Introduction 3. Materials and methods 3.1. In silico studies on CDK2 3.1.1. Molecular docking 3.1.2. Molecular dynamics simulation 3.2. In vitro CDK2 inhibition assay 3.3. In vitro evaluation of antimetastatic potential on B16F10 cell line 3.3.1. Determination of cell viability by MTT assay 3.3.1.1. Preparation of solutions for compounds for MTT assay 3.3.1.2. MTT assay 3.3.2. Preparation of dilutions for colony formation assay, wound scratch assay, and Leighton tube studies 3.3.3. Colony formation assay 3.3.4. Wound healing assay 3.3.5. Determination of cellular morphology by Leighton tube assay 4. Results and Discussion 4.1 In silico studies on CDK2 4.1.1. Molecular docking 4.1.1.1. Validation of docking protocol 4.1.1.2. Docking studies of synthetic flavones 4.1.2. Molecular dynamics simulation 4.2. In vitro CDK2 inhibition assay 4.3. In vitro studies on B16F10 cell line 4.3.1. Synthesized flavones exert cytotoxicity & inhibit the proliferation of B16F10 cells 4.3.2. Synthesized flavones hamper the formation of colonies in B16F10 melanoma 4.3.3. Synthesized flavones inhibit cellular migration in wound scratch assay 4.3.4. Synthesized flavones initiate changes in the morphology of B16F10 cells 5. Conclusion CDK2 Inhibition and antimetastatic effect of flavones 665

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Background: EGFR (Epidermal Growth Factor Receptor) and CDK2 (Cyclin Dependent Kinase 2) are important targets in the treatment of many solid tumors and different ligands of these receptors share many common structural features. Objective: The study involved synthesis of benzamide-substituted chalcones and determination of their antiproliferative activity as well as preliminary evaluation of EGFR and CDK2 inhibitory potential using both receptor binding and computational methods. Methods: We synthesized 13 benzamide-substituted chalcone derivatives and tested their antiproliferative activity against MCF-7, HT-29 and U373MG cell-lines using Sulforhodamine B Assay. Four compounds were examined for activity against EGFR and CDK2 kinase. The compounds were docked into both EGFR and CDK2 using Glide software. The stability of the interactions for most active compound was evaluated by Molecular Dynamics Simulation using Desmond software. Molecular Docking studies on mutant EGFR (T790M, T790M/L858R, and T790M/C797S) were also carried out. Results: From the SRB assay, we concluded that compounds 1g, and 1k were effective in inhibiting the growth of MCF-7 cell line whereas the other compounds were moderately active. Most compounds were either moderately active or inactive on U373 MG and HT-29 cell line. Compounds 1g and 1k showed good inhibitory activity against CDK2 kinase while 1d and 1f were moderately active. Compounds 1d, 1f, 1g, and 1k were moderately active against EGFR kinase. Molecular docking reveals involvement of one hydrogen bond with Met793 in binding with EGFR however; it was not stable during simulation and these compounds bind to the receptor mainly via hydrophobic contacts. This fact also points towards a different orientation of the inhibitor within the active site of EGFR kinase. Binding mode analysis for CDK2 inhibition studies indicate that hydrogen bonding interaction with Lys 33 and Leu83 are important for the activity. These interactions were found to be stable throughout the simulation. Considering the results for wild-type EGFR inhibition, the docking studies on mutants were performed and which indicate that the compounds bind to the mutant EGFR but the amino acid residues involved are similar to the wild-type EGFR and therefore, the selectivity seems to be limited. Conclusion: These benzamide-substituted chalcone derivatives will be useful as lead molecules for the further development of newer inhibitors of EGFR and/or CDK2 kinases.

Twelve hits with best obtained docking scores ranging from -10.48 to -7.23 kcal/mol and mimicking characteristic type I inhibitor interactions were identified which could be probable inhibitors of VEGFR-2.

A total of 95 crystal structures of CDK2 were selected after considering criteria such as resolution and absence of missing residues in the active site; and subjected to cross-docking. 14 out of 95 crystal structures exhibited docking accuracy for greater than 70% of ligands at RMSD cut off 2Å in the cross- docking studies. These 14 crystal structures were selected for the second part of the study, which included validation using DUD sets and enrichment calculations. 8 out of 14 crystal structures possessed the enrichment factor of >10 at 1% of the ranked database. ROC-AUC, AUAC, RIE, and BEDROC were calculated for these 8 crystal structures. 2WXV produced maximum BEDROC (0.768, at α=8) and RIE (11.22). 2WXV as a single initial crystal structure in the virtual screening protocol is likely to produce more accurate results than any other single crystal structure.