IN SILICO SCREENING AND IN VITRO ASSAY OF CYCLINDEPENDENT KINASE 4 (CDK4) AND CYCLIN-DEPENDENT KINASE 6 (CDK6) INHIBITORS

• Main Author: Made Pitri Susanti, Ni
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66695
• Institution: Institut Teknologi Bandung

Cancer is one of the leading causes of death worldwide, therefore research to find the accurate treatment continues to be developed. The characteristic of cancer is uncontrolled cell growth due to its ability to maintain chronic proliferation. In normal tissues, the production and release of growth-promoting signals that regulate the cell cycle can be well controlled to ensure homeostasis of normal tissue numbers and functions. Therefore, controlling cell proliferation through intervention in the cell cycle can be one of the efforts in the treatment of various types of cancer such as breast cancer. Cyclin-dependent kinase (CDK), cyclins, and endogenous CDK inhibitors (CKI) are the main components that regulate the cell cycle. CDK which belong to the serine/threonine kinase enzyme group play a role in each stage of the cell cycle. CDK4 and CDK6 are activated by cyclin D after a mitogenic signal in the G1 phase. Phosphorylation of the retinoblastoma protein (Rb) initiates further stimulation of the release of other cyclins to activate other CDKs at later stages of the cell cycle. Endocrine resistance is also associated with overexpression of cyclin D. Moreover, inhibition of CKI can trigger hyperactivity of CDK4 and CDK6. Inhibition of CDK4 and CDK6 results in the absence of cyclin expression associated with later stages of the cell cycle resulting in complete cessation of the cycle. The CDKs have many cellular functions with structural similarities, threrefore selectivity is needed in their inhibition. In addition to efforts to increase the effectiveness of therapy, prevention of unwanted effects due to blockade of other CDKs is also needed. Oral CDK4 and CDK6 inhibitors with acceptable clinical activity and toxicity profile have been approved by the US Food and Drug Administration (FDA) for breast cancer therapy. Therefore, this study was conducted to identify potential CDK4 and CDK6 inhibitory compounds through a combination of in silico and in vitro methods. The study began with pharmacophore modeling through molecular alignment of three CDK4/6 inhibitor compounds that have been approved by the Food and Drug Administration (FDA) for clinical use. The next stage is in silico screening to obtain candidate compounds in the ZINC15 compound database using a validated pharmacophore model followed by an interaction study of candidate compounds with CDK4 and CDK6 targets through molecular dockings. The hit compound obtained from in silico screening was then analyzed for stability of its interaction with the target through molecular dynamics simulation for 200 ns and the binding free energy was calculated using the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method. The hit compounds were selected based on the results of molecular dynamics simulations followed by in vitro assay to confirm their selectivity and inhibitory activity on CDK4 and CDK6. The in silico screening stage resulted in eight hit compounds which were selected based on the docking score and their interaction with the target. Based on the interaction stability and binding free energy, four compounds were selected, namely ZINC585292724 (ZN09), ZINC585292587 (ZN07), ZINC585291674 (ZN06), and ZINC585291474 (ZN05), and were then studied in the next stage. In vitro assay was carried out in two steps, selectivity assay using a single compound concentration of 1 µM, and inhibitory activity assay by determining IC50 using ten concentrations of compound. Selectivity and inhibitory activity assay on CDK6 were carried out using the Kinase Selectivity Profiling System: CMGC2 + ADP-Glo TM Assay, while for CDK4 was performed using the CDK4 assay kit and Kinase-Glo ® Max Lunimescence Kinase Assay. The results of the selectivity assay showed that the four compounds had good selectivity towards CDK6. Compounds ZINC585292724 (ZN09) and ZINC585291674 (ZN06) were classified as having strong activity with remaining enzymatic activity of 18 and 12%, respectively, while compounds ZINC585292587 (ZN07) and ZINC585291474 (ZN05) were classified as having moderate activity with remaining enzymatic activity of 35 and 23%, respectively. The results of the inhibitory activity assay showed that the four compounds had inhibitory activity on the nano molar scale. Compounds ZINC585291674 (ZN06) and ZINC585292724 (ZN09) had the first and second best inhibitory activities on both targets. Compound ZINC585291674 (ZN06) has IC50 values on CDK4 and CDK6 of 185.14 and 111.78 nM, respectively, while compound ZINC585292724 (ZN09) has IC50 of 286.75 and 196.25 nM, respectively. Based on the results of selectivity and its inhibitory activity, the compound ZINC585291674 (ZN06) can be recommended as a candidate for CDK4 and CDK6 inhibitors. This compound can be used as a lead compound in an effort to develop compounds with better inhibitory activity.