Cancer is a broader term reflecting a group of diseases which result in the abnormal growth and division of cells inside the human body. Subsequent to cancer development, the affected cells lose their normal function and continue to grow indefinitely spreading a larger area gradually. The notable causes to the development of cancer can be attributed to genetic heterogeneity, malnutrition, environmental hazards, etc. [1] [2] . Currently, cancer affects the people of both less and more developed country with more recorded incidents in female than in male. The increasing occurrence of cancer is subjected to increased risk factors such as, smoking, physical inactivity, overweight and changing reproductive pattern in most of the cases. Almost 14.1 million new cancer cases and 8.2 million deaths were reported worldwide only in 2012. And the trend is shifting toward the less developed country day by day [3] . Sophisticated treatments like chemotherapy, surgery, radiation therapy and stem cell therapy display a great percentile of recovery but there is always a growing demand of new medication since the available treatments are not accessible to every person due to higher cost. Moreover, these treatments often involve a range of short and long term health effects which again discourage most of the cancer patients [4] . Many natural compounds from medicinal plants have been reported to have anticancer property against variety of cell lines and they exploit this role with different mechanisms [5] . Clitoria ternatea is a medicinal herb that contains alkaloids, tannins, saponins, anthocyanins, cardiac glycosides, etc., which provides potential health benefits to consumers. Its major phytochemicals of potent therapeutic value include Kaempferol, Myricetin, P-Hydroxycinnamic acid, Quercetin, Beta-sitosterol, Anthoxanthin glucoside, Tannic acid, Taxaxerol, etc. Aqueous extract of seeds from this plant has already been shown to have cytotoxic activity in laboratory experiment [6] [7] [8] .

Cyclin dependent kinases (CDKs) are specific serine/threonine kinases which contribute to the cell cycle progression by phosphorylating and inactivating Retinoblastoma (Rb) protein. Rb protein usually resides inside the cell forming a complex with a transcription activator called E2F which has at least five DNA binding domains and takes part in progressing the dividing cell from G₁ phase to S phase (Figure 1). In the complex form, Rb represses the activity of E2F protein which is the case when the cell is in resting sate [9] . Several mitogens released from the upstream signaling pathway activate Cyclin D which forms complex with CDK-4/6 and helps in its activation. In the active state, CDK-4/6 phosphorylates Rb and partially inactivates it facilitating the release of E2F from the complex [10] [11] . Once E2F is released from the complex, it becomes activated and carries out the events required for the G₁ to S phase transition in the cell cycle. E2F also promotes the activation of Cyclin E-CDK-2 complex which in turn contributes in the phosphorylation of Rb in a feedback loop and thus prolongs the E2F activity [12] .

Inside the cell both types of CDKs are repressed by inhibitors comprising proteins from INK4 (inhibitor of CDK-4) and CKI (cyclin-dependent kinase inhibitor) families and this contributes to the decision of the cell to undergo cell division or not. CDK-4/6 is inactivated by inhibitors like p15/p16 and CDK-2 is inactivated by the p21/p27 inhibitory proteins [13] . The dysregulated hyperactivity of CDK due to the mutation in CDKs or their inhibitor encoding genes can lead to uncontrolled cell growth which characterizes the cancer. Different types of CDKs have been reported to be associated with different forms of cancer in which they lack the ability to bind inhibitor and ultimately become resistance [14] [15] .

Computational drug design is a widely accepted technique for new lead discovery. Virtual screening technique reduces both time and cost of the drug discovery expenditure. More than 50 drugs have been designed and repurposed with the aid of these computational simulation tools and many of them received FDA approval for marketing like Raltegravir, Saquinavir, Nelfanavir, Itraconazole etc. Molecular docking tries to predict the pose, interaction and conformation of a ligand molecule within the binding site of a target molecule, usually a large macromolecule. After estimating the type of interactions, the software assigns scoring function to each of the bound ligands with specified algorithm which reflects the binding affinity. The lowest score of binding represents the most favorable interaction between ligand and receptor molecule [16] [17] [18] .

The safety and efficacy testing of a candidate drug molecule is a major concern in clinical and preclinical trial. In silico approaches to assess the drug features has enabled the test to be carried out in a much simpler way where in vitro and in vivo assessment of safety and toxicity is time consuming and costly. ADME/T testing provides information regarding the drug feature like adsorption, distribution, metabolism, excretion and toxicology information inside human body. Moreover, these approaches help in generating data about the extent of drug absorption inside the body, blood brain barrier permeability, susceptibility to biodegradation, mutagenicity, carcinogenicity etc. [19] [20] .

This study has been designed to investigate the inhibitory potentiality of four phytochemicals: Kaempferol, Myricetin, P-Hydroxycinnamic acid and Quercetin (Figure 2) from Clitoria ternatea against CDK-2 and CDK-6 (Figure 3) in cancer cell and to assess their physicochemical, pharmacokinetic and pharmacodynamic properties inside biological system.

Three dimensional structures of CDK-2 (PDB Id: 3EZV) and CDK-6 (PDB Id: 1XO2) were downloaded in PDB format from protein data bank (www.rcsb.org). The protein was then processed and refined utilizing the Protein Preparation Wizard in Maestro Schrödinger v11.8. Bond orders were assigned, hydrogens were added to heavy atoms. All the waters were deleted from the molecules and selenomethionines were converted to methionines. Finally, the structure was optimized and then minimized using built-in default force field OPLS_2005. Minimization was performed setting the greatest substantial particle RMSD (root-mean-square-deviation) to 30 Å and any outstanding water under 3H-bonds to non water was again erased during the minimization step.

The 3D conformations of Kaempferol (PubChem CID: 5280863), Myricetin (PubChem CID: 5281672), P-Hydroxycinnamic acid (PubChem CID: 637542) and Quercetin (PubChem CID: 5280343) were downloaded from PubChem (https://www.pubchem.ncbi.nlm.nih.gov/). These structures were then processed prepared using the LigPrep of Maestro Schrödinger. Minimized 3D structures of ligands were generated using Epik2.2 and within pH 7.0 ± 2.0 in the suite. Minimization was again carried out using OPLS_2005 force field which generated maximum 32 possible different rearranged spatial conformations (stereoisomers) depending on available chiral centers for each of the ligand molecules.

Grid usually restricts the active site to specific area of the receptor protein for the ligand to dock specifically within that area. In Glide, a grid was generated using default Van der Waals radius scaling factor 1.0 and charge cutoff 0.25 which was then subjected to OPLS_2005 force field for the minimized structure. A cubic box was generated around the active site (reference ligand active site) of target molecules. Then the grid box volume was adjusted to 14 × 14 × 14 for docking to be carried out.

SP adaptable glide docking was carried out using Glide in Maestro Schrödinger. The Van der Waals radius scaling factor and charge cutoff were set to 0.80 and 0.15 respectively for all the ligand molecules under study. Final score was assigned according to the pose of docked ligand within the active site of the receptor molecules. The docking result is summarized in Table 1. 2D and 3D representation of ligand-receptor interaction are summarized in Figure 4 and Figure 5 respectively.

The molecular structures of every ligands were analyzed using SWISSADME server (http://www.swissadme.ch/) in order to confirm whether the ligands follow Lipinski’s rule of five or not. Physicochemical properties of ligand molecules were calculated using OSIRIS property explorer (https://www.organic-chemistry.org/prog/peo/). The result of drug likeness property analysis is summarized in Table 2.

The ADME/T for each of the ligand molecules was carried out using an online based server ADMET-SAR (http://lmmd.ecust.edu.cn/admetsar1/predict/) to predict their various pharmacokinetic and pharmacodynamic properties including blood brain barrier permeability, human abdominal adsorption, AMES toxicity, Cytochrome P (CYP) inhibitory capability, carcinogenicity, mutagenicity, Caco-2 permeability etc. The result of ADME/T for all the ligand molecules is represented in Table 3.

All the selected ligand molecules docked successfully with both CDK-2 and CDK-6. Kaempferol, Myricetin, P-Hydroxycinnamic acid and Quercetin docked with CDK-2 with −7.872 Kcal/mol, −8.137 Kcal/mol, −7.149 Kcal/mol and −8.298 Kcal/mol binding energies respectively (Table 1). Kaempferol formed total 1 hydrogen bond withVal64, Myricetin formed total 4 hydrogen bonds-2 with Leu83, 1 with Asp86 and another 1 with Asp145, P-Hydroxycinnamic Acid formed 2 hydrogen bonds with Leu83 and Asp145 and Quercetin also formed 2 hydrogen bonds with Val64 and Glu81 within the binding site of CDK-2 structure backbone. Kaempferol, Myricetin, P-Hydroxycinnamic acid and Quercetin interacted with 8, 9, 7 and 9 amino acid residues respectively in total within the binding pocket of CDK-2 target molecule.

All the selected molecules exhibited a slightly lower binding energy and hence higher affinity for CDK-6 than CDK-2. Kaempferol, Myricetin, P-Hydroxycinnamic acid and Quercetin docked with CDK-6 with −9.012 Kcal/mol, −9.622 Kcal/mol, −7.103 Kcal/mol and −8.559 Kcal/mol binding energies respectively (Table 1). Kaempferol formed 6 hydrogen bonds-2 with Val101, and 1 with Lys43, Glu61, His100 and Gln149 each in the binding site of CDK-6. Myricetin formed 5 hydrogen bonds-2 with Val101 and 1 with Lys43, Glu61 and Asp104 each. P-Hydroxycinnamic formed 4 hydrogen bonds (2 with Val101 and 1 with Lys43 and Asp163 each) and Quercetin formed 3 hydrogen bonds with Glu21, Val101 and Asp163 within the binding site of CDK-6. Moreover, Kaempferol, Myricetin, P-Hydroxycinnamic acid and Quercetin interacted with 11, 14, 5 and 9 amino acid residues respectively in total within the binding pocket of CDK-6 target molecule.

Kaempferol, P-Hydroxycinnamic acid and Quercetin followed Lipinski’s rule of five with respect to with respect to molecular weight (acceptable range: ≤500), number of hydrogen bond donors (acceptable range: ≤5), number of hydrogen bond acceptors (acceptable range: ≤10), lipophilicity (expressed as LogP,

acceptable range: ≤5) and molar refractivity (40 - 130) without any violation (Table 2) [24] . But, myricetin violated the rule of hydrogen bond donors (6) exceeding the acceptable range. Myricetin possesses the largest polar surface area or topological polar surface area (151.59) and p-hydroxycinnamic acid possesses lowest area (57.53). Kaempferol and quercetin encompass a moderate topological polar surface area 107.2 and 127.4 respectively. Kaempferol has the lowest LogS value of −3.31 whereas p-hydroxycinnamic acid shows the largest value of −2.02 among all the ligand molecules. Myricetin and quercetin exhibit slightly similar LogS values of −3.01 and −3.16 respectively. Both quercetin and myricetin showed satisfied drug likeness and drug score than other two ligand molecules.

The result of ADME/T test of selected ligand molecules is summarized in Table 3. Kaempferol and P-Hydroxycinnamic acid are capable of penetrating blood brain barrier but other two ligands are not. All the ligand molecules are highly absorbable in human intestinal tissue. Only P-Hydroxycinnamic acid is biodegradable in biological medium. No ligand molecules showed mutagenicity and AMES toxicity and hence carcinogenicity test is not required. P-Hydroxycinnamic acid might induce type III acute oral toxicity whereas others may capable of inducing type II. Both myricetin and quercetin are inhibitors of Cytochrome CYP450 1A2 and CYP450 3A4. Kaempferol inhibits another 2 cytochromes-CYP450 C9 and CYP450 C19 in addition to CYP450 1A2 and CYP450 3A4. However, P-Hydroxycinnamic acid is non-inhibitors of every cytochromes that are summarized in Table 3.