Prediction of Binding Mode of Secondary Metabolites in Apium graveolens to Bcl-2

Developing new cytotoxic agent which has minimal effect against normal cell is required to reduce the side effects obtained from the existing chemotherapy agents. Celery (Apium graveolens) showed several pharmacology activities, including anti-cancer. This study was conducted to predict and visualize the binding mode of ten secondary metabolites in A. graveolens, i.e., apigenin, caffeic acid, kaempferol, limonene, shikimic acid, chlorogenic acid, ascorbic acid, quercetin, eugenol and ferulic acid against Bcl-2. Navitoclax was employed as reference. Molecular dokcing simulation was performed using AutoDoc Vina v.1.5.6. The results showed that the interaction enery (Ei) ranged from -4.20 to -6.01 kcal/mol, whereas inhibition constant (Ki) were 40.15 to 842.29 μM. Kaempferol showed the best affinity to Bcl-2 (Ei=-6.01 kcal/mol; Ki=24,21 μM). Hydrogen bonds were bond between kampferol and Lys20, Ser102, and Arg103, amino acid residues in Bcl-2.In conclusion,


Introduction
A cancer cell is a mutated abnormal cell, which proliferate progressively and become predominantly malignant. Variety of chemotherapy agents act to inhibit cell proliferation and malignancy. 1 Developing new cytotoxic agent which has minimal effect against normal cell is required to reduce the side effects obtained from the existing chemotherapy agents. Thus, developing new chemotherapy agents from herbal medicine is a potential alternative in order to reduce the side effects. 2 Secondary metabolites which exist in celery plants, such as apigenin, caffeine, kaempferol, limonene, cycmic acid, chlorogenic acid, ascorbic acid, quercetin, eugenol and ferulic acid exhibited antitumor and cytotoxic activity. 3 Moreover, apigenin inhibited human lung cancer (NCI-H460) cells proliferation and induced apoptotic by increasing Bax and caspase-3 expression and decreasing Bcl-2 Bcl-2 is a family of Bcl-2 protein regulator, which inhibits apoptotic activity. 5 Bcl-2 is a homodimer form, consists of monomer A and B. The expression of Bcl-2 will increase in lung cancer patient due to the inhibition of apoptotic activity. 6 The high level of Bcl-2 will protect the cells through the early death of apoptotic. Thus, Bcl-2 is an anti-apoptotic protein, which is classified as an oncogene. 7 The purpose of this study was to predict and visualize the binding mode of ten secondary metabolites in A. graveolens, i.e., apigenin, caffeic acid, kaempferol, limonene, shikimic acid, chlorogenic acid, ascorbic acid, quercetin, eugenol and ferulic acid against Bcl-2.

Materials
3D structure of Bcl-2 complexed with navitoclax (indol), resolution 2.07 Å (PDB   The maximum number of cycles indicated the maximum number of rounds required by a compound to achieve the most stable conformation with minimum global energy or potential energy. The arrangement of geometrical optimization condition utilized the 0.1 kcal/Åmol of root mean square gradient (RMSG) value, which indicated an increasing amount of energy detected due to stable conformation changes in the compound. 11 The optimization results of the six compounds exhibited the difference value of 0.00000, which indicated all optimized compounds have reached the most stable conditions with the lowest energy levels, where the conformer potential energy equals to the minimum global energy (the minimum point on the potential energy curve). The volume of Bcl-2 active site was 2156.54 Å 3 , thus all ligands were predicted could occupy the active site, since all of the ligands have smaller volume.
Furthermore, molecular properties of the ligands were analyzed with quantitative structure-activity relationship (QSAR) using Portable HyperChem Professional 7.03. Analysis of the molecular properties included the basic parameters of analysis, such as molecular mass, molecular volume, coefficient partition (log P), geometric optimization energy, and electrostatic potential.
The Lipinski's Rule of Five is a method for evaluating drug-likeness, where the rule specifies drug molecules must have a relative molecular mass less than 500 dalton, log P value less than five, a coefficient partition value between -2 to 5 and have ten maximum hydrogen bonds donor. Drugs with molecular weight over 500 daltons are more difficult to penetrate through lipid bilayers and tend to undergo first pass metabolism. 12 In this study, all ligands qualified for Lipinski's Rule of Five. The higher of the log P, the more hydrophobic or lipophilic the compounds, hence it will be more easily to penetrate the cell membrane. 13 The most hydrophilic compound was caffeic acid, while, the most hydrophobic was limonene. Redocking of navitoclax crytstal into its origin location in Bcl-2 resulted in Ei= -11.24 ± 0.08 kcal/mol, Ki=0.006 ± 0, 001 µM. The The re-docking results had 66.67% similarity compared to the origin.

Conclusion
Secondary metabolites in A.graviolen were predicted could interact with amino acid resiues in BCl-2 binding pocket. Their binding modes were similar to navitocalax. Kaempferol showed the best interaciton.