Phytopharmacology 2011, 1(4) 95-100

Molecular insights probing Bismurrayaquinone A as an angiogenesis inhibitor via inhibition of VEGFR-2 Kinase domain
Hung-Ta Zhong1, Yan-Zhen Yu1,*, Cristina Velasco2
Department of Biology, Zhong Shan University, Guangzhou, China. Laboratorio de Investigaciones Biomdicas, Fundacin IMABIS, Hospital Universitario Virgen de la Victoria, Mlaga, Spain
2 1

*Corresponding Author: Email: zhonght698@ymail.com

Received: 24 June 2011, Revised: I7 July 2011, Accepted: 18 July 2011

Abstract Angiogenesis is a key process in tumor growth and survival. Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) kinase inhibition is one of the well established strategies to promptly tackle tumor growth by suppression of angiogenesis. In the current study an alkaloid Bismurrayaquinone A was isolated from Murraya koenigii. Structure-based virtual screening indicated its strong potential as a VEGFR-2 kinase domain inhibitor, which was validated using its experimental inhibitory effects on VEGFR-2 kinase and Human umbilical vein endothelial cells (HUVECs). Molecular interactions of Bismurrayaquinone A were studied at molecular level using molecular docking studies inside ATP binding pocket of VEGFR-2 kinase, which supported its experimental results Significant inhibitory activity of Bismurrayaquinone A on VEGFR-2 kinase (IC50: 126.020.02 nM) and HUVECs (IC50: 39.14.170.06 nM) showed as new angiogenesis inhibitor of natural origin. Keywords: Angiogenesis, Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2), Human umbilical vein endothelial cells (HUVECs), Docking

Introduction Angiogenesis refers to generation of new blood vessels from existing vasculature. It is the key factor in development and progression of various human diseases, including cancer, where it is necessary for the growth, spread and survival of tumors (Harris et al., 2008). Angiogenesis is involved in metastasis (uncontrolled spread of tumor cells) by supplying oxygen, nutrients, and related growth factors to small tumors and removing the waste products of metabolism. Tumors that lack an adequate vasculature become necrotic or apoptotic leading to their limited growth. Thus, new means to retard angiogenesis have shown promise as potential cancer therapies and inhibition of the vascular endothelial growth

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Figure 1. Bismurrayaquinone A isolated from Murraya koenigii

factor (VEGF) signaling pathway has emerged as one of the most promising new approaches for cancer therapy (Harris et al., 2008, Adham et al., 2010). VEGF is secreted by tumors and induces a mitogenic response through its binding to one of three tyrosine kinase receptors (VEGFR-13) on nearby endothelial cells. Thus inhibition of this signaling pathway should block angiogenesis and subsequent tumor growth. There is much evidence that direct inhibition of the kinase activity of VEGFR-2 will result in the reduction of angiogenesis and the suppression of tumor growth. (Ferrara et al., 2003; Prewett et al., 1999; Chekler et al., 2008). A number of small molecule structural classes have been reported as potent inhibitors of VEGFR-2 in vitro or have demonstrated antiangiogenic. In order to discover new smallmolecule inhibitors of VEGFR-2, we have designed a study to explore an alkaloid Bismurrayaquinone A isolated from a sponge Hyrtios erectus guided by computer-aided virtual screening. Materials and Methods Extraction and isolation Fresh aerial parts of the plant (Murraya koenigii) were collected locally and was identified and deposited in herbarium at Department of Biology, Zhong Shan University, Guangzhou, China. Ariel parts of the plant were dried and grinded that finally yielded in 5.9 kg powder. Powdered material was extracted with methanol that provided crude methanolic extract (283 g). Various subfractions were obtained via liquid-liquid extraction of crude extract with organic solvents viz. n-hexane, dichloromethane, ethyl acetate and distilled water. Ethyl acetate fraction (53 g) was load for column chromatography (silica gel, Merck) and eluted with increasing polarity from 100 % n-hexane to 100 % Chloroform gradient. Furthermore polarity was increased with methanol-gradient, which exhausted the column using 17 % methanol-chloroform as mobile phase. Sub fraction MK-C-13 was further purified using 6 % methanol-chloroform gradient that afforded a compound (14 mg). Spectral data of the compound was matched with data cited in literature that lead to identification as Bismurrayaquinone A (Ito et al., 1993). VEGFR-2 kinase inhibition Recombinant VEGFR kinase protein was purchased from Upstate Biotechnology (Lake Placid, USA). The enzyme selectivity screen was performed with a tyrosine kinase assay kit (Invitrogen, PanVera Co., USA) based on fluorescence polarization detection. 96

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Reactions were performed in 96-well polystyrene round-bottomed plates in a final volume of 100 mL. Reaction mixtures contained 20 mM HEPES (pH 7.4), 5 mMMgCl2, 2 mM MnCl2, 50 mM Na3VO4, 200 ng/mL enzyme, 20 mM ATP, and 1 ng/mL poly(Glu,Tyr) 4:1. One hundred compounds at concentrations of 10 and 1 mM were tested. After 1 h of incubation, the reactions were terminated by adding a 6 mM EDTA solution; anti-phosphotyrosine antibody, PTK green tracer, and FP dilution buffer mixtures were then added. The fluorescence polarization values were measured after 30 min at room temperature, using a Victor D fluorescence reader (BioTek, USA). Kinase inhibition analysis was performed by using Prism 5.0 (GraphPad Software Inc., USA). VEGF-stimulated proliferation of HUVECs Bismurrayaquinone A was evaluated for its inhibitory effect on cell proliferation. Cell proliferation was measured using 5-bromo-2-deoxyuridine (BrdU) incorporation method using commercially available kits (Roche Diagnostics, USA). Briefly, HUVEC were seeded in a medium containing 5% FBS in type 1 Collagen-coated 96-well plates and incubated overnight at 37C, 5% CO2. The medium was aspirated from the cells, and various concentrations of kinase inhibitors in serum-free medium were added to each well. After 30 min, VEGF (10 ng ml-1) was added to the wells. Cells were incubated for an additional 72 h and BrdU (10 M) was added during the last 1824 h of incubation. Data were fitted with a curve described by the equation, y=Vmax (1-(x/(K+x))), where K is equal to the IC50 valve (Dev ET AL., 2004).

Figure 2. Molecular interactions of Bismurrayaquinone A inside ATP-binding site of VEGFR-2 Kinase. Polar hydrogens were omitted for clarity.

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Figure 3. A closer view of Bismurrayaquinone A inside ATP-binding site of VEGFR-2 Kinase. Hydrogen bonding of Bismurrayaquinone A with Cys 919, Lys868 and Cys 1045 are visible. Polar hydrogens were omitted for clarity. Molecular modeling studies FRED 2.1 (McGann et al., 2003) was used in this study to dock the OMEGA pregenerated multi-conformer library mentioned above. FRED 2.1 strategy is to exhaustively dock/score all possible positions of each ligand in the binding site. The exhaustive search is based on rigid rotations and translations of each conformer within the binding site defined by a box. FRED filtered the poses ensemble by rejecting the ones that clash with the protein (LOX) or that does not have enough contacts with the protein. The final poses can then be scored or re-scored using one or more scoring functions. In this study, the smooth shapebased Gaussian scoring function (shapegauss) was selected to evaluate the shape complementarily between each ligand and the binding pocket. Default FRED protocol was used except for the size of the box defining the binding sites. In an attempt to optimize the docking-scoring performance we performed exhaustive docking with shapegauss applying the "Optimization" mode. The "Optimization" mode involves a systematic solid body optimization of the top ranked poses from the exhaustive docking. 3 different boxes were explored for VEGFR-2 kinase (PDB ID: 2XIR). Three different simulations were carried out with an added value of 8 around the reference ligand. Results & Discussion A quinine derivative was isolated from crude extract of Murraya in order to develop potential anticancer lead compound targeting angiogenesis. Bismurrayaquinone A showed

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Figure 4. Hydrophobic (yellow areas) and hydrophilic (blue areas) surfaces of inside ATPbinding pocket of VEGFR-2 Kinase. Molecular shape and electrostatic environment matches. Bismurrayaquinone A inside ATP-binding site of VEGFR-2 Kinase. promising inhibitory profile in vitro VEGFR-2 kinase inhibitory assay as predicted by molecular docking studies. The isolated compound revealed considerable VEGFR-2 kinase inhibitory activity (IC50: 126.020.02 nM). Bismurrayaquinone A also showed significant inhibition of VEGF-stimulated proliferation of HUVECs (IC50: 39.14.170.06 nM). Significant effects of Bismurrayaquinone A was could be correlated to its significant molecular interactions with its target protein. Structural insights of the best pose of Bismurrayaquinone A significant molecular interactions with important amino acid residues lining ATP binding pocket of VEGFR-2 (Figure 2 and 3). On one side quinine ring occupied the flat hydrophobic cleft surrounded by Ala 866, Phe918, Cys919, Leu 840 and Leu1035 while on the other quinine ring (perpendicular to first one) deeply penetrated the bent and deeper ATP binding pocket (Figure 4). Shape and electrostatic features of Bismurrayaquinone A seems to be matched with VEGFR-2 binding pocket. Hydrogen bonding played critical role in favorable molecular interactions between ligand and its protein target. Oxygen of quinine moiety was found to be interacting with Cys919 inside the flat hydrophobic cleft covering ATP-binding pocket (Figure), via hydrogen bonding at distance of 2.98 A. Furthermore hydrogen further reinforced this molecular contact between thiol group of Cys1045 (3.04 A) with the second quinine moiety deeper inside ATP Binding pocket. Interestingly, amino group of Lys868 also revealed a favorable contact via hydrogen bonding (3.11 A) with carbonyl moiety of second quinine group. Apart from hydrogen bonding, favorable hydrophobic contacts were also observed. However no clear - interactions were detected. Critical hydrogen bonding interactions and supporting electrostatic and steric interactions between Bismurrayaquinone A and VEGFR-2 could be the possible reasons for its considerable inhibitory potential. This study indicated considerable potential of Bismurrayaquinone A to be further modified and developed as potential class of new and better VEGFR2.inhibitors. Extensive studies including structure and ligand based optimization and 3D-

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Zhong et al. QSAR techniques could be utilized to discover better chemotherapeutic agents targeting angiogenesis to overcome tumor growth, development and resistance in challenging conditions associated with metastatic cancers.

References Adham SA, Sher I, Coomber BL. (2010). Molecular blockade of VEGFR2 in human epithelial ovarian carcinoma cells. Laboratory Investigation. 90: 709-723. Chekler ELP, Katoch-Rouse R, Kiselyov AS, Sherman D, Ouyang X, Kim K, Wang Y, Hadari YR, Doody JF. (2008) Synthesis and evaluation of heteroaryl-ketone derivatives as a novel class of VEGFR-2 inhibitors. Bioorganic & Medicinal Chemistry Letters 18 43444347. Ferrara N, Gerber HP and LeCouter J. (2003) The biology of VEGF and its receptors. Nature Medicine 9: 669-76. Harris PA, Boloor A, Cheung M, Kumar R, Crosby RM, Davis-Ward RG, Epperly AH, Hinkle KW, Hunter RN 3rd, Johnson JH, Knick VB, Laudeman CP, Luttrell DK, Mook RA, Nolte RT, Rudolph SK, Szewczyk JR, Truesdale AT, Veal JM, Wang L, Stafford JA. (2008). Discovery of 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfonamide (Pazopanib), a novel and potent vascular endothelial growth factor receptor inhibitor. Journal of Medicinal Chemistry 51: 4632-4640. Ito C, Thoyama Y, Omura M, Kajiura I and Furukawa H. (1993). Alkaloidal constituents of Murraya koenigii. Isolation and structural elucidation of novel binary carbazolequinones and carbazole alkaloids. Chemical and Pharmaceutical Bulletin. 41: 2096-2100. Dev IK, Dornsife RE, Hopper TM, Onori JA, Miller CG, Harrington LE, Dold KM, Mullin RJ, Johnson JH, Crosby RM, Truesdale AT, Epperly AH, Hinkle KW, Cheung M, Stafford JA, Luttrell DK and Kumar R. (2004) Antitumour efficacy of VEGFR2 tyrosine kinase inhibitor correlates with expression of VEGF and its receptor VEGFR2 in tumour models. British Journal of Cancer 91: 1391-1398. McGann MR, Almond HR, Nicholls A, Grant JA and Brown FK. (2003). Gaussian docking functions. Biopolymers 68:76-90. Prewett M, Huber J, Li Y, Santiago A, O'Connor W, King K, Overholser J, Hooper A, Pytowski B, Witte L, Bohlen P and Hicklin DJ. (1999) Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Research 59:5209-5218.

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