Molecular docking studies of quercetin and its analogues against human inducible nitric oxide synthase (1).pdf
RESEARCH Open Access Molecular docking studies of quercetin and its analogues against human inducible nitric oxide synthase Salam Pradeep Singh* and Bolin Kumar Konwar Abstract Nitric oxide synthases (NOS) catalyze to produce nitric oxide (NO) from L-arginine. The isoform of NOS i.e. inducible nitric oxide synthases (iNOS) expression is observed in various human malignant tumors such as breast, lung, prostate and bladder, colorectal cancer, and malignant melanoma. Also an increased level of iNOS expression and activity has been found in the tumor cells of gynecological malignancies, stroma of breast cancer and tumor cells of head and neck cancer. Because of its importance in causing tumors and cancer, iNOS enzyme has become a new target in finding novel inhibitors as anti cancer agents. The present work focuses on the molecular docking analysis of quercetin and its analogues against iNOS enzyme. Earlier there are reports of quercetin inhibiting iNOS enzyme in certain experiments as anti cancer agent. But the clinical use of quercetin is limited by its low oral bioavailability and therefore needed its molecular modification to improve its pharmacological properties. In the present study ten analogues of quercetin were found to be docked at the active site cavity with favorable ligand-protein molecular interaction and interestingly from the ADME-Toxicity analysis these analogues have enhanced pharmacological properties than quercetin. Keywords: Inducible iNOS, Molecular docking, Molecular modification, Cancer, Analogues Background Nitric oxide synthases (NOS) (EC 1.14.13.39) are a family of enzymes that catalyze producing nitric oxide (NO) from L-arginine. It is an important cellular signaling molecule, having a role in various cellular processes. The free radical (NO) is an important effector molecule in the nervous, immune and cardio vascular systems (Garthwaite and Boulton 1995; MacMicking et al. 1997; Michel and Feron, 1997). Mammals contain three isoforms of NOS that pro- duce NO and citrulline by catalyzing NADPH and O2 dependent oxidation of L-arginine (Griffith and Stuehr 1995; Marletta et al. 1997). Two isoforms of NOS are expressed in cells such as neurons (nNOS) and endothe- lium (eNOS) which are activated by Ca+2 dependent cal- modulin (CaM) binding and the third inducible isoform (iNOS) is induced by cytokines which binds CaM inde- pendently (Ghosh et al., 1999). The iNOS isoform is a homodimer (Michal 1999) and the iNOS gene is located on chromosome 17 (Xu et al. 1994). iNOS exerts its func- tions independent of Ca+2 while calmodulin remains non- covalently bound to the iNOS complex and forms an essential subunit of the isoform (Knowles and Moncada 1994, Cho et al. 1992). Regulating NO production via iNOS necessarily occurs during transcription and transla- tion, for once active, iNOS synthesizes large amounts of NO until substrate depletion (Hickey et al. 2001). The role of NO affects the expression and activity of oncogenes, which are vital to the cell cycle and apoptosis (Forrester et al. 1996; Messmer et al. 1994; Sandau et al. 1997). For- rester et al. observed an up regulation of the tumor sup- pressor gene p53 after the exposure of cells to NO donors which might be a reaction due to NO mediated DNA damage (Forrester et al. 1996). Also, the p53 gene is an important inhibitor for iNOS expression as it regulates NO production by a negative feedback loop mechanism. The non-mutant p53 protein (wild-type form) binds to a site on the iNOS gene, preventing its transcription (Brennan and Moncada 2002). Thus, suggesting the wild- type p53 is vital for the control of NO mediated * Correspondence: salampradeep@gmail.com Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India a SpringerOpen Journal © 2012 Singh and Konwar; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Singh and Konwar SpringerPlus 2012, 1:69 http://www.springerplus.com/content/1/1/69 genotoxicity (Forrester et al., 1996). Certain experiments with mutant p53 animal tumors have found out there is an increase in NOS activity in such cancers which grew faster with greater angiogenic potential. Thus, promoting cancer progression by providing a selective growth advan- tage to tumor cells (Ambs et al. 1998a). NO could also be shown to activate p53 resulting in anti-carcinogenic effects, mutagenic and increase cancer risk (Goodman et al. 2004, Rao 2004). The multifactorial process involved in carcinogenesis requires mutations in somatic cells and subsequent alterations of morphology and growth pattern, eventually resulting in transformation, local invasion, and metastasis (Lirk et al. 2002). The expression of iNOS can be observed in a various human malignant tumors such as breast (Vakkala et al. 2000), lung (Marrogi et al. 2000), prostate (Aaltoma et al. 2001; Aaltomaa et al. 2000; Uotila et al., 2001) and bladder (Swana et al. 1999; Hayashi et al. 2001), colorectal cancer (Kojima et al. 1999), and malig- nant melanoma (Massi et al. 2001). However, there are many conflicting reports that increased levels of iNOS are not a ubiquitous finding in human cancer and its expres- sion depends on the histological type or grade of the tumor and the tumor stage (Crowell et al. 2003; Kinaci et al. 2012). Various studies have also found out the expression and the activity of iNOS in human cancer (Weiming et al. 2002; James et al. 2003). An increased level of iNOS expression and activity has been found in the tumor cells of gynecological malignancies, (Thomsen et al., 1994) in the stroma of breast cancer, (Thomsen et al. 1995) and in the tumor cells of head and neck cancer (Gallo et al. 1998; Franchi et al. 2002). Several studies have reported an increase of iNOS expression in tumor tissue when compared with normal mucosa (Ambs et al. 1998b; Ambs et al. 1999; Ropponen et al. 2000; Yagihashi et al. 2000; Kojima et al. 1999; Hao et al. 2001). The present work aims on molecular docking analysis of iNOS enzyme against a class of flavonoid (quercetin and its analogues) which is present in fruits, vegetables, leaves and grains and is reported to have effective anti-cancer property. Scientists have long considered quercetin and flavonoids present in fruits, vegetables, leaves and grains important in cancer prevention. There are also reports of lower risk of cancer in people who eat more fruits and vegetables. (Verschoyle et al. 2007; Rietjens et al. 2005; van der Woude et al. 2005; Chen et al. 2001). Interestingly, quercetin inhibiting against iNOS as anti cancer agents has been reported by García-Mediavilla et al. and Raso et al. (García-Mediavilla et al. 2007; Raso et al. 2001). But the clinical use of quercetin is limited by its low oral bio- availability (Peng et al. 2008) and therefore compels its molecular modification to enhance its pharmacological properties. In the present study the best docking hit analo- gues were undergo ADME–Toxicity prediction (absorp- tion, distribution, metabolism, and toxicity) to evaluate its pharmacological properties to be an orally active com- pound. Here in the present work, we are reporting for the first time the analogues of quercetin as iNOS inhibitors with enhanced pharmacological properties. Results and discussion Molecular docking analysis Quercetin (3,3’,4’,5,7-pentahydroxylflavone) is a plant derived flavonoid which is present in the plant kingdom as a secondary metabolite. It is the most well defined group of polyphenolic compounds (Murakami et al., 2008). The flavonoids contain a basic skeleton of Table 1 Docking score of the top docking hits and quercetin SN Ligand MolDocka Rerankb Interactionc Internald HBonde LE1f LE3g 1 5281604 −129.14 −104.75 −148.27 19.14 −11.81 −5.61 −4.55 2 5315126 −122.90 −102.63 −146.11 23.21 −15.38 −4.55 −3.80 3 9818879 −133.99 −95.04 −150.44 16.46 −9.33 −5.58 −3.96 4 5481966 −122.87 −93.67 −141.73 18.86 −2.43 −4.55 −3.47 5 5282154 −116.71 −93.58 −135.98 19.27 −14.02 −4.86 −3.90 6 13964550 −113.94 −93.40 −130.62 16.68 −4.56 −5.18 −4.25 7 5281691 −124.63 −92.63 −144.57 19.94 −7.95 −5.42 −4.03 8 11834044 −116.92 −91.50 −140.67 23.75 −13.46 −5.08 −3.98 9 6477685 −130.50 −91.09 −144.13 13.63 −4.18 −5.67 −3.96 10 Quercetin −77.29 −65.79 −97.17 19.88 −8.42 −3.51 −2.99 a - Moldock score is derived from the PLP scoring functions with a new hydrogen bonding term and new charge schemes. (Thomsen and Christensen 2006). b - The rerank score is a linear combination of E-inter (steric, Van der Waals, hydrogen bonding, electrostatic) between the ligand and the protein, and E-intra. (torsion, sp2-sp2, hydrogen bonding, Van der Waals, electrostatic) of the ligand weighted by pre-defined coefficients. (Thomsen and Christensen 2006). c - The total interaction energy between the pose and the protein (kJ mol−1). d - The internal energy of the pose. e - Hydrogen bonding energy (kJ mol−1). f - Ligand Efficiency 1: MolDock Score divided by Heavy Atoms count. g - Ligand Efficiency 3: Rerank Score divided by Heavy Atoms count. Singh and Konwar SpringerPlus 2012, 1:69 Page 2 of 10 http://www.springerplus.com/content/1/1/69 site was set inside a restriction sphere of radius 15 Å (X 0.28, Y 99.79, Z 8.70) using MVD. The 85 analogues retrieved from the NCBI PubChem database were imported in the Molegro Virtual Docker (MVD). Bond flexibility of the compounds was set along and the side chain flexibility of the protein for the active site residues (Trp372, Glu377, Trp463, Phe476) was set with a tolerance of 1.10 and strength of 0.90 for docking simulations. RMSD threshold for multiple cluster poses was set at 2.00 Å. The docking algorithm was set at a maximum iteration of 1,500 with a simplex evolution size of 50 and a minimum of 10 runs were performed for each compound. The best pose of each compound was selected for the subsequent ligand–protein interaction energy analysis. Molecular docking was carried out using Molegro Virtual Docker. MVD is based on a differential evolution algorithm; the solution of the algorithm considers the sum of the intermolecular interaction energy between the ligand and the protein and the intramolecular interaction energy of the ligand. The docking energy scoring function is based on the modified piecewise linear potential (PLP) with new hydrogen bonding and electrostatic terms included. Full description of the algorithm and its reliabi- lity compared to other common docking algorithm is described by Thomsen et al. (Thomsen and Christensen 2006). ADME-toxicity prediction ADME-Toxicity for the top docking hits and quercetin was predicted using QikProp (Schrödinger 2012). Qik- Prop predicts physically significant descriptors and pharmaceutically relevant properties of organic mole- cules, either individually or in batches. QikProp provides ranges for comparing a particular molecule’s properties with those of 95% of known drugs. In the present study QikProp properties and descriptors such as apparent MDCK cell permeability (QPPMDCK), IC50 value for blockage of HERG K+ (QPlogHERG), Caco-2 cell perme- ability (QPPCaco), Lipinski rule of five (Lipinski et al. 1997; Lipinski 2008), brain/blood partition coefficient (QPlogBB), human oral absorption on 0 to 100% scale (Percent Huma- nOralAbsorption), aqueous solubility (QPlogS) for the top docking hits and quercetin was predicted to obtain the ADME properties of the compounds. Additionally LD50 mouse and probability of health effects predictions for the top docking hits were calculated using ACD/ I-Lab 2.0 (Advanced Chemistry Development, Inc 1994 ) which is a web-based service that provides instant access to spectral and chemical databases, and predicts properties including physicochemical, ADME, toxicity characteristics. Also a comparative analysis were performed for LD50 mouse (intraperitoneal, oral, intravenous, subcuta- neous) and probability of health effect of blood, cardiovas- cular system, gastrointestinal system, kidney, liver and lung for the top docking hits. Abbreviations NO: Nitric oxide; NOS: Nitric oxide synthases; iNOS: Inducible Nitric oxide synthase; eNOS: Endothelium Nitric oxide synthases; (nNOS): Neurons Nitric oxide synthase; CaM: Ca+2 –dependent calmodulin; MVD: Moegro Virtual Docker; ADME–Toxicity: Absorbtion distribution metabolism excretion and toxicity; NCBI: National Centre for Biotechnology Information; ACD: Advance Chemistry Development. Competing interest The authors declare that they have no competing interests. Author’s contribution SPS carried out the molecular docking studies and ADME-toxicity analysis. SPS and BKK conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Figure 6 Comparative analysis on probability of health effect on blood, cardiovascular system, gastrointestinal system, kidney, liver and lung for Compound ID (5281604, 5315126, 9818879, 5481966, 5282154, 13964550, 5281691, 11834044, 6477685 and quercetin). Singh and Konwar SpringerPlus 2012, 1:69 Page 8 of 10 http://www.springerplus.com/content/1/1/69 Acknowledgement SPS thanks Vice-Chancellor, Tezpur University, Assam, India for the necessary support. Received: 16 October 2012 Accepted: 11 December 2012 Published: 17 December 2012 References Aaltoma SH, Lipponen PK, Kosma VM (2001) Inducible nitric oxide synthase (iNOS) expression and its prognostic value in prostate cancer. Anticancer Res 21:3101–3106 Aaltomaa SH, Lipponen PK, Viitanen J, Kankkunen JP, Ala-Opas MY, Kosma VM (2000) The prognostic value of inducible nitric oxide synthase in local prostate cancer. 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Title: Molecular docking studies of quercetin and its analogues against human inducible nitric oxide synthase ​
Authors: Salam Pradeep Singh, Bolin Kumar Konwar
Publisher: SpringerPlus
Date: December 17, 2012 ​
Abstract: Nitric oxide synthases (NOS) catalyze to produce nitric oxide (NO) from L-arginine. ​ The isoform of NOS i.e. ​ inducible nitric oxide synthases (iNOS) expression is observed in various human malignant tumors such as breast, lung, prostate and bladder, colorectal cancer, and malignant melanoma. ​ Also an increased level of iNOS expression and activity has been found in the tumor cells of gynecological malignancies, stroma of breast cancer and tumor cells of head and neck cancer. ​ Because of its importance in causing tumors and cancer, iNOS enzyme has become a new target in finding novel inhibitors as anti cancer agents. ​ The present work focuses on the molecular docking analysis of quercetin and its analogues against iNOS enzyme. ​ Earlier there are reports of quercetin inhibiting iNOS enzyme in certain experiments as anti cancer agent. ​ But the clinical use of quercetin is limited by its low oral bioavailability and therefore needed its molecular modification to improve its pharmacological properties. ​ In the present study ten analogues of quercetin were found to be docked at the active site cavity with favorable ligand-protein molecular interaction and interestingly from the ADME-Toxicity analysis these analogues have enhanced pharmacological properties than quercetin. ​