Identification of PKC-directed ligands from different sources
3.2 Experimental procedures
3.2.1 Collection of heterocyclic molecules from chemical libraries: Zinc database (http://zinc.docking.org/) is a good source of drugs and heterocyclic compounds with their detailed 3- Dimensional (3-D) structures. This database was chosen to select the subset of all drug-like compounds.
The 3-D structures of these molecules (approximately 8,000 molecules) were downloaded in mol2 format.
The mol2 structures were then converted to PDB format and used for docking to the C1b domain of PKC- α as the drug target.
3.2.2 Collection of phytochemicals from medicinal plants found in north-eastern (NE) India: The list of plants present in north-eastern India with known medicinal properties was collected through literature search. A comprehensive list of these plants is given in Appendix I and names of selected plants is given in Table 3.1. The phytochemicals present in each plant were collected from Dr.Duke’s phytochemical and ethnobotanical databases (https://phytochem.nal.usda.gov/phytochem/search). A unique set of phytochemicals representing all plants was prepared. Then, amongst all the phytochemicals, we selected only the phytochemicals with a previously known medicinal property. Finally, ~100
phytochemicals were selected for our study. The 3-D structures of all these phytochemicals were downloaded from various libraries (PubChem, zinc database, ChemSpider, etc.). Finally, all the structures were converted to PDB format with the help of Open Babel software (openbabel.org/).
Table 3.1: List of selected medicinal plants present in north-eastern India S no. Botanical Name Common Name Ethnobotanical uses
1. Camelia sinensis Tea Used as a beverage. Also used as a carminative, CNS stimulant and also possess anti-cancer property.
2. Coffea arabica L Coffee Used as a beverage. Also used as a cardiotonic, CNS stimulant, treatment of asthma and headache.
3. Curcuma longa Indian Saffron;
Turmeric
Key spice of many South Asian and Middle Eastern cooking. Extracts from turmeric have antifungal and antibacterial properties.
4. Curcuma xanthorrhiza Temu Lawak; Javan Turmeric
It is used as a spice. Useful for treating digestive disorders and anti-inflammation.
5. Zingiber officinale Ginger Used as a spice. It is used to treat digestive disorders, treatment of fever, nausea and vomiting and possess anti-microbial property.
6. Capsicum annuum Paprika; Green Pepper; Cherry Pepper; Bell Pepper;
Cone Pepper; Sweet Pepper
Used as a spice. It is used in combination homeopathic and natural preparations. It is used as a relief of oral discomfort or toothache, external analgesia, as a digestive aid, in menstrual conditions, and in cosmetics as cleansers and bath products.
7. Arnica montana Arnica; Dagtutunu Used in the treatment of fever, as a cardiotonic and in the treatment of cancer.
8. Acacia nilotica Babli; Gumarabic Used as a decongestant, treatment of gall- bladder, spleen and as an anti-cancer agent.
9. Hibiscus sabdariffa Roselle; Oseille De Guinee; Vinagrillo;
Maravilla; Kerkedeh;
Chai Kujarat; Afrika Bamyasi
Used as a flavouring agent. Also used to lower pressure in hypertension.
10. Nicotiana tabacum Tabigh; Tobacco;
Tabaco
Used for curing toothache, antidote to bee-sting, centipede bites,scorpion-bites. Used in asthma.
Used as Parasiticide, anti-cancer agent and CNS stimulant.
Selected list of plants with medicinal value selected from Dr. Duke’s database. See Appendix 1 for full list.
3.2.3 Molecular Modeling and Docking Studies: The NMR 3-D structure of the C1b domain of PKC- α (PDB ID: 2ELI) was downloaded from Protein Data Bank in PDB format. The average structure was drawn taking the rmsd values of all the conformations. The 3-D structures of the ligands were downloaded from Zinc database. The virtual docking experiment was performed by AutoDock 4.1 program suite of MGL Tools 1.5.4 software (Morris et al., 2009). For PKC-α, polar hydrogen, Kollman charges, and atomic solvation charges were defined, and a grid of 0.375Ǻ was centred around the potential ligand binding
Chapter 3|81 pocket of the C1b domain using the autogrid module of autodock 4.1. Next, the Grid Parameter File (GPF) was defined. For the ligand molecules polar hydrogens, atomic charges, and flexible torsions were accordingly defined. A number of docking parameters such as the number of generations, energy evaluation, and the GA run were set as 27000, 2500000 and 100 respectively. Genetic algorithm was used to perform docking simulations. Binding energy obtained from docking experiments are reported in kcal/mol.
3.2.4 In-silico agonist competition assay: The top-hit molecules from initial docking experiments were used for the agonist competition assay. Agonist competition gives us a real idea to what extent the candidate ligands can mimic the binding pattern of a well-known agonist such as PMA. Hence, ligand molecules were docked again but this time the molecules were docked against the structure of PMA bound C1b domain complex (C1b-PMA) instead of the native C1b domain. The receptor and ligand preparations, grid and docking parameters remained the same as for initial docking using the native C1b domain of PKC. The results of competition docking experiments were taken into consideration for selection of the ligands. Final lists of selected molecules were prepared by subtracting binding energy of competition assay from the binding energy of docking results with the native C1b domain of PKC-α.
3.2.5 In-silico toxicity determination of heterocyclic compounds: Heterocyclic compounds are known in their potential carcinogenic hazard towards mammalian life, whereas phytochemicals are of no concern in this aspect as they are readily available in edible beverages, fruits, vegetables and plants. The toxicity and carcinogenic potential of the top hit heterocyclic compounds was determined to select the non- carcinogenic and non-toxic molecules for further studies. The toxicity and mutagenic potentials of the top-hit heterocyclic molecules were predicted by ToxPredict (toxpredict.org/)(Willighagen et al., 2011).
Toxpredict (www.toxpredict.net) is a server that is used to estimate the chemical hazard of any chemical structure. ToxPredict was utilized to calculate the carcinogenic and mutagenic potential of the heterocyclic compounds. Tox Predict utilizes open Tox API-v1.1 web services to estimate the chemical hazard any chemical structure. ToxPredict can also predict if any compound is non-carcinogenic in single-cell, rat and hamster based carcinogenicity models.
3.2.6 Analysis of PKC-ligand docked complexes and interaction analysis: After the docking simulation was over, each of the PKC-ligand molecular models was taken for analysis. The PKC-ligand molecular models were analyzed in PyMOL v0.99 (Schrodinger, 2010) and interaction analysis was performed by LigPlot+ software (Wallace et al., 1995). The PKC residues were numbered as per the convention followed in the PDB of PKC 3-D structure.