential of this indigenous shrub. Ca2+ ion mediates insulin gene

activation and expression via CREB (Calcium Responsive Element Binding Protein)

and is responsible for release and breakdown of stored insulin (Veiga et al., 2006).

Alkaloidal and steroidal content of this plant has been the major factor

responsible for hypoglycemic activity (Adebajo et al., 2006). Coagulin L from fruits

of W. coagulans has been reported to have antihyperglycemic activity in rats (Maurya

et al., 2008). The median effective dose of coagulanolide isolated from the fruits of

W. coagulans was determined in streptozotocin-induced diabetic rats and was

compared to the antidiabetic drug metformin (Maurya et al., 2008). Also treatment

with W. coagulans dried fruit extract for 4 weeks significantly reversed the

hyperglycemic levels in streptozotocin-induced diabetic rats comparable to the drug

glipizide (Datta et al., 2013).

2.2.8 CNS activity

The extracts from various parts of both the plants of withania have the

capacity to modulate neurotransmitters. Bhatnagar et al. (2009) observed that the

extracts of withania work as a suppressor of corticosterone secretion and activates

choline acetyltransferase, which in turn increases the levels of serotonin in

hippocampus. The bioactive metabolites isolated from withania was found to be

effective in alleviating disorders associated with central nervous system such as

anxiety, depression, epilepsy and catalepsy (Subramanian and Sethi, 1971; Budhiraja

et al., 1977; Bhattacharya et al., 1997; Jain et al., 2001; Dhuley, 2001; Naidu et al.,

2006). Withanolide VI and withanolideA isolated from W. somnifera induces neurite

outgrowth in cultured neurons and in rodents injected with Aβ 25-35 (Kuboyama et

al., 2002). Recently Sehgal et al. (2012) revealed that the semi purified root extracts

of W. somnifera reversed behavioural deficits, accumulation of β-amyloid peptides

(Aβ), plaque pathology and oligomers in the brains of Alzheimer’s diseased mice

24

subjects by enhancing low density lipoprotein receptor proteins in the brain

microvessels.

2.2.9 Cardiovascular effect

W. somnifera when tested in experimental rats, was reported to exert a strong

cardioprotective effect on isoprenaline-induced myonecrosis (Mohanty et al., 2004).

Maintenance of myocardial antioxidant status and augmentation of endogenous

antioxidants contributes to its cardioprotective effect of W. somnifera at 50 mg/kg

dose. Also a new withanolide (3β-hydroxy-2, 3-dihydrowithanolide F) with its unique

chemical structure similar to aglycones of cardiac glycosides has been isolated from

W. coagulans fruits. This withanolides upon administration to dogs produced a

moderate fall of blood pressure which was induced by atropine (Budhiraja et al.,

1983).

2.3 Analytical tools employed to study plant secondary metabolites

Plants synthesize a wide range of phytochemicals that are useful in the

maintenance of health and vitality of humans. These include primary and secondary

metabolites with their unique functions and metabolic activities. The herbal extracts,

singularly and in combinations, contain myriad of such compounds in which no single

active constituent is responsible for the overall efficacy. The quality assurance and

quality control still remains a challenge because of the high variability of chemical

constituents involved. Due to natural variability in plant material, chemical analysis is

a great challenge and requires special approaches.

2.3.1 Qualitative and quantitative analysis

Secondary metabolite analysis can either be qualitative or quantitative in

nature. Qualitative analysis is used to identify a characteristic compound or a

metabolite species present in the mixture. Qualitative analysis is less instructive and

has to be done as a routine. The classical qualitative analysis scheme for identification

purpose has been around for well over 100 years, but still continues to be an important

part of any analytical training. This is because it offers an effective means for

presenting descriptive nature and illustrates important compositions.

25

In quantitative analysis, we are interested in the relative amount of certain

secondary metabolite present in the mixture. Knowing the composition of a sample is

very important and there are several ways that have been done to make it possible.

Also it is necessary to develop methods in estimations of active constituents or marker

compounds as the quantitative target to assess the inherent and authenticity of quality.

Planar chromatography is the most versatile option required for the identification tests

for the quality control of the herbal drug. In its traditional form, thin layer

chromatography is frequently used for analysis of crude plant extractions and has a

long record in almost all pharmacopeia for its use in analysis of plant extracts. There

are other analytical techniques such as HPLC and HPTLC that can ascertain the

presence of certain compounds in plants and also quantify them more precisely

(Sreekumar and Ravi, 2007).

2.3.2 High performance thin layer chromatography

High-performance-thin-layer-chromatography (HPTLC) is an advanced form

of instrumental TLC which not only include the use of high performance adsorbent

layers (silica material with refined uniform particles of approximately 5 µm in

diameter compared to 12 µm in TLC), but also adopts instrumentation such as

advanced development chambers. It usually implies a standardized methodology for

development, optimization and documentation after a proper validation of methods.

The HPTLC technique is applied in qualitative and quantitative separation of

compounds of a mixture, where the quantitative mode operates in a more optimized

way and hence applicable in the assay of compounds in a sample.

There are several advantages of using HPTLC for analysis of compounds

compared to HPLC and spectrometric titrations (Sethi, 1996; Kalasz and Bathori,

2001). They are (Shewiyo et al., 2012)

The process of separation is easy to follow: especially with coloured

compounds.

Several samples can be separated in parallel on the same silica plate resulting in

high through-put and a rapid low cost analysis.

Specific and sensitive colouring agents can be applied to detect the separated

spots.

Two-dimensional separations can be performed easily.

26

HPTLC can combine and consequently use varied modes of evaluation,

allowing compounds identification with different colours or light-absorption

characteristics.

Contact detection allows microbial activity in the spots to be assessed and

radiolabelled compounds to be monitored.

The TLC plates are disposable and therefore regeneration and clean-ups is not

required.

The development and detection of the separated spots on a plate are different

processes in time and therefore, after separation, the plates can be stored for a

long time and detection can be performed at a later stage.

Literature search reveals a huge number of published papers describing

various usage of the HPTLC technique in many analytical fields. To emphasize on the

importance, opportunities and challenges of the HPTLC technique, older review

papers are available in the literature (Kalasz and Bathori, 2001; Nyiredy, 2002; Poole,

2003). Kaale et al. (2011) recently reported the availability of a large number of

studies on HPTLC methods that have been developed, validated and applied in

pharmaceutical analysis of an active ingredient. HPTLC serves a convenient tool in

analysing the distribution pattern of phytoconstituents, which is unique for each plant.

HPTLC fingerprinting can be applied for authentication of herbal extracts and

formulations. Various workers developed HPTLC protocol for analysis of

phytoconstituents in crude drugs such as bergenin, gallic acid and catechine in

Bergenia lingulata and Bergeniacilliata (Dhalwal et al., 2008). HPTLC technique is

widely employed in pharmaceutical companies in process development, detection and

identification of adulterants in herbal products. It is also used in the quality control of

biological matrices such as whole plant, leaves, flowers and health foods and herbal

formulations (Soni and Naveed, 2010).

HPTLC method has been developed by Sharma et al. (2007) for estimation

of withanolide A and withaferin A in different plant parts such as root, stem, leaf and

fruit of two morphotype of W. somnifera. Patel et al. (2009) developed fingerprint

profile and analysed the withaferin A in old and young roots after complete ethyl

acetate extraction. Distinct HPTLC fingerprint profile was developed for standard

drug and for young and old root extracts by using toluene:ethyl acetate:acetone (2:3:3)

27

as mobile phase. The fingerprinting of hydroalcoholic extracts of withania

formulations has been done using HPTLC in an attempt to understand the heavy metal

content and confirmed its prescription to be safer (Chandran et al., 2010). Recently,

Jirge et al. (2011) developed and validated a HPTLC protocol to determine variations

by herbal manufacturers of Ashwagandha formulations. They employed HPTLC

standardization of four Ayurvedic formulations for their withaferin A and β-sitosterol-

D-glucoside content and their variation in the same plant product. A selective HPTLC

analytical method has also been developed for fingerprinting and determination of

constituents in extracts of Ashwagandha root and their polyherbal formulation (Alam

et al., 2012). Natural populations of W. somnifera and W. coagulans from Iran were

analysed for their withaferin A content by means of HPTLC finger print and was

found out their accumulation were higher in aerial parts than in root extracts. The

result also showed a high level of variations in the Iranian natural populations of

withania, which can be utilised for conservation and breeding programs of a selective

morphotype to improve withaferinA production (Mirjalili et al., 2009b). Similarly,

HPTLC method standardization and withanolide A quantification has been carried out

in field roots of W. coagulans obtained from different geographical locations of Iran.

A morphotype has been identified with increased phytoconstituents and withanolides

content for their in vitro propagation (Preethi et al., 2014).

2.4 In silico analysis

2.4.1 ADME predictions

Computational approach is one of the fastest and newest developing

technique in pharmacokinetics, ADME (absorption, distribution, metabolism and

elimination) predictions, drug discovery and toxicity analysis (Boobis et al., 2002).

In silico analysis of pharmacokinetic and phytochemical parameters are primarily

based on ADMET (absorption, distribution, metabolism, excretion and toxicity) and

Lipinski’s rule of five. ADME describes the potential utility of compounds as drug

leads. Unlike in vivo and in vitro ADME assays, in silico ADME prediction is

particularly cheap and efficient in search of a great number of compounds by

screening libraries and virtual molecules prior to their synthesis. Studies have

demonstrated that initial screening of drug candidates for their ADME properties is a

successful approach to enhance the drug quality (Di and Kerns, 2008). Thus, the

in silico ADME prediction plays a crucial role in facilitating pharmaceutical

28

companies to wisely select drug candidate’s prior expensive clinical trials. The

ADME data can also be used to identify potential liabilities, structural modifications,

select compounds for experimental studies, prioritize lead compounds, and diagnose

in vivo assay results (Di and Kerns, 2008).

ADME-Tox properties can be classified into two categories namely, the

“physiological” and “physicochemical” categories. The physiological ADME-Tox

properties can be further grouped into in vitro ADME-Tox properties (such as MDCK

permeability and Caco-2 permeability, liver microsomes, etc.) and in vivo

pharmacokinetic properties (such as human intestinal absorption – HIA, plasma

protein binding – PPPB, oral bioavailability –F, urinary excretion, area under the

plasma concentration –AUC, total body clearance –CI, elimination half time –t1/2 and

volume of distribution) are governed by many factors. On the other hand, the

physicochemical property which includes logarithm of octanol-water partition

coefficient –logP, aqueous solubility, and logarithm of octanol-water distribution

coefficient (logD) are governed by simple physicochemical laws (Wang and Hou,

2009). In the last few decades, several new ADME-Tox models have been published

and many new software packages and databases have emerged to theoretically

estimate these parameters for a given chemical structure. Also many in silico

approaches for predicting ADME properties of drug compounds for their chemical

structure have been initiated and developed, ranging from database approaches such

as QSAR and 3-dimensional QSAR to structure based methods such as pharmacore

modelling and ligand-protein docking (Yamashite and Hashida, 2004). Schrödinger’s

QikProp is an extremely fast ADME predicting program with following benefits:

Wide range of prediction – QikProp predicts the widest varieties of

pharcological properties such as octanol/water and water/gas coefficient, logPs,

logS, logKhsa, logBB, Caco-2 and MDCK cells permeabilities, overall CNS

activity, oral absorption level, log IC50 for HERG K+ channel blockage.

Accurate ADME predictions – QikProp predicts the ADME properties based on

3D molecular structure; unlike fragment based methods. Therefore, they provide

equally accurate results in predicting molecular properties from a molecules

scaffold structures compared to the novel scaffolds of a well-known drug

analogue.

29

Jorgensen rule of three and Lipinski rule of five – QikProp has ability to

accurately check for Jorgensen rule of three and Lipinski rule of five violations

to provide an at-a-glance measure of compounds drug characteristics.

Similarity check – QikProp automatically identifies molecular similarity

between a submitted ligand and compounds of 1700 molecular databases and

user-specified libraries.

Lead generation – QikProp rapidly screens compound libraries for hits and

identifies molecules with user defined properties that fall inside the normal

range of known drugs. Thus making it simpler to filter the candidates with

suitable and unsuitable ADME properties.

Lead optimization – QikProp plays important roles in lead optimization by

analysing within defined classes of compounds as well as by identifying the

compounds to eliminate because they exhibit extreme values of predicted

properties.

Improving the accuracy level – QikProp computes around twenty physical

descriptors thereby improving the predictions by fitting to additional or

proprietary experimental data and generating alternate QSAR models.

Easy-to-use-interphase – QikProp accepts a wide variety of input formats,

including MDL SD files, Maestro files and PDB files. Calculations are easily set

up and results can be plotted and analysed using Maestro interface.

ADME screening provided peer analysis for final selection of potential drug

candidates from the compound library generated for nevirapine and 47 nevirapine

structural derivatives (Sengupta et al., 2008). In a study of Das et al. (2011), the best

fit ligands benzoxazinone were subjected to in silico ADME screening and was

concluded that the series has the significant potential for type 2 diabetes based on

ADME screening and docking analysis and thus benzoxazionepharmacore could be

used for further development. Withanolides were also screened for their ADME

properties before their molecular docking against the bacterial and viral components

to study their novelty and recommendation for future drug candidature against the

infections caused by these components (Santhi and Aishwarya, 2011; Regon et al.,

2014). In another study, ADME analysis was done using QikProp software before

molecular dock analysis of withaferin A and withanone to prove their efficiency as

small molecule drugs (Vaishnavi et al., 2012).

30

2.4.2 Molecular docking

In recent years, the search for novel drugs has evolved from a trial and error

process into computer based approaches. In structure-based design, the structures of

known target proteins are used in order to discover the novel compounds with

therapeutic relevance. This approach can be accomplished by molecular docking and

involves the formation of protein-ligand complexes (Holger et al., 2001). Docking is

frequently used to predict the binding orientation of small drug candidates into their

appropriate protein targets in order to predict the activity and affinity of the drug

molecule. Hence docking plays an important role in rational designing of drugs. The

development of docking methods is also concerned with making the right assumptions

of target proteins and drug candidates and therefore finding the acceptable

simplification by providing sufficient accuracy and predictive model for protein-

ligand interactions (Abraham et al., 1998). Given the structure of a protein and ligand,

the task is to predict the structure of the resulting complex from their interactions.

During the course of this process, the protein and the ligand adjust their conformation

to achieve an overall “best-fit” and this kind of conformation adjustment in their

overall binding is referred to as “induced-fit” (Wei et al., 2004). In their bound

conformation, the ligand exhibits chemical and geometric complementarily, both of

which are essential for successful drug activity.

The molecular docking tool has been generated to obtain a preferred

geometry of interaction of the receptor-ligand complexes having minimum interaction

energies and based on different scoring functions viz. dock score, sum of steric and

electrostatic fields. Some of the important protein-ligand docking tools used are listed

in Table 2.4. Accuracy and efficiency of the geometric modelling of the dock,

depends on the scoring function. The scoring functions used in the molecular docking

have been adapted from salvation and entropy of the dock complex. The challenge of

the lead-generation phase of the protein-ligand docking is to quickly screen millions

of possible compounds that fit particular receptors with high specificity and affinity.

The set of ligands thus selected can be screened further using more involved

computational technique such as free-energy perturbation theory or in experimental

assays.

31

Table 2.4: Characteristics of current open access protein-ligand docking tools

S.No. Program Designer/Company

License terms Docking approach Supportedplatforms

Scoring function Reference

1 AutoDock D.S. Goodsell andA.J. OlsonThe ScrippsResearch Institute

Free foracademic use

Genetic algorithmLamarckian geneticalgorithmSimulated annealing

Unix, Mac, OSX,Linux, SGI

AutoDock (force-field methods)

Good sell andOlson, 1990

2 DOCK I. KuntzUniversity ofCalifornia, SanFrancisco

Free foracademic use

Shape fitting (spheresets)

Unix, Linux, Sun,IBM AIX,, OSX,Mac, Windows

ChemScore, GB/SAsolvation scoring

Kuntz et al.,1982

3 FRED OpenEyeScientificSoftware

Free foracademic use

Shape fitting(Gaussian)

Unix, Linux, SGI,Mac, IBM AIX,OSX, Windows

ScreenScore, PLP,Gaussian shapescore, user defined

Schulz-Gaschand Stahl, 2003

4 FlexX T. Lengauer andM. RareyBioSolveIT

Commercialfree evaluation(6 weeks)

Instrumentalconstruction

Unix, Linux, Sun,SGI, Windows

FlexXScore, PLP,ScreenScore,DrugScore

Rarey et al.,1997

5 GOLD CambridgeCrystalographicData Centre

Commercialfree evaluation(2months)

Genetic algorithm Linux, SGI, IBM,Sun, Windows

GoldScore.ChemScore userdefined

Jones et al.,1997

6 Glide Schrodinger Inc. Commercial Monte Carlo sampling Unix, Linux, IBM,AIX, SGI,

GlideComp,GlideScore

Friesner et al.,2004

7 LigandFit Accelrys Inc. Commercial Monte Carlo sampling Linux, SGI, IBMAIX

LigScore, PMF, PLP Venkatachalamet al., 2003

32

2.4.3 Scoring function

Scoring functions are fast and approximate mathematical methods used to

predict the strength of the interactions between two molecules after docking. Most

commonly, one of the molecules is small and organic drug compound and the second

is a biological target such as protein receptor (Jain, 2006). Scoring function is also

been developed to predict the strength of other types of interactions such as protein-

protein or protein-DNA (Robertson and Varani, 2007). The scoring function takes a

docked pose as input and returns the strength in numbers indicating the likelihood that

the pose represents favourable binding interactions. Most of the scoring functions are

physics based molecular mechanics estimating the force fields. Scoring methods

ranges from molecular mechanics force fields such as OPLS, AMBER or CHARMM

through empirical free energy scoring function or knowledge based functions. The

docking methods utilize the scoring functions in one of two ways. The first approach

utilizes the full scoring function to rank the protein ligand complex formed. The

system is then modified by the search algorithm and the same scoring function is

again implemented to rank the new structure (Taylor et al., 2002).

2.4.4 Target proteins

Proteins involved in cell cycle

Cell cycle deregulation is a distinguished hallmark of tumour cells (Stewart

et al., 2003). Normal cells possess the ability to arrest cell cycle after DNA damages

to maintain genome integrity whereas tumour progressing cells are characterised of

deregulation of cell cycle whereby the damaged DNA possessing cells proceed to

undergo DNA replication and cell division, resulting in an unrestrained cell

proliferation. Cancers such as breast, lung and gastric cancer are known to exhibit

uncontrolled cell growth and division. The best parameter to judge the efficacy of

anti-cancer therapies is through their ability to arrest cell cycle. It is essential to

identify and eliminate cells proliferating inappropriately and therefore cell cycle

regulators play a vital role in tight check of cell cycle (Meikrantz and Schlegel, 1995;

King et al., 1996).

The timing and order of events involved in cell cycle are monitored during

cell cycle check points that occur at G1/S phase boundary and during G2/M phase

33

transitions (Murray and Hunt, 1993). These check points ensures critical events after a

particular phase of a cell cycle is completed and before a new phase is been initiated,

thereby preventing the formation of abnormal cells. It is at these checkpoints that the

cell determines and chooses the machinery for its own beneficial. The cell cycle

control system depends on three protein families: the cell division cycle 25 protein

(Cdc25), the cyclins and the cyclin dependent protein kinases (CDKs). Arresting the

cell cycle involves down regulation of these proteins. In cancer, mutations are

observed in genes encoding CDK, CDK-activating enzymes, cyclins, and other check

point proteins (Sherr, 1996; Mc Donald and el Deiry, 2000). The cell cycle phases and

the mediators involved in them are presented in Figure 2.4.

Figure 2.4: Cell cycle phases and components. Upon proliferative stimuli, the D-cyclin level increases and forms complexes with CDK4/6, leading to phosphorylationof Rb. Cyclin E along with CDK2 further phosphorylates and results in transitionfrom G1 to S phase. Cyclin A binds with CDK2 in S phase and CDK1 in G2/M phase.In M phase, CDK1 is in complex with cyclin B (Bjorner, 2013).

The cell division cycle 25 proteins (Cdc25) are phosphatases that activate

the CDKs and cyclins which in turn regulate the progression of cell cycle.

Mammalian cells express three Cdc25 - Cdc24A, Cdc25B, Cdc25C of which Cdc25A

mainly controls G1/S transitions and, Cdc25B and Cdc25C predominantly activates

G2/M progression (Molinari et al., 2000; Mailand et al., 2000; Donazelli and Draetta,

2003; Santamaria et al., 2007). It is now evident that all the three Cdc25 isoforms

cooperate to play essential roles in spatial and temporal regulation of the CDKs

during various stages of the cell cycle (Boutros et al., 2006; Boutros et al., 2007;

Rudolph, 2007; Lavecchia et al., 2009). Cdc25s have been associated to undergo

34

oncogenic transformation and elevated expression of Cdc24A and Cdc25B at both

mRNA and protein level has been reported in a wide variety of primary human

cancers with poor prognosis such as breast cancer (Galaktionov et al., 1995), prostate

(Ngan et al., 2003), colon cancer (Takemara et al., 2000; Hernandez et al., 2001) and

lung cancers (Wu et al., 1998; Sasaki et al., 2001). Enhanced expression of Cdc25s in

tumours correlates with specific clinical and pathological features resulting in more

aggressive tumours and a short disease free survival (Boutros et al., 2006; Boutros

et al., 2007). The inhibition of Cdc25 phosphatases may thus contribute as the novel

approach in the development of anticancer therapeutics.

Selective inhibitors of Cdc25 with potent and reversible potential should

facilitate the elucidation of biological functions for these enzymes. Furthermore,

potential inhibitors prove to be useful in targeted cancer therapies due to oncogenic

nature of Cdc25A and Cdc25B in cancer cell lines (Brisson et al., 2004). Although

structures of the catalytic domains of these Cdc25 have been available (Fauman et al.,

1998; Reynolds et al., 1999), very little studies have been carried out of its interaction

with the small molecule inhibitors. Synthetic compounds based on phosphatase

inhibitors of Cdc25 are either not potent or fail to enter the cells effectively. Inhibitors

derived from natural sources such as menadione, dysidiolide, dnacin B1 and

coscinosulfate were found to form irreversible adducts with Cdc25 (Lyon et al.,

2002). Withanolides being a natural compound with proven track record of

therapeutic properties can be used as a one such potential inhibitor.

Cyclin is an essential cell cycle regulator in all eukaryotes. Cyclin binds to

the cyclin-dependent kinase (CDKs) to form Cyclin/CDK complex which then

activates and phosphorylates a critical set of proteins to set the succeeding events into

motion that defines mitosis (Smits and Medema, 2001). These events include nuclear

membrane breakdown, assembly of spindle apparatus, chromosomal condensation and

segregation of sister chromatids (Sanchez and Dynlacht, 2005). Given the central role

of cyclins in cell division, its deregulation not surprisingly contributes to cancer.

Cyclins are overexpressed in several human carcinomas and level of expression of

cyclins correlates with the aggressiveness of the tumour (Ikuerowo et al., 2006). Thus

cyclin B serves as effective targeted cancer therapeutics (Yuan et al., 2004).

Inhibition of cyclin expression either by antisense methodology or by antibody

35

microinjection lengthens the G1 phase duration and causes reduction in proliferation

as demonstrated in nude mice (Arber et al., 1997).

The Cyclin Dependent Kinases (CDKs) holoenzymes are a family of

serine/threonine kinases which play a major role in regulating the cell cycle

progression in all eukaryotic organisms (Nilsson and Hoffman, 2000). As their name

suggest, their activity is controlled in part by regulatory subunits called Cyclins that

bind and activate and provides substrate specificity for their catalytic partners (Sherr

and Roberts, 1999). Inhibitory phosphorylation on N-terminal tyrosine and threonine

residues maintains CDK complexes in an inactive state (Parker and Worms, 1992;

Squire et al., 2005). The structure-function relationship of cyclin-CDK complexes

have been extended further by mutational analysis in the N-terminal helix of the

mitotic cyclin by Goda et al. (2001). This active site is highly conserved in all known

mitotic cyclins.

In order to keep the cells in a controlled the cell cycle progression in a

controlled state, the cyclins and CDKs are tightly regulated through inhibition,

subcellular localization and degradation. The CDK inhibitors (CKI) bind CDKs alone

or in combination with cyclins thereby regulates the activity of CDKs. These

inhibitors are in turn regulated by both intercellular and extracellular signals. For

example, p21 is transcriptionally regulated by p53 tumor suppressor and induced upon

DNA damage (el-Deiry et al., 1993). Genes such as c-myc, are known to inhibit or

activate cell proliferation by affecting the formation of CDK complexes.

Proteins involved in apoptosis

Apoptosis or the programmed cell death is a physiological homeostatic

mechanism and is critically important for the survival of multicellular organisms

(Lockshin and Zakeri, 2007). As a result of this process, unwanted cells are

eliminated in a well-organised sequential process, characterised by various

morphological and biochemical changes such as pyknosis, mitochondrial membrane

permeability, plasma membrane blebbing, and activation of caspase cascades

(Elmore, 2007). Activation of apoptosis is mainly mediated through intrinsic

mitochondrial pathway and extrinsic death receptor pathway which involve a variety

of caspase family members (Hengartner, 2000; Fulda and Debatin, 2006). The

regulation of Bcl-2 family members dissipates the mitochondrial membrane potential

36

resulting in the release of proapoptotic proteins such as cytochrome C and apoptosis

inducing factor from the intermembrane space into the cytosol. Following this, the

apoptosome, a complex that turns out from the interacting cytochrome C and

apoptosis protease-activating factor, results in activation of caspases. Activation of

various caspases subsequently cleaves poly-(ADP-ribose) polymerase (PARP),

ultimately leading to apoptosis mechanism.

A rational approach to treat cancer is to selectively eliminate the proliferating

tumor cells via apoptosis and spare quiescent and terminally differentiated cells

(Schwartz and Shah, 2005; Petrelli and Giordano, 2008). The failure to control cancer

cells associated with apoptosis induction has been considered to be a critical cause of

resistance against cancer therapies (Fulda and Debatin, 2006). The entire apoptosis

mechanism is discussed in detail in Figure 2.5.

Figure 2.5: Apoptosis pathways. The extrinsic cell death pathway mediated by TNFreceptor superfamily is initiated by the recruitment of adapter proteins, like FADD(Fas associated death domain), via DD (death domain), which in turn binds to deatheffector domain containing caspases. Formation of this death inducing signallingcomplex (DISC) leads to activation of caspase-8/10 which then activates caspase-3. Inintrinsic mitochondrial pathway, proapoptotic Bcl-2 family members, Bak and Baxtranslocates to the mitochondria where it forms an oligomeric pore in the outermitochondrial membrane. This releases cytochrome c and other pro-apoptotic factorsinto the cytosol. This mechanism triggers the assembly of apoptosome from Apaf-1,caspase-9 and ATP as a third component. Subsequently apoptosome activatescaspase -3, leading to cell death (Kalimuthu and Kwon, 2013).

37

In mammalian cells, five antiapoptotic proteins Bcl-2, Bcl-XL, Bcl-w, MCL1

and A1 antogonises the proapoptotic functions of BAX and BAK (Youle and Strasser,

2008). Overexpression of Bcl-2 and Bcl-XL enhances the cell survival by suppression

of apoptosis (Yang and Korsmeyer, 1996; Boise et al., 1995; Reed, 1995; White,

1996). Unlike Bcl-2 and Bcl-XL, over expression of BAK and BAX accelerates cell

death (Oltvai et al., 1993). Both groups of proteins are proposed to regulate apoptosis

by means of homo and hetero dimerization (Sedlak et al., 1995; Yang et al., 1995).

The structures of pro and antiapoptotic proteins are illustrated in Figure 2.6.

Figure 2.6: Pro and anti-apoptotic proteins involved in the apoptosis mechanism.The hypothesis is that upon engagement of BH3-only proteins termed as “activators”,notably Bim and Bid at the trigger site of BAX leads to major conformational changeincluding allosteric release of its C-terminal helix and exposure of its BH3 domain foractivation and oligomerization within the mitochondrial membrane (Shamas-Dinet al., 2013).

The B-cell lymphoma 2 (Bcl-2) family proteins is been discovered in many

types of cancer cells and promotes cell survival (Vaux et al., 1988) leading to

impaired apoptosis, a critical step in tumor developments (Hanahan and Weinberg,

2000). The pro- and anti-apoptotic family members heterodimerize each other and

seemingly titrate their functions (Oltvai et al., 1993). Mutagenesis studies established

that BH1, BH2 and BH3 domains strongly influences these homo and

heterodimerizations (Yin et al., 1994; Chittenden et al., 1995). Proapoptotic proteins

allow its insertion into the groove of the BH3 domains of the anti-apoptotic proteins

(Muchmore et al., 1996). The fourth “BH4 site” is the N-terminal helix is conserved

38

for inhibition of apoptosis and binding of drug molecules at this site results in

suppressing their inhibitory profile and supports the function of other pro-apoptotic

proteins under tumerogenesis. The BH4 region of the Bcl-2 binds with regulatory

domain of the inositol 1,4,5 triphosphate (IP3) receptor which in turn controls the

calcium efflux from the endoplasmic reticulum, thereby inhibiting the initiation phase

of calcium mediated apoptosis (Rong et al., 2009).

Bcl-2 and Bcl-XLare the best model to be screened under the category of

anti-apoptotic proteins since it has a higher global flexibility with pliable binding

pockets within the BH3 domain compared to the deeper hydrophobic pockets of other

anti-apoptotic proteins that restrict the binding to specific BH3 domain containing

proteins (Lee et al., 2009). In fact, the Bcl-2 coding nucleotides are currently being

tested in clinical trials for various cancer treatments (Rudin et al., 2002). In addition,

by utilising the structures of Protein-Protein complexes, small molecule inhibitors of

Bcl-2 have been designed (Petros et al., 2004). Wang et al. (2000) were the first to

report small molecule inhibitors of Bcl-2 by building the modelled Bcl-2 in the form

of peptide complex. Subsequently they employed a computational docking strategy to

screen around 193,833 compounds from the available compound libraries. In another

study using computer-based screening, around 206,876 organic compounds were

searched from National Cancer Institute 3D database to identify the potential binders

of Bcl-2 (Enyedy, 2001).

BAK and BAX are the two main pro-apoptotic proteins considered to be

essential gateway for apoptosis mechanism (Wei et al., 2001) and when deleted, give

rise to severe developmental defects (Lindsten et al., 2000). In normal cells, BAX is

largely cytosolic and translocate to mitochondrial membrane only upon receiving

apoptotic stimulus (Wolter et al., 1997; Edlich et al., 2011). Cytosolic BAX

comprises a globular bundle of nine helices and the last helix is assumed to regulate

the BAX activity as it neither anchors the BAX to the mitochondrial membrane nor

resides in a hydrophobic groove on the surface of the cytosolic BAX (Suzuki et al.,

2000). Defining how a BAX metamorphoses from an inert cytosolic monomer to

cytotoxic mitochondrial membrane perforating oligomer has been deemed the “holy

grail” of apoptosis research (Youle and Strasser, 2008). This pivotal mechanism is

poorly understood because no structure of any activated form of BAX is available.

39

However, the mechanism by which BAX are activated in the first place has been the

theme of much debate (Leshchiner et al., 2013).

Poly (ADP-ribose) polymerases (PARPs) are nuclear protein enzymes

involved in synthesis of poly (ADP ribose) in DNA damages and repairing of single-

strand breaks (SSBs) through base excisional repair (BER) (Do and Chen, 2013).

Escalating the DNA damages and to deteriorate the DNA repair system are the other

important features leading to genomic instability which provides valuable clues in the

rational development and exploitation of inhibitor drugs in clinical setting and

designing of a new therapeutic approach in cancer. The human genome integrity is

constantly under stress from both endogenous insults such as reactive oxygen species

and exogenous insults such as chemotherapeutic agents. Cellular response depends

upon the magnitude of insult and if the damage is extensive and irreparable, cell death

occurs.

Catalytic domain of PARP is highly conserved and forms the active site

(Ame et al., 2004; Otto et al., 2005). Under normal condition, the inactive PARP

resides in the nucleoplasm and once the DNA breaks are introduced, synthesis of poly

(ADP-ribose) takes place at the sites of breakage. In vitro studies indicates that

PARP-1 binds tightly to DNA nick which activates its catalytic domain inducing poly

(ADP-ribosyl)ation. This allows for the recruitment of DNA repair proteins such as

DNA polymerase, DNA ligase and scaffolding proteins (El-Khamisy et al., 2003;

Houtgraaf et al., 2006). More recent studies by Helleday et al. (2011) proposed that

PARP inhibitors results in trapping of PARP from DNA repairing and stalling of

replication forks. A considerable effort is centred in order to manipulate DNA damage

responses to selectively induce death in tumor cells (Helleday et al., 2008).

Accordingly, PARP inhibitors that compete with β -NAD+ at their active site are

arisen as a new potential therapeutic strategies as chemo-and radio-potentiation for

cancer treatments (Rouleau et al., 2010).

Proteins involved in Inflammation

Inflammation is an adaptive response triggered by noxious conditions and

stimuli such as tissue injury and infection (Kumar and Cotran, 2003; Majno and Joris,

2004). Inflammation is a complex process mediated by immune cells such as

macrophages and monocytes (Chen et al., 2005) which results in restoration of

40

damaged tissue structure and function (Lawrence et al., 2002). It is generally thought

that a controlled inflammatory response is beneficial by providing protection against

infections and tissue damage, but become detrimental if deregulated to cause septic

shocks. Acute inflammatory response involves the coordinated delivery of blood

components to the site of infection, causing increased swelling, temperature, redness

and pain (Kumar and Cotran, 2003; Koh et al., 2010). It is well known that,

macrophages along with neutrophils and dendritic cells play important roles in innate

immune reactions (Carralot et al., 2009). The key inflammatory mediators such as

cyclooxygenase-2 (COX-2), nitric oxide (NO), nitric oxide synthase (iNOS) and

prostaglandins (PGE2) and proinflammatory cytokines such as interleukins (IL) and

tumor necrosis factor (TNFα) are released from activated macrophages (Nakagawa

et al., 2012). Lipopolysaccharide (LPS), a cell wall component of Gram-negative

bacteria is reported to activate these macrophages and triggers a series of signalling

cascades which leads to activation of MAPKs and NF-kB pathways (Zhang and

Dong, 2005). Herein, LPS induced macrophages is a well-established model to study

innate immune responses (Aderem and Ulevitch, 2000).

The main effect of these mediators is to elicit inflammatory exudate locally

and prevent its access to extravascular tissues through post capillary venules. The

activated blood vessel tissue allows selective extravasation of neutrophils and

prevents the exit of erythrocytes. This selective permeability is afforded by ligation of

endothelial cell integrins with selectins and chemokine receptors on leukocytes (Pober

and Sessa, 2007). When the neutrophils becomes activated upon reaching the afflicted

tissue site either by direct contact with the pathogens or through actions of cytokines

secreted by infected cells, they releases toxic components of their granules such as

reactive oxygen species (ROS) and reactive nitrogen species (RNS) and attempts to

kill the invading agents. These highly potent effectors released from the neutrophils

do not discriminate between pathogens and host targets, leading to unavoidable

collateral damage to the host tissues (Nathan, 2002). A successful acute inflammatory

response results in the expulsion of infectious agents followed by a repair phase

mediated mainly by macrophages recruitment (Serhan and Savil, 2005). The switch in

lipid mediators from pro-inflammatory prostaglandins to lipoxins with anti-

inflammatory potential is crucial for the transition to resolution from inflammation.

Lipoxins inhibits the neutrophil recruitment and engage monocytes to remove dead

41

cells and initiate tissue remodelling (Serhan and Savil, 2005). Protectins and resolvins

contribute another class of lipid mediators, as well as growth factor produced by

macrophages and transforming growth factor β possess crucial role in resolution of

inflammation and initiation of tissue repair (Serhan, 2007).

The MAP kinases (MAPK) comprises of large enzyme family in both

prokaryotes and eukaryotes. These are serine/threonine kinases that act by modulating

the activity of cellular proteins such as cell surface receptors, other kinases enzymes,

transcription factors and structural proteins (Davis, 1993). They catalyse the transfer

of the terminal phosphoryl group of ATP to their appropriate protein substrates. It has

been recognised for many years that protein phosphorylation regulates many cellular

functions such as metabolism, survival, movement, division and death. Therefore, any

process that disrupts normal phosphorylation disrupts the function of cell and results

in diseases (Cohen, 2002). Several individual MAPK signal transduction pathways

have been characterised of which one pathway leads to activation of the extracellular

signal regulated protein kinases (ERK) which plays important roles in regulating

cellular responses and growth factors (Blumer and Johnson, 1994). The ERK MAP

kinases are activated in a wide variety of cell types by varying stimuli (Cobb et al.,

1991; Pearson et al., 2001). Boulton et al. (1991) coined the acronym ERK for

extracellular signal regulated protein kinase and cloned the cDNA of rat ERK.

ERK1/2 have small amino-terminal lobe and large carboxyterminal lobe with several

conserved helices and strands as described by Knighton et al. (1991). The small lobe

consists of five antiparallel sheets with conserved glycine rich (GxGxxG) ATP-

phosphate binding loop. The large C-terminal lobe is mainly helical with four short

conserved strands that contain most of the catalytic residues associated with

phosphoryl transfer from ATP to ERK substrate (Taylor and Kornev, 2011). Early

studies indicated that these enzymes are activated upon cellular stimulation by

bradykinin, fibroblast growth factors, epidermal growth factors, platelet derived

growth factors and nerve growth factors (Cobb et al., 1991). Then ERK1/2 are also

known to be activated by cytokines, osmotic stress and transmembrane receptors

proteins (Raman et al., 2007). The Ras-Raf-MEK-ERK signalling cascade is

dysregulated in a variety of diseases such as brain injury, cancer, inflammations,

cardiac hypertrophy and diabetes (Muslin, 2008; Tidyman and Rauen, 2009; Tanti

and Jager, 2009; Montagut and Settleman, 2009; Chico et al., 2009; Kim and Choi,

42

2010). Owing to the importance of ERK1/2 signalling cascade, they are known to

represent bonafide drug targets with considerable attention from large cadre of

biomedical scientists (Cohen, 2002).

A second MAPK pathway is regulated by changes in extracellular stimuli’s

induced by physical or chemical changes or by proinflammatory cytokines leading to

activation of transcription factors of c-Jun (Derijard et al., 1994; Kallunki et al.,

1994). The JNKs are a larger group of protein kinases and can be expressed in 10

different isoforms. They are activated by dual phosphorylation by MAPK kinases

MKK4 and MKK7 on specific threonine and tyrosine within their

activation/phosphorylation loop (Davis, 2000). Activation of JNK by extracellular

stimuli such as cytokines or stress leads to the phosphorylation of several transcription

factors and cellular substrates implicated in cell survival and proliferation and mRNA

stabilization. Since these pathways are implicated in a variety of disease states, JNK

constitutes valuable targets for drug discovery and development (Manning and Davis,

2003; Weston and Davis, 2007). JNK interacting protein 1 (JIP1), enhances JNK

signalling by creating proximity effect between the JNK and upstream kinases. An

oligopeptide corresponding to an 11 amino sketch (153-163) is known to inhibit JNK

activity by competing with JIP protein (Whitmarsh et al., 1998; Barr et al., 2002).

Recently, small molecules which mimic JIP serves as substrate-competitive inhibitors

have been reported and inhibit the phosphorylation of JNK substrates both in vitro

and in vivo in a dose dependent manner (Stebbins et al., 2008).

A third MAPK pathway leads to p38 pathway activation after varying stimuli

such as UV light, proinflammatory cytokines, microbial pathogens and increased

extracellular osmolarity (Han et al., 1994; Rouse et al., 1994; Freshney et al., 1994;

Raingeaud et al., 1995). p38 in mammals are in four forms: α, β, γ and δ and among

all p38α is best characterised and expressed in most cell types. p38 was initially

identified as a 38 kDa polypeptide with tyrosine phosphorylation in response to

endotoxins and shocks (Han et al., 1994). The amino-acid sequence identity shows

that p38α and p38β are 75% identical whereas p38δ and p38γ are 61% and 62%

identical, respectively. MAPK family members have marked sequence homologies

with around >40% sequence identity. Nonetheless, there are specificities found

around upstream activators, the MAPK kinases (Crews et al., 1992; Yan et al., 1994;

Derijard et al., 1995; Han et al., 1996; Raingeaud et al., 1996; Cuenda et al., 1996)

43

and the substrates activated by the active MAPKs (Derijard et al., 1994; Kallunki et

al., 1994; Sanchez et al., 1994; Raingeaud et al., 1995; Wang and Ron, 1996).

The nuclear factor-kB (NF-kB) family proteins are essential for immunity,

inflammation, cell proliferation and cell death (Baeuerle and Henkel, 1994; Barnes

and Karin, 1997; Bours et al., 2000; Karin et al., 2002). NF-kB exists in a latent state

in the cytoplasm and requires activation of signalling pathways. Such NF-kB

activating pathways are triggered by diverse extracellular stimuli leading to

phosphorylation and subsequent proteasome mediated degradation of inhibitory

mediators, the inhibitor of NF-kB (IkB) proteins (Karin and Benneriah, 2000).

Therefore, a key step for controlling NF-kB activity is by regulating IkB-NF-kB

interaction. Almost all signals leading to NF-kB activation converge on the activation

of a high molecular weight complex containing a serine specific IkB kinase (IKK).

IKK targets IkBα for ubiquitination and degradation. This IkB protein masks the

nuclear localization signals (NLS) located on each subunits of NF-kB to prevent its

translocalization. The IkBα protein is divided into three parts: an N-terminal domain

that integrates activating signals together, a central part with ankyrin repeats involved

in contact and inhibition of NF-kB subunits, and a C-terminal PEST region, rich in

proline, glutamic acid, serine, and threonine that regulates the half-life of the

molecule. IkB not only interferes with nuclear translocation of NF-kB but also has the

ability to displace NF-kB bound DNA (Ghosh et al., 1995). In response to diverse

stimuli, the inhibitory subunit IkB is phosphorylated and then undergoes degradation

to free NF-kB. Most of the stimuli such as LPS, pro-inflammatory cytokines and

ionizing radiations converge to IKK protein complexes in activating signal

transduction (Karin, 1999). After phosphorylation and degradation of IkB by means

of IKKs, leads to translocation of NF-kB to the nucleus to activate transcription of

target genes (Pahl, 1999). The gene coding for IkB is among the first to be transcribed

by NF-kB and newly synthesized IkB molecules migrates to the nucleus to turn off

NF-kB dependent transcription (Sachdev et al., 1998). Then, with the help of nuclear

export sequences, the newly formed NF-kB/IkB complexes returns back to cytoplasm

(Gilmore, 1999). The UV irradiation induces the activation of NF-kB after

phosphorylation and degradation of IkB apparently without the involvement of IKK

complexes (Li and Karin, 1998). Cell reoxygenation and oxidative stress also leads to

NF-kB activations (Imbert et al., 1996).

44

2.5 Toll-like receptors (TLRs) in inflammation

Deciphering the signalling pathways under normal and disease states remains

a major challenge. Toll-like receptors (TLRs) sense the invading pathogens and play

crucial roles in activation of innate and adaptive immunity. Recent studies of Toll-like

receptors (TLRs) signalling during bacterial infections revealed the molecular

mechanisms by which bacterial components induce innate defence and

proinflammatory responses (Jo et al., 2007; Basu et al., 2012). TLR mediated innate

immune responses are mainly regulated by mitogen activated protein kinase (MAPK)

and nuclear factor NF-kB pathways (Jo et al., 2007; Yuk and Jo, 2011). Both these

signalling pathways play major roles in the activation of antimicrobial responses and

in the generation of effector molecules during infections (Schorey and Cooper, 2003).

Moreover, reactive oxygen species (ROS), primarily derived from NADPH oxidases

(NOX), are important in controlling and shaping the key signalling network systems

during inflammatory responses (Bae et al., 2011; Brune et al., 2013).

Figure 2.7 illustrates different TLR’s involved in signaling cascades. Some

TLRs share common ligands, and heterodimerization between the ligands is common

(Huang et al., 2011). Therefore, it is necessary to have a sound knowledge of how this

excessive TLR activation disrupts immune homeostasis and results in the

development of inflammatory and autoimmune diseases. The list of endogenous and

exogenous ligands of different TLRs is represented in Table 2.5. TLRs are found to

be conserved evolutionarily from wide array of plants to mammals. Toll means

“great” in German and Anderson et al. (1985) coined it for a protein which played a

critical role in the early development of Drosophila embryos. Later it was found that

this protein also played an essential role in host innate immunity against fungal

infections in adult flies (Anderson et al., 1985; Lemaitre et al., 1996). Since then,

research interest has been focused on TLRs activation and functioning. All TLRs

show similar domain architecture comprising with an extracellular leucine-rich

domain sensing the pathogens, a Toll-interleukin 1 receptor (TIR) domain which

mediated the downstream signal transduction and a single transmembrane helix

(Basith et al., 2011). On the whole, 13 TLRs have been discovered until date and is

found to have common role in immunity. These TLRs differ in their ligand

specificity, cell localization, usage of adaptor proteins and cellular responses (Iwasaki

and Medzhitov, 2004). However, TLRs are not the only group of receptors for

45

invading pathogens, the system also possess certain non-TLRs such as cyctosolic

DNA receptors (Lee and Kim, 2007), nucleotide-binding domain and leucine-rich

repeat containing gene family (NOD receptors) (Martinon et al., 2007) and retinoic-

acid-inducible gene-I- (RIG-I) like receptors (RLRs) (Yoneyama and Fujita, 2009).

Figure 2.7: Various TLRs and their locations. The cell membrane bound TLRs useMyD88 as adaptor protein for signal transduction. TLR4 alone uses three or moreadaptors apart from MyD88 to elicit antiviral responses. Endosome located TLRs alsouse MyD88 except, TLR3, which uses TRIF, the universal adaptor protein recruitedby TLR3 and TLR4. All these pathways converge on TRAF6 (Tumor necrosis factorreceptor associated factor 6), and activates NF-κB, which in turn translocate tonucleus for expression of various genes involved immune responses (Krishnan et al.,2012).

Toll-like receptors (TLRs) were identified by means of genetic analysis as

the sensors that detect the released ligands. First LPS (Poltoraket al., 1998), then

DNA, RNA, flagellin and lipopeptides of microbes were shown to be ligands of

TLRs. Since TLRs are capable of recognizing molecules derived from bacteria,

viruses, fungi and protozoa, they are able to sense most of the infections which we

might ever encounter and their sensing role is indispensable. Other sensors are

inadequate to cope with wide array of infections in the absence of TLR signaling.

Experimental mice lacking MyD88 and TRIF cannot generate signals via any of the

TLRs were found to rarely survive the weaning age without supplementation of

46

proper antibiotic (Hoebe et al., 2003). Even mutations of individual TLRs such as

TLR2 (Takeuchi et al., 2000), TLR4 (Poltorak et al., 1998) and TLR9 (Tabeta et al.,

2004) cause quite obvious susceptibility to infectious agents and redundancy in

system to detect pathogenic microbes. Conventional adjuvants lead to strong adaptive

immune responses in the absence of TLR signaling (Gavin et al., 2006). This

redundancy is also seen in initiation of adaptive immune responses witnessed in germ

free mice when encountered with enteric microbes, which occurs if TLR signaling is

abolished by mutations (Slack et al., 2009). Thus TLRs although mediate adjuvant

effects, adaptive immune responses can none the less be perfect in their absence.

Table 2.5: Endogenous and exogenous ligands for TLRs

TLRLigands

Endogenous Exogenous

1 - Triacyl lipopeptide

2 Necrotic cells, HSPs (HSP-60, HSP-70,Gp-96), Biglycans

Peptidoglycans andlipoproteins

3 Self-messenger RNA Double-stranded RNA

4

Extra domain A-containing fibronectin,Fibrinogen, Polysaccharide fragments ofheparin sulphate, Oligosaccharides ofHyaluronic acid, Β-Defensin 2, Oxidized low-density lipoprotein, HGPs, Neutrophilelastase, High mobility group box 1 protein,Biglycans

LPS and Taxol

5 - Flagellin

6 - Diacyl lipopeptide

7 - Single-stranded RNA

8 - Single-stranded RNA

9 Chromatin-IgG complex Unmethylated CpG DNA

10 - Unknown

11 - Urapathogenic E. coli

47

2.5.1 Toll-like receptor (TLR) 4 signalling

LPS is a key component of gram negative bacterial cell wall and is composed

of three structural elements: a core oligosaccharide, a lipid component and an

O-specific chain with repeating sequences polysaccharides, which are responsible for

the proinflammatory properties of LPS (Alexander and Rietschel, 2001). The LPS

binding to the endothelial cells surface results in endothelial activation (Hack and

Zeerleder, 2001). LPS also activates macrophages to stimulate the produce

proinflammatory components which in turn modulates the endothelial function.

Collectively, these processes initiate a parallel cascade of events contributing to the

clinical manifestations of sepsis. The TLRs are pattern recognition receptors classified

based on the homology of the cytoplasmic domain (Slack et al., 2000). To date, there

have been 10 TLRs identified in humans (Chuang and Ulevitch, 2000; Du et al.,

2000; Chuang and Ulevitch, 2001) and TLR4 was established as the LPS signalling

receptor. The first host protein involved in the LPS recognition is LPS-binding protein

(LBP) (Schumann et al., 1990). LBP is an acute-phase protein which recruits LPS to

the cell surface by binding to LPS and forms a ternary complex with LPS receptor

molecule, CD14 (Schumann et al., 1990). Formation of LPS and CD14 complex

facilitates LPS transfer to LPS receptor complex composed of TLR4 and MD2

(da Silva Correia et al., 2001). MD2 is a secreted glycoprotein and functions as an

indispensable extracellular adaptor molecule in LPS initiated signalling events by

aiding ligand recognition (Nagai et al., 2002; Visintin et al., 2003). The TLR4

signalling cascades following LPS binding is enhanced by homodimerization of the

receptor and subsequent recruitment of Toll/IL-1 receptor (TIR) domain-containing

adaptor molecules (TIRAP) with cytoplasmic domains of the receptor (Zhang et al.,

2002; Lee et al., 2004). These adaptors include myeloid differentiation factor 88

(MyD88), TIR-containing adaptor inducing IFNb (TRIF) also called TIRAP-1 and

TRIF-related adaptor molecule (TRAM) also called TIRAP-2 (Akira and Takeda,

2004). Activation of TLR4 stimulated MyD88-dependent and independent pathway

which involves activation of NF-kB and mitogen-activated protein kinases (MAPKs)

(Figure 2.8). These pathways and activation regulates the balance between cell

viability and inflammation.

48

Figure 2.8: MyD88 dependent signalling pathway and activation of NF-kB andMAPKs. LPS binds to TLR4 receptor consisting of soluble CD14 and MD2 and as aresult, recruitment of adaptor proteins MyD88 and TIRAP takes place. Then IRAKproteins interacts with the receptor complex. IRAK1 recruits and activates TRAF6leading to downstream activation of MAPKs and IKKs. Activation of IKK complexresults in phosphorylation and degradation of IkB, permitting translocation of NFkBand expression of pro-inflammatory cytokines.

2.5.2 NF-kB and Mitogen-activated protein kinases activation

MyD88 originally cloned as an adaptor molecule possess a C-terminal TIR

domain and an N-terminal death domain (DD) (Wesche et al., 1997). During MyD88-

dependent signalling, MyD88 is recruited to the TLR4 through interaction with the

TIR domain of TLR4 (O’Neill et al., 2003). This multiplex in turn facilitates the

recruitment of IRAK1 and IRAK4 (Wesche et al., 1997; Li et al., 2002). The binding

of IRAK4 with the receptor complex facilitates the IRAK1 to transphosphorylate,

inducing IRAK1 kinase activity (Burns et al., 2003). The autophosphorylation of

IRAK1 results in the binding of TNF receptor-associated factor-6 (TRAF6) (Cao

et al., 1996). TRAF6 then becomes activated and associates with TAB2 which

activates the TAK1 (transforming growth factor-b-activated kinase) which is

constitutively associated with its adaptor protein TAB1 (Shirakabe et al., 1997;

49

Ninomiya-Tsuji et al., 1999; Yamaguchi et al., 2008). TAK1 acts as a common

activator of both NF-kB and MAPKs consisting of extracellular signal-regulated

kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 (Wang et al., 2001). NF-kB

activation starts with the assembly of high molecular weight protein complex known

as signalosome. This complex is made up of inhibitory-binding protein kB kinases,

IKKa and IKKb, along with a scaffolding protein known as NF-Kb essential

modulator (NEMO) (Wang et al., 2001). IKK activation involves TRAF interacting

protein with a forkhead-associated (FHA) domain (TIFA) protein (Ea et al., 2004).

TIFA then promotes oligomerization of TRAF6 and facilitates downstream activation

of NF-Kb (Ea et al., 2004). Activation of IKKs leads to phosphorylation of inhibitors

of NF-Kb (IkB) family, resulting in ubiquitin proteasome mediated degradation of

IkB members, thus permitting the release and translocation of activated NF-kB (Chen

et al., 1995). In addition to the activation of NF-kB, activation of TAK1 also leads to

activation of MAPKs (O’Neill et al., 2003).

2.5.3 Chronic Inflammation

Inflammation is the physiologic response to tissue injury caused by infection,

wounding and chemical damage (Philipa et al., 2004). Acute inflammation is the base

line of defence response, but chronic inflammation has been found to mediate a wide

array of inflammatory diseases (Kao et al., 2009). The host system resists infection in

many different ways and in one way or the other, must discriminate from self and

nonself molecular components. One example is downregulation of class I MHC

molecules during viral infections, a change that is perceived by NK cells.

Discrimination of self-nonself may also depend upon concomitants of infection. This

includes changes in expression of molecules indicative of ‘stresses’. Microbial

protease initiates proteolytic cascades that culminate innate immune response

(Ferrandon et al., 2007). But the most basic and broadly applied system for self-

nonself discrimination depends upon receptors susceptible to molecular signatures

unique to microbes.

Activated immune cells (macrophages, eosinophils, neutrophils, monocytes

and phagocytes) secrete increased amounts of inflammatory mediators such as

reactive oxygen species (ROS), nitric oxide (NO) and pro-inflammatory cytokines

(Block and Hong, 2005; Kim and de Vellis, 2005; Yoon et al., 2009; Tremblay et al.,

50

2011). Although macrophage activation is essential for host defence mechanisms,

aberrant activation of macrophages can lead to disastrous outcomes of inflammatory

diseases such as sepsis, inflammatory bowel and autoimmune diseases (Wyss-Coray

and Mucke, 2002; Block et al., 2007). Normal inflammatory responses are self-

limited by a process involving down-regulation of pro-inflammatory mediators and

increase of anti-inflammatory medators (Kim et al., 2008). However, chronic

inflammations leads to excess release of pro-inflammatory mediators including

COX-2, iNOS and cytokines such as IL-1, IL-6 and TNF-α. During this process, the

activation of immune cells upregulates inflammation (Rietchel et al., 1994; Tao et al.,

2009).

Figure 2.9: Roles of TLRs in inflammation and anti-inflammation. TLRactivation has both negative and positive effects. Over activation of TLRs leads tovarious inflammatory diseases such as sepsis and inflammatory bowel diseases.Positive effects of TLRs are essential in bridging the connection of innate andadaptive immune responses (Krishnan et al., 2012).

Receptors of innate immune system are activated by lipopolysaccharide

(LPS), a microbial component and a key molecule involved in the initiation of sepsis

syndrome (Medzhitov, 2001). However, activation of TLRs at excessive levels can

disrupt immune homeostasis and results in chronic inflammations. Figure 2.9

presents the positive and negative effects of TLR activations and their subsequent

inflammatory cascades. Chronic inflammation is the continued presence of

pro-inflammatory factors at higher levels than baseline and many folds lower than

acute inflammation. Inflamed tissues at chronic levels are characterised by the

51

presence of infiltrating macrophages and lymphocytes, fibrosis, abundant blood

vessels and often tissue necrosis (Nathan, 2002; Sarkar and Fisher, 2006). Sepsis is

the leading cause of mortality in critically ill patients suffering from chronic

inflammation (Angus et al., 2001). The sepsis develops as a result of systemic

inflammatory response due to severe bacterial infection. In sepsis condition, immune

responses gets hyper activated and leads to excessive production of proinflammatory

cytokines leading to cellular injury (Pinsky, 2004). Thus, regulation of macrophage

activation may be a valuable therapeutic target for various inflammatory diseases.