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Protease activated receptor-2 (PAR2): possible target of phytochemicals a

Kavita Kumari Kakarala & Kaiser Jamil

a

a

Centre for Biotechnology and Bioinformatics (CBB), School of Life Sciences, Jawaharlal Nehru Institute of Advanced Studies (JNIAS), 6th Floor, Buddha Bhawan, M.G. Road, Secunderabad 500003, Andhra Pradesh, India Accepted author version posted online: 11 Nov 2014.Published online: 13 Dec 2014.

Click for updates To cite this article: Kavita Kumari Kakarala & Kaiser Jamil (2014): Protease activated receptor-2 (PAR2): possible target of phytochemicals, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2014.986197 To link to this article: http://dx.doi.org/10.1080/07391102.2014.986197

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Journal of Biomolecular Structure and Dynamics, 2014 http://dx.doi.org/10.1080/07391102.2014.986197

Protease activated receptor-2 (PAR2): possible target of phytochemicals Kavita Kumari Kakarala* and Kaiser Jamil Centre for Biotechnology and Bioinformatics (CBB), School of Life Sciences, Jawaharlal Nehru Institute of Advanced Studies (JNIAS), 6th Floor, Buddha Bhawan, M.G. Road, Secunderabad 500003, Andhra Pradesh, India Communicated by Ramaswamy H. Sarma

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(Received 18 August 2014; accepted 6 November 2014) The use of phytochemicals either singly or in combination with other anticancer drugs comes with an advantage of less toxicity and minimal side effects. Signaling pathways play central role in cell cycle, cell growth, metabolism, etc. Thus, the identification of phytochemicals with promising antagonistic effect on the receptor/s playing key role in single transduction may have better therapeutic application. With this background, phytochemicals were screened against proteaseactivated receptor 2 (PAR2). PAR2 belongs to the superfamily of GPCRs and is an important target for breast cancer. Using in silico methods, this study was able to identify the phytochemicals with promising binding affinity suggesting their therapeutic potential in the treatment of breast cancer. The findings from this study acquires importance as the information on the possible agonists and antagonists of PAR2 is limited due its unique mechanism of activation. Keywords: Protease activated receptors; breast cancer; signal transduction; docking; phytochemicals

Introduction Due to the alarming increase in the number of breast cancer cases, combined with drug resistance and heterogeneity in the subtypes of breast cancer, targeted therapy is not always useful in the treatment of breast cancer. Therefore, the search for novel targets and their validation is an important area, which is of great interest to clinicians. In this context, interesting experimental results have established the correlation between altered expression of protease-activated receptor 2 (PAR2) and breast cancer cell proliferation and metastasis (Hjortoe et al., 2004; Jaber et al., 2013; Matej, Mandáková, Netíková, Poucková, & Olejár, 2007; Morris et al., 2006; Parisis, Metodieva, & Metodiev, 2013; Su et al., 2009). It has also been shown that PAR2 peptide agonists facilitate breast cancer cell chemokinesis through the G (alpha)-cSrc-JNK-paxillin signaling pathway (Su et al., 2009). The various events that could be triggered by PARs were explained in detail in the recent review of Gieseler, Ungefroren, Settmacher, Hollenberg, and Kaufmann (2013); PAR signaling leads to activation of NF-κB, protein kinase C, phosphatidylinositol 3-kinase pathways, MAPK signaling, etc. These signaling events result in the fast cellular release of agonists like prostaglandins or EGF-receptor (EGFR) ligands which trigger other receptors by an autocrine or paracrine mechanism, signaling an intracellular kinase pathway (e.g. Src-family tyrosine kinase) resulting in the transactivation of ion channels or toll-like receptors (Gieseler et al., 2013). Thus, PAR2 *Corresponding author. Email: [email protected] © 2014 Taylor & Francis

appears to be a master regulator, controlling various signaling events. However, drug development targeting of this receptor has not gained much attention, as PAR2 activation has not been understood completely (Bohm et al., 1996; Coughlin & Camerer, 2003; Gieseler et al., 2013; MacFarlane, Seatter, Kanke, Hunter, & Plevin, 2001). Further, the structure and ligand-based drug design efforts have not progressed as the crystal structure of PAR2 is not yet available and due to the absence of information on the possible endogenous ligands. But, studies with proteolytic enzyme trypsin (natural agonist) (Matej et al., 2007), synthetic peptides (Su et al., 2009), peptide-mimetic antagonist K-12940 and K-14585 (Kanke et al., 2009) have been useful to understand PAR2 function to some extent. However, due to poor bioavailability and rapid degradation, these peptides were not useful for in vivo studies. Recently, studies using intracellular Ca2+ release as an indication for PAR2 activation and vice versa, using synthetic agonist GB110 and antagonist GB88, have been reported (Barry, Le, & Fairlie, 2006; Barry et al., 2010; Suen et al., 2012). Beyond this point, not much progress is recorded in identification of agonist/antagonist. Thus, research leading to the identification of compounds with inhibitory effect on PAR2 signaling will have therapeutic application in breast cancer treatment. Currently, breast cancer cure is focused on phytochemicals, as they are pharmacologically safe, moreover

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K.K. Kakarala and K. Jamil

the importance of phytochemicals-based therapy is strengthened by the fact that most of the successful anticancer drugs are derived from natural products (Schmidt, Ribnicky, Lipsky, & Raskin, 2007). Further, what is more interesting is that most of the phytochemicals are known to interfere with signaling mechanism, thereby arresting the tumor growth; curcumin, black raspberry extract, neem leaf extract etc. have been proved to regulate a number of potential molecular targets like NFκB, p53, Akt, Nos3, Erk1/2, SOD2, p50, p65, TNFα, IAP1, IAP2, and Survivin (Aravindan, Natarajan, Herman, Awasthi, & Aravindan, 2013). Most of the other phytochemicals were also reported to exhibit anticancer properties by modulating signal transduction pathways (Khag, Ansari, & Khan, 2013; Vadodkar, Suman, Lakshmanaswamy, & Damodaran, 2012; Venugopal & Liu, 2012). PAR2 expression has been established to regulate signal transduction pathways apart from cross talking with other membrane receptors. The binding affinity of these phytochemicals was studied using flexible docking methodology (IFD) followed by post-docking analysis with Prime/MMGBSA, to estimate free energy of ligand binding, thereby interpreting the binding affinity of these ligands much more accurately. Qikprop study was undertaken to analyse the drug-like property of phytochemicals and structural interaction fingerprint (SIFt) analysis to identify the important residues in the binding site of PAR2 homology model. The docking result of the PAR2-EGCG complex, which showed highest binding affinity in our study, was verified using molecular dynamics studies of PAR2-EGCG complex for 20 ns. Therefore, this study and its results are a step ahead toward the design of pharmacologically safe drugs toward this promising receptor. Materials and methods The phytochemicals with proven antibacterial, antifungal, antiprotozoal, and antiviral activities were selected from the database of Potential “Antibiotics” from flowering plants developed (maintained by Bioinformatics Infrastructure facility Center by Department of Biotechnology P. G. & Research Department of Microbiology and Biotechnology Presidency College (Aut.) Chennai, India. (http://www.bifcpresidency.tn.gov.in/potential%20antibiot ics.html) (Table 1). The 3D structure of phytochemicals, which were screened, were downloaded from PubChem (http://www.ncbi.nlm.nih.gov/pccompound) (Figure 1(a) and (b)). Protein preparation PAR2 homology model was obtained using methodology published by our group recently (Kakarala, Jamil, & Devaraji, 2014). The homology model of PAR2 was pre-

pared using protein preparation wizard of Schrödinger (Schrödinger Suite 2012 Protein Preparation Wizard; Epik version 2.3, Schrödinger, LLC, New York, NY, 2012; Impact version 5.8, Schrödinger, LLC, New York, NY, 2012; Prime version 3.1, Schrödinger, LLC, New York, NY, 2012). The protein preparation wizard prepares the structures by adding missing hydrogen atoms and correcting bond order assignments, charge states, and orientation of various groups. This process includes three main steps: first one includes preprocessing where the structure is assigned bond orders, by adding hydrogens and the creation of disulfide bonds. The next step is the optimization of hydrogen bonding network by reorientation of hydroxyl groups, water molecules, and amide groups of asparagine and glutamine, predicting protonation states of histidine (His), aspartic acid (Asp), glutamic acid (Glu), and tautomeric states of histidine (His). This process improves charge–charge interactions with neighboring groups. Finally, the protein was minimized using OPLS_2005 Force field. Ligand preparation The structures of phytochemicals (ligands) (Table 1, Figure 1(a) and (b)) were prepared with LigPrep (LigPrep, version 2.5, Schrödinger, LLC, New York, NY, 2012). This application consists of a series of steps that perform conversions from 2D into 3D, apply corrections to the structure, generate ionization states at biological pH and possible tautomers, optimize the geometries followed by minimization of the ring conformations. Prepared ligands were used for further docking analysis. Induced fit docking Induced fit docking method is based on a flexible docking protocol, which accommodates changes in conformation of the receptor and accounts for small backbone relaxations as well as side-chain conformational changes (Farid, Day, Friesner, & Pearlstein, 2006; Sherman, Beard, & Farid, 2006; Sherman, Day, Jacobson, Friesner, & Farid, 2006). It was implemented using Schrödinger Suite 2013 (Schrödinger Suite 2013-2 Induced Fit Docking protocol; Glide version 6.0, Schrödinger, LLC, New York, NY, 2013; Prime version 3.3, Schrödinger, LLC, New York, NY, 2013). Induced fit docking combines rigid receptor docking and protein structure refinement using Prime (Jacobson et al., 2002, 2004). The residues chosen for a receptor grid generation were Tyr 310 (6.59), Tyr 344 (7.53), Phe 244 (5.39), His 227 (ECL2), and Asp 228 (ECL2) which are the corresponding activesite residues identified in PAR1, which was used as a template in our study (Zhang et al., 2012). Briefly, it involves following steps. Step1: initial Glide docking parameters were set by fixing the receptor van der Waals

PAR2: possible target of phytochemicals

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Table 1.

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Description of phytochemicals used for induced fit docking studies against PAR2 (http://www.bifcpresidency.tn.gov.in/).

S. No.

Name of the phytochemical

1. 2.

-(-) Epigallocatechin3-gallate Mangiferin

3. 4.

Oleuropein Xanthohumol

5.

Curcumin

6.

Glabridin

7.

Andrographolide

8.

Octadecanoic acid

9.

Capsaicin

10.

Pinoresinol

11.

Scopoletin

12.

Totarol

13.

Piperine

14.

Sesquiterpenes

15. 16.

Thymol Quinidine

17.

Justicidin A

18.

Beta-bisabolene

19.

Trans-ferulic acid

20.

Nitidine

21.

Berberine

Description -(-) Epigallocatechin-3-gallate (EGCG) is an antioxidant flavonoid. EGCG is present in green tea, black tea, Oolong tea, dark chocolate, and cocoa Present in high concentrations in young leaves (172 g/kg), bark (107 g/kg), old leaves (94 g/kg) of Mangifera indica, and (Mango tree) Present in the leaf extract of Syringa oblata Lindl var.alba (chao yang ding xiang) A prenylflavonoid derived from the female flowers of the hops plant Humulus lupulus (common hop or hop) It is obtained from Curcuma longa (Turmeric plant) which is the source of the spice turmeric, ground turmeric, curcumin powder Glabridin is a polyphenolic flavonoid and a main constituent in the hydrophobic fraction of licorice extract root of Glycyrrhiza glabra (licorice) Andrographolide is a diterpenoid, and is very bitter compound. It is present in the leaves of bark Andrographis paniculata Nees Kalmegh (Hindi), Chuanxinlian (Chinese) Octadecanoic acid is a long-chain fatty acid consisting of 18 carbon atoms without double bonds. It is obtained from natural animal and vegetable fats, mainly for the production of stearate The chemical compound capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the active component of chili peppers, which are plants belonging to the genus Capsicum. Capsaicin is a pungent element in a variety of red peppers that are widely used as food additives and considered to be an antimicrobial factor Pinoresinol is a lignan, obtained from woody or fibrous plants Forsythia suspense (weeping forsythia), Sesamum indicum seeds Scopoletin is present in highest levels in the leaves of Huosang (164.24 μg/g) while it is lowest in the leaves of Dahongpi (25.1 μg/g) Totarol is present in high concentrations (approximately 5% by mass on a dry basis) in the heartwood of Totara Podocarpus Totara (Pouakani Tree) and in much lower concentrations (less than 1% by mass) in other Podocarpus, Dacrycarpus, Cupressus and Juniperus species It is obtained from the Fruit of Piper Nigrum (black pepper) or Piper longum (long pepper). Piperine has been used in some forms of traditional medicine and as an insecticide. Piperine is soluble in water, alcohol, ether or chloroform It is obtained from Artemisia tridentata (Big Sagebrush) containing sesquiterpene lactones which are sesquiterpenoids (built from three isoprene units) and contain a lactone ring, hence the name It is obtained from oil of Thymus vulgaris also known as 2-isopropyl-5-methylphenol Quinidine is a pharmaceutical agent that acts as a class I antiarrhythmic agent (Ia) in the heart. It is a stereoisomer of quinine, originally derived from the bark of the cinchona tree Justicidin A is an arylnaphthalide lignin. It is obtained from plants like Justicia hayatai var.decumbens, Justicia procumbens Linn.var.leucantha Honda, Justicia procumbens Linn. Haplophyllum cappadocicum Spach. Haplophyllum tuberculatum (Forsk.) Beta-bisabolenes are a group of closely related natural chemical compounds which are classified as sesquiterpenes. Bisabolenes are present in the essential oils of a wide variety of plants including cubeb, lemon, and oregano It is present in the seeds of plants such as rice, wheat, oats, as well as in coffee, apple, artichoke, peanut, orange, and pineapple. Trans-ferulic acid is a hydroxycinnamic acid, a type of organic compound. It is an abundant phenolic phytochemical found in plant cell wall components such as arabinoxylans as covalent side chains, belong to the family of hydroxycinnamic acid Nitidine is found in the roots of Toddalia asiatica (Toddalia) and have been isolated from folliage and wood Berberine, a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids It is obtained from Plant Berberis vulgaris (Barberry), Berberis aquifolium (Oregon grape), Berberis aristata (Tree Turmeric)), Hydrastis canadensis (Goldenseal), and Phellodendron amurense

scaling and ligand van der Waals radii to 0.5 along with the option to remove side chains of the residue during initial docking. The maximum number of poses to be retained after initial standard precision docking in Glide (Friesner et al., 2004; Halgren et al., 2004) was limited to 20. Step 2: after this initial docking phase, Prime 3.3 was used to refine the side chains for the residues within

5 Å of ligand. The refined complexes were ranked according to Prime calculated energy (molecular mechanics and solvation), and those within 30 kcal/mol of the minimum energy structure were used in the last step of the process i.e. redocking Glide XP method. Step 3: from the filtered poses, the ligands were redocked using extra precision docking (XP) in Glide (Friesner et al.,

K.K. Kakarala and K. Jamil

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Figure 1. (a) and (b) 2D structures of phytochemicals. The 2D structures of the phytochemicals (Table 1) were obtained from Pubchem.

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PAR2: possible target of phytochemicals 2006) (Schrödinger Suite 2013-2 Induced Fit Docking protocol; Glide version 6.0, Schrödinger, LLC, New York, NY, 2013; Prime version 3.3, Schrödinger, LLC, New York, NY, 2013). The best poses were carefully observed and selected based on the position and orientation of functional group within the binding cavity, the Glide scores [Glide score (G-score) in kcal/mol is calculated as follows: G-Score = H bond + Lipo + Metal + Site + .130 Coul + .065 vdW − Bury P − RotB.where Hbond = Hydrogen bonds, Lipo = hydrophobic interactions, Metal = metal binding term, Site = polar interactions in the binding site, vdW = van der Waals forces, Coul = coulombic forces, Bury P = penalty for the buried polar group, RotB = freezing rotatable bonds] and the relative energy of interaction calculated by IFD score (IFD score = GlideScore + .05 × Prime eEnergy) (Schrödinger Suite 2013-2 Induced Fit Docking protocol; Glide version 6.0, Schrödinger, LLC, New York, NY, 2013; Prime version 3.3, Schrödinger, LLC, New York, NY, 2013). Prime/MM-GBSA binding-free energy calculation Post-docking analysis done using Prime/MM-GBSA method was used to predict the free energy of ligand binding of the receptor–ligand complex. Prime MMGBSA (Lyne, Lamb, & Saeh, 2006) combines OPLS molecular mechanics energies, an SGB solvation model for polar solvation, and a nonpolar solvation term composed of the nonpolar solvent accessible surface area and van der Waals interactions. The total free energy of binding was then expressed as: ΔG bind = G complex − (G protein + G ligand), where G = MME (molecular mechanics energies) + GSGB (SGB solvation model for polar solvation) + GNP (nonpolar solvation) (Prime, version 3.3, Schrödinger, LLC, New York, NY, 2013). Qikprop properties QikProp was used to calculate absorption, distribution, metabolism, and excretion properties of the phytochemicals used for present study. The program analyzes druglike properties of the ligands. A total of 44 properties could be predicted including physical descriptors and physicochemical properties (Jorgensen & Duffy, 2002, Biologics Suite 2013-3: BioLuminate, version 1.3, Schrödinger, LLC, New York, NY, 2013). SIFt studies were performed for analyzing PAR2ligand docked complexes. This study is useful in the identification of important residues in the active site of PAR2 model. The residues involved in hydrogen bond donor, hydrogen bond acceptor, and hydrophobic interactions were analyzed (Deng, Chuaqui, & Singh, 2004).

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Molecular dynamics simulation PAR2– Epigallocatechin-3-gallate (EGCG) The protein–EGCG complex was prepared using the system builder tool in Maestro 9.5 (Desmond Molecular Dynamics System, version 3.1, D.E. Shaw Research, New York, NY, 2012, Maestro-Desmond Interoperability Tools, version 3.1, Schrödinger, New York, NY, 2012), compiled for Maestro with the OPLS-AA 2005 force field (Bowers et al., 2006; Shivakumar et al., 2010). The system was prepared by embedding PAR2-EGCG structure in POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) lipid bilayer, solvating the membrane by TIP4P explicit water. The system was neutralized by the addition of .15 mol/L sodium and chloride ions with 10 Å distance of buffer on all directions. Equilibration was performed at constant pressure and temperature (NPT ensemble; 300 K, 1.01325 bar) using NOSEHoover temperature coupling and isotropic scaling for 20 ns. Trajectories after every 4.8 ps were recorded. Variations in the energy and root mean square deviation (RMSD) of the complex in each trajectory were analyzed with respect to simulation time (Desmond Molecular Dynamics System, version 3.1, D. E. Shaw Research, New York, NY, 2012, Maestro-Desmond Interoperability Tools, version 3.1, Schrödinger, New York, NY, 2012). Energy variation, RMSD and root mean square fluctuations (RMSF) of PAR2–EGCG complex were analyzed with respect to simulation time. The inter-molecular interactions of PAR2–EGCG complex were assessed for stability of the docking complex.

Number system of amino acids The numbers within brackets in the following sections indicate the Ballesteros–Weinstein numbering (Ballesteros, Weinstein, & Stuart, 1995) and the numbering of residues corresponds to the number assigned by Schrödinger software as described in our recent publication (Kakarala et al., 2014).

Results In this study, we screened some of the phytochemicals to be able to draw some conclusion on binding affinity of these compounds with PAR2. Phytochemicals were chosen as they are known to protect against cancer by interfering with the signalling cascade leading to proliferation and metastasis (Chen, 2014; Lee, Bode, & Dong, 2011; Singh & Shankar, 2011). The binding affinity of these phytochemicals was studied using induced fit docking method (Section 2). The binding affinity was evaluated based on the Glide scores, IFD scores, and values of free energy of ligand binding.

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K.K. Kakarala and K. Jamil

Of the various phytochemicals screened (Table 1, Figure 1(a) and (b)), Epigallocatechin-3-gallate (EGCG), mangiferin, oleuropein, xanthohumol, curcumin, and andrographolide showed promising Glide scores (−19.457012, −18.01732, −15.855193, −13.2726, −12.857, −11.727302) and free energy of ligand binding assessed by Prime/MMGBSA method (−128.894, −107.393, −109.052, −113.294, −103.958, −112.826, −111.249) (Table 2). Interestingly, glabridin showed a promising Glide score (−11.824998) but relatively less binding energy (−98.442). The other phytochemicals totarol, piperine, sesquiterpines, and quinidine showed relatively lower Glide scores (−9.867857, −9.592357, −9.52168, −8.514775) but high binding affinity (−112.826, −105.165, −105.405, and −105.941) evaluated by Prime/MMGBSA (Table 2). Curcumin showed hydrogen bonding interaction with residues Y82 (1.39), S337 (7.46) and M159 (3.36) and π bond interaction with Y82 (1.39) and F155 (3.32) respectively (Figure 2(a) and (b)). Interestingly, binding pose of berberine, when analyzed, showed no interacting residue of PAR2, but Prime/MMGBSA values which measure the free energy of ligand binding indicates that its position may be stabilized by hydrophobic interactions of Y82 (1.39), V85 (1.42), Y156 (3.33) and Y326 (7.35), respectively (Figure 3(a) and (b)). The phytochemicals pinoresinol, thymol, scolpoletin, trans-Ferulic acid, justicidin_B, and beta-bisabolene may not be good antagonists inspite of having good docking scores (−10.225623, −9.090869, Table 2.

−8.003067, −8.87476, −8.87476) as their free energy of ligand binding was low as calculated by Prime/ MMGBSA (−89.296, −70.262, −55.418, −49.571, −75.548, −80.973). However, nitidine and berberine showed high binding energies (−97.441, −118.121) inspite of showing low docking scores (−7.943283, −6.79681) (Table 2). Qikprop analysis The phytochemicals used in our study were evaluated for their drug like property using Qikprop (Table 3). Epigallocatechin-3-gallate, mangiferin showed two violations of Lipinski’s rule of five and Jorgensen rule of three (Jorgensen & Duffy, 2002). Oleuropein showed violation of 3 to Lipinski’s rule of five and 2 violations of Jorgensen’s rule of three. Totarol, beta-bisabolene showed one violation of rule of five. Sesquiterpenes and octadecanoic acid, showed one violation of Lipinski’s rule of five (Ro5)and Jorgensen rule of three (Ro3), whereas thymol, scopoletin, quinidine, trans-Ferulic acid, justicidin_B, capscicin, glabardin, curcumin, andrographolide, piperine, transferulic acid showed a zero violation of Lipinski’s rule and was predicted to have high percentage of human absorption by Qikprop analysis (Section 2). SIFt (Deng et al., 2004) was used for analyzing protein ligand binding interactions of phytochemicals and PAR2 complex (Section 2). On the basis of this interaction study, it was observed that the residues Y82 (1.39),

Glide score, induced fit docking score and MMDBSA values of phytochemicals screened.

S. No.

Pubchem ID and entry name

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15 16. 17. 18. 19. 20. 21

CID_65064_Epigallocatechin-3-gallate (EGCG) CID_12306736_Mangiferin CID_5281544_Oleuropein CID_639665_Xanthohumol CID_2889-curcumin CID_124052_Glabridin CID_5318517_Andrographolide CID_445639_octadecanoic acid CID_1548943_Capsaicin CID_17750970_Pinoresinol CID_5280460_Scopoletin CID_92783_Totarol CID_638024_Piperine Sesquiterpenes CID_6989_Thymol CID_441074_Quinidine CID 442882 JUSTICIDIN_B CID_10104370_Beta-bisabolene CID_445858_trans-ferulic acid CID_4501_Nitidine CID_2353_Berberine

Glide G-score

IFD score

MMGBSA dG_bind (kcal/mol)

−19.457 −18.017 −15.855 −13.273 −12.857 −11.825 −11.727 −10.878 −10.400 −10.225623 −8.402892 −9.867857 −9.592357 −9.52168 −9.090869 −8.514775 −8.87476 −8.87476 −8.003067 −7.943283 −6.79681

−567.0666 −560.6741 −560.5267 −560,878 −558.863 −557.728 −55,688 −560.7934 −560.260 −553.2016 −553.5559 −557.6899 −554.7683 −554.8394 −553.5147 −553.5261 −546.0013 −556.3576 −551.6231 −549.8292 −550.8384

−128.894 −107.393 −109.052 −113.294 −112.826, −98.442 −111.249 −103.958 −117.127 −89.296 −55.418 −112.826 −105.165 −105.405, −70.262 −105.941 −75.548 −80.973 −49.571 −97.441 −118.121

Notes: The binding affinity of phytochemicals was studied using induced fit docking module of Schrodinger 2013 (a flexible docking program) and the free energy of binding was assessed using PRIME/MMGBSA method. The receptor ligand free energy calculated by this method was reported to correlate well with experimental binding affinity. The rows shown in bold are phytochemicals with ≥2 violations of Lipinski’s rule Phytochemicals showing ≤2 violations are shown in italics The phytochemicals which showed no violations of Lipinski’s rule are shown with normal font.

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PAR2: possible target of phytochemicals

Figure 2. (a) Binding pose of PAR2 complexed with curcumin. Hydrogen bond interaction between Y82 (1.39), S337 (7.46), and M159 (3.36) is shown. The red dashed lines indicates hydrogen bonds. The important residues involved in binding are displayed in ball and stick format. (b) Lig interaction plot. Ligand interaction plot shows hydrogen bonding interaction between Y82 (1.39), S337 (7.46), and M159 (3.36), π–π interaction with Y 82 (1.39), F 155 (3.32), and Cucurmin is depicted. Hydrogen bonding interactions are depicted with pink arrows and π–π interactions are depicted with a green line.

S124 (2.53), K131 (2.60), N158 (3.35), S162 (3.39), and N303 (6.62) may act as hydrogen bond donor residues. Further, the phytochemicals, scopoletin, piperine, and xanthohumol showed maximum interactions with these

residues (Figure 4). Among these residues, Y82 (1.39), K131 (2.60), and N303 (6.521) showed maximum interactions with these ligands. SIFt analysis could not detect any consensus in hydrogen bond acceptor residue

K.K. Kakarala and K. Jamil

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Figure 3. (a) Binding pose of PAR2 complexed with berberine. The binding pose of berberine showed no hyrogen bonding but was stabilized possibly through hydrophobic interaction with residues Y82 (1.39), V85 (1.42), Y326 (7.35), and Y156 (3.33) of PAR2 model. (b) Ligand interaction plot shows that berberine is positioned between residues Y326 (7.35), Y156 (3.33), Y82 (1.39), and V85 (1.42) may stabilize the ligand through hydrophobic interaction.

interactions; however, residues M159 (3.36) and H227 (ECL2) were observed in such interactions (Figure 5). The residues L78 (1.35), Y82 (1.39), V85 (1.42), V125 (2.54), F128 (2.57), L151 (3.28), I152 (3.29), F155 (3.32), Y156 (3.33), M159 (3.36), Y160 (3.37),

I163 (3.40), L164 (3.41), M166 (3.43), V206 (4.56), L210 (4.60), L235 (ECL2), L236 (ECL2), F244 (5.39), L247 (5.42), V251 (5.46), F252 (5.47), P255 (5.50), A256 (5.51), L292 (6.41), M294 (6.43), Y295 (6.44), F299 (6.48), L306 (6.55), Y323 (7.32), Y326 (7.35), and

PAR2: possible target of phytochemicals Table 3. S. No.

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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Qikprop properties of the phytochemicals screened. QPlog Po/we

QPlog HERGf

QPP Cacog

QPlog Khsah

% HABi

8.75

−.24

−5.72

1.018

−.44

0

2

2

7 6 2 2 2 3 1 2 2 1 1 0 0 1 1 0 0

13 17.4 4 7 3 8.1 2 4 6.4 4 .75 4.5 0 .75 5.45 6.0 0

−1.68 −.49 4.085 2.997 4.021 1.856 5.900 3.673 3.108 .952 5.137 3.176 5.142 3.299 3.438 2.844 6.189

−4.85 −6.15 −6.09 −6.31 −5.66 −4.38 −3.612 −4.31 −5.14 −3.75 −3.71 −4.582 −2.98 −3.65 −5.308 −5.217 −4.07

4.93 6.026 294.8 326.5 1157 252.3 250.527 914.2 2055 927.3 4999 3597 9906 3695 634.947 3190.361 9906

−.9 −1 .587 −.03 .707 −.09 .758 .18 .175 −.5 1.137 −.040 .912 .057 .269 −.76 1.015

3.576 0 95.06 89.49 100 80.8 91.468 100 100 85.63 100 100 100 100 100 100 100

2 3 0 0 0 0 1 0 0 0 1 0 1 0 0 0 1

2 2 1 0 0 0 1 0 1 0 0 0 1 0 0 0 2

2

3.5

1.372

−2.239

63.656

−.611

67.255

0

0

Pubchem ID and entry namea

MWb

HBcD HBdA

CID_65064 Epigallocatechin-3gallate CID_12306736 Mangiferin CID_5281544_Oleuropein CID_639665 Xanthohumol CID_2889Curcumin CID_124052 Glabridin CID_5318517_Andrographolide CID_445639 Octadecanoic acid CID_1548943 Capsaicin CID_17750970 Pinoresinol CID_5280460 Scopoletin CID_92783 Totarol CID_638024 Piperine Sesquiterpenes CID_6989 Thymol. CID_441074 Quinidine CID 442882 Justicidin_B CID_10104370_Betabisabolene CID_445858 Trans-ferulic acid

458.4

8

422.3 540.5 354.4 368.4 324.4 350.5 282.465 305.4 358.4 192.2 286.5 285.342 204.4 150.2 324.422 364.354 204.4 194.187

RO5j RO3k

a

Pubchem ID and compound name. Molecular weight (

Protease activated receptor-2 (PAR2): possible target of phytochemicals.

The use of phytochemicals either singly or in combination with other anticancer drugs comes with an advantage of less toxicity and minimal side effect...
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