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Identification of potential anti-inflammatory bioactive compounds from

Withania somnifera (L.) Dunal: Molecular docking studies using Glide

Santhi N , Rajeshwari M, Kalaiselvi Senthil and Parvatham R.
Department of Biochemistry, Biotechnology and Bioinformatics
Avinashilingam University for Women, Coimbatore 641 043, India
Non-steroidal anti-inflammatory drugs and COX-2 inhibitors bind to COX-2 and provide
relief from the symptoms of pain and inflammation. However, they lack anti-thrombotic
activity and hence lead to cardiovascular and renal liabilities apart from gastrointestinal
irritation. J Martel-Pelletier et al. reported that dual 5-LOX/COX inhibitors are potential
new drugs to treat inflammation. They act by blocking the formation of both
prostaglandins and leucotrienes but do not affect lipoxin formation. Such combined
inhibition avoids some of the disadvantages of selective COX-2 inhibitors and spares the
gastrointestinal mucosa. The search can be focused on plant natural products that may
offer treatment for inflammation than currently used drugs.
As an attempt to identify such natural alternates with anti-inflammatory activity, a set of
57 compounds from Withania somnifera, were docked against homology modeled human
5-LOX/COX-2 enzymes using GLIDE 5.0. Among the compounds analysed, five
alkaloids, namely, Withasomine (Glide score of 7.96 for COX-2 and 5.05 for 5-LOX);
Cuseohygrine (Glide score of 6.75 for COX-2 and 6.16 for 5-LOX); anahygrine (Glide
score of 6.72 for COX-2 and 5.81 for 5-LOX); 3-tropyltigloate (Glide score of 6.08
for COX-2 and 4.14 for 5-LOX) and tropine (Glide score of 5.71 for COX-2 and 4.48
for 5-LOX) were identified as potent and selective inhibitors of 5-LOX/COX-2 compared
to currently available drug molecules. Rofecoxib, Celecoxib, Lumiracoxib and
Valdecoxib showed inhibitory activity only for COX-2 (Glide score was 6.92, -5.54, 5.75, and -6.44) and not with LOX-5. These alkaloids from W. somnifera are potential
lead compound for developing new anti-inflammatory drug.

Withania somnifera (L) Dunal of (Solanaceae), commonly called Ashwagandha is an
Ayurvedic Indian medicinal plant, which has been widely used as a home remedy for
several ailments. A methanolic extract of the parts of Withania somnifera had antiinflammatory activities comparable to that of hydrocortisone sodium succinate[1]. An
80% ethnolic extract of Withania somnifera displayed significant anti-inflammatory
activity on carrageenan-induced paw edema [2, 3].

The principle bioactive compounds

of W. somnifera are steroidal lactone compouds known as withanolides and withanolide

glycosides and alkaloids [4,5]. They are classified according to their structural skeleton
[6] and the structural variation is responsible for the wide array of pharmacological
Inflammation is a complex process occurring through a variety of mechanisms,
leading to changes in local blood flow and the release of several mediators. COX-2 is
expressed after an inflammatory stimulus and releases metabolites that are used to induce
pain and inflammation.

During normal physiology, COX-2 levels are undetectable

whereas during periods of acute and chronic inflammation, the level of COX-2 is
significantly higher. NSAIDs (Non-Steroidal Anti-inflammatory Drugs) exhibit their
effect through inhibition of cyclooxygenase (COX) enzymes by blocking the synthesis of
prostaglandins from arachidonic acid [7]. Conventional NSAIDs are profoundly used in
the treatment of wide variety of inflammatory conditions and they act by inhibition of
cyclooxygenase (COX), the enzyme involved in the biosynthesis of prostaglandins,
prostacylins and thromboxanes from arachidonic acid [8,9].
It has been shown recently that the COX enzyme exists in two different isoforms COX1 and COX-2. COX-1 primarily responsible for cytoprotection and COX-2, the inducible
form is associated with inflammation. COX-1 enzyme is responsible for maintaining
homeostasis (gastric and renal integrity), whereas COX-2 induced the inflammatory
symptoms in response to inflammatory stimuli [10]. The two COX isoforms are 60%
homologous. The ability to inhibit one isoform selectively is attributed to the different

amino acids at position 523, isoleucine in COX-1 and valine in COX-2 [11]. Therefore,
selective COX-2 inhibition would reduce the undesired side effects such as gastrointestinal disorders, ulcers and renal failure [12]. In addition, there is a growing body of
evidence the complex pathway of arachidonic acid metabolism in inflammation involves
a variety of mediators other than the COX, all of which have a role in the overall process.
Leucotrienes, which are the second main family of arachidonic acid derivatives, are
synthesized from the activity of 5-lipoxygenase (5-LOX) and have a major role in the
inflammatory process (Fig.1).

As a consequence, there has been a drive for the

developments of new dual 5-LOX/COX inhibitors are now being studied as potential new
drugs, post-COX-2 selective inhibitors, to treat the inflammatory processes.

Fig. 1. Products and enzymes of Arachidonic acid metabolism involved in inflammatory

Traditional synthesis of a series of new compounds utilizing combinatorial
chemistry and high-throughput screening can be carried out at high cost and also are time
consuming whereas on the other hand, docking various ligands to the protein of interest
followed by scoring to determine the affinity of binding and to reveal the strength of
interactions has become increasingly important in the contest of drug discovery.

As bioactive compounds from W. somnifera have been shown to possess antiinflammatory activity in some earlier studies [4, 13], it was considered worthwhile to
study the interaction of withanolides and alkaloids with both 5-LOX/COX and compared
with existing drug molecules by molecular docking studies.
Retrieval and preparation of 5-LOX/COX (Target protein) of human
There is no binding mechanism or any docking studies done for Human 5LOX/COX until now although we have effective inhibitors for COX. This is due to the
lack of an appropriate crystal structure.

Therefore, we have developed 3D-model

structure using homology modeling method. BLAST (blastp) search was employed to
search the relevant template for COX-1, COX-2 and 5-LOX of human protein. Fig. 2
shows the sequence alignment between the target protein (human COX-1, COX-2 and 5LOX) and the template. The MODELLER module was used to develop the homology
model [15, 16] and the final 3D model was validated by PROCHECK [17]. Fig. 4, 5 and
For GLIDE calculations, the modeled structures were imported to Maestro
(Schrodinger) and the energy was minimized using protein preparation wizard by
applying OPLS-AA forcefield (19).

The refinement procedure is recommended by

Schrodinger because Glide uses the full OPLS-AA force field at an intermediate docking
stage and is claimed to be more sensitive to geometrical details than other docking tools.
Water molecules were removed and H atoms were added to the structure. The most
likely positions of hydroxyl and thiol hydrogen atoms, protonation states and tautomers
of His residues, Chi flip assignments for Asn, Gln and His residues were selected by the
protein assignment script shipped by Schrodinger. Minimizations were performed until
the average root mean square deviation of the non-hydrogen atoms reached 0.3A. The
ligand binding pocket of the modeled structure was predicted by SiteMap of Schrodinger
packages (Table 1)
Data collection and development of databases
We have collected the structures of withanolides and alkaloids of W. somnifera









Our 5-LOX/COX inhibitor database comprises 38

drug molecules and 57 bioactive compounds (Fig.4) from W. somnifera. Each structure
was assigned an appropriate bond order using LigPrep (Schrodinger). The inhibitors
were converted to maestro format and optimized by means of MMFF94 force field using
default settings [20]. The collected structures (ligands) were prepared using LigPrep 2.2
for further studies. 62 structures for drug molecules and 359 structures for bioactive
compounds were generated by LigPrep.
Molecular docking
Glide docking and scoring function
Glide calculations were performed with Impact version v18007 (Schrdinger, Inc.) [21].
It performs grid-based ligand docking with energetics and searches for favourable
interactions between one or more typically small ligand molecules and a typically larger
receptor molecule, usually a protein [22]. Schrdinger recommends the performance of
test calculations with different scaling factors for the van der Waal radii of the receptor


atom, because steric




otherwise be

overemphasized, leading to rejection of overall correct binding modes of active

compounds. After ensuring that the protein and ligands were in the correct form for
docking, the receptor-grid files were generated using a grid-receptor generation program.
To soften the potential for non-polar parts of the receptor, we scaled van der Waal radii
of receptor atoms by 1.00 with a partial atomic charge of 0.25. A grid box was
generated at the centroid of the active site amino acid residues [23] and the ligands to be
docked were selected from the workspace.
The LigPrep generated structures which have satisfied drug-like properties were
docked into the modeled human COX-1, COX-2 and 5-LOX active site region using
GLIDE docking software. GLIDE dock score which distinguishes molecules based on
their interacting ability is calculated for all molecules.
The ligands were docked with the active site using the Glide Standard Precision
docking procedure. Then the ligands with best Glide score were subjected to xtra
precision docking algorithm. Glide generates conformations internally and passes these

through a series of filters. The first places the ligand centre at various grid positions of a 1
grid and rotates it around the three Euler angles. At this stage, crude score values and
geometrical filters weed out unlikely binding modes. The next filter stage involves a gridbased force field evaluation and refinement of docking solutions including torsional and
rigid body movements of the ligand. The OPLS-AA force field is used for this purpose. A
small number of surviving docking solutions can then be subjected to a Monte Carlo
procedure to try and minimize the energy score. The final energy evaluation is done with
GlideScore and a single best pose is generated as the output for a particular ligand.
GScore = a * vdW + b * Coul + Lipo + Hbond + Metal + BuryP + RotB + Site

where, vdW => van der Waal energy; Coul => Coulomb energy; Lipo => lipophilic
contact term; HBond => hydrogen-bonding term; Metal => metal-binding term; BuryP
=> penalty for buried polar groups; RotB => penalty for freezing rotatable bonds; Site =>
polar interactions at the active site; and the coeffi cients of vdW and Coul are: a = 0.065,
b = 0.130.
ADME screening
The QikProp program [24] was used to obtain the ADME properties of the ligands. It
predicts both physically significant descriptors and pharmaceutically relevant properties.
All the analogues were neutralized before being used by Qikprop. The neutralizing step is
essential, as QikProp is unable to neutralize a structure and no properties will be
generated in the normal mode. The program was processed in normal mode, and
predicted 44 properties for the 57 molecules, consisting of principal descriptors and
physiochemical properties with a detailed analysis of the log P (Octanol/Water), QP%,
and log HERG. It also evaluates the acceptability of the analogues based on Lipinskis
rule of 5 (Lipinski 2001), which is essential for rational drug design.
To find the potential inhibitors of all the three anti-inflammatory drug target protein 5LOX/COX, all the bioactive compounds from W. somnifera and currently using drug
molecules (ligands) collected were docked into the active site. The docking results of
this ligand are given in table 2, 3 and 4. The ranking of ligand is based on the Glide
score. Except the drug molecules viz., celecoxib, valdecoxib, etoricoxib, lumiracoxib,

rofecoxib and licofelone all the other drug molecules showed interaction with both 5LOX/COX.

(Table 2).

Salicylic acid, acetaminophen and salsalate showed almost

similar Glide score for both 5-LOX and COX.

Among the bioactive compounds from W. somnifera, only the alkalods showed
interaction with both 5-LOX and COX protein (Table 3). Whereas, the other compounds
withanolide, withaferin and withanosides showed interaction only with COX and showed
no interaction with 5-LOX protein. Hence, it was not given in the table form. Among
the alkaloids: withasomine, cuseohygrine and anahygrine showed better inhibitory
activity to both 5-LOX and COX. The other three alkaloids showing anti-inflammatory
activity are anahygrine, 3-tropyltgloate and tropinine. Improvement in docking score was
observed for COX-1 and 5-LOX protein. (Table 4).
Binding mode
Active site of 5-LOX offers two different binding modes for these compounds. All the
ligands were docked deeply within the binding pocket region forming interaction with
receptor site amino acids. All the ligands both drug and alkaloids interact with same
binding site aminoacids of COX-1. A similar result was observed for COX-2. Whereas
5-LOX showed different binding mode for drug and alkaloids. Table 1

Predicted ADME properties

We analysed 44 physically relevant properties of bioactive compounds from Withania
somnifera, among which were molecular weight, H-bond donors, H-bond acceptors, Log
P (octanol/water), log P MDCK, log kp (skin permeability), humoral absorption and their
position according to Lipinskis rule-of-five (Tables 4,5,6,7,8 and 9). Lipinskis rule of 5
is a thumb to evaluate drug likeness, or determine if a chemical compound with a certain
pharmacological or biological activity has properties that would make it a orally active
drug in humans.

The rule describes molecular properties important for a drugs

pharmacokinetics in the human body, including its ADME. However, the rule does not
predict if a compound is pharmacologically active (Lipinski 2001). In this study, all the
alkaloids showed allowed values for the properties analysed and exhibited drug-like
characteristics based on Lipinskis rule-of-five. Three compounds of withanolides and 14
compounds of withaferin deviate Lipinskis rule-of-five.

Molecular docking of ligands with target proteins are routinely and extensively used to
reduced cost and time of drug discovery. The alkaloids: withasomine, cuseohygrine and
anahygrine was docked deeply within the binding pocket region forming interaction with
binding site residues of both 5-LOX and COX protein of human. These compounds were
considered as novel inhibitors of 5-LOX and COX and as promising lead-compounds for
developing new anti-inflammatory drugs.

Further, work can be extended to study the receptorligand interactions experimentally
and evaluation of their inhibitory activity would help in designing new anti-inflammatory