<p>The present study was aimed to identify the novel compounds and explore the mechanism of the compounds from the library against prostate cancer by targeting the CDK6 signaling. The CDK6 signaling is involved in the cell cycle regulation and gain of function mutation paves the way for tumorigenesis in various cancers including the prostate. The virtual screening was performed against the CDK6 protein using Enamine compound library (Phenotypic screening library) containing 5760 compounds to obtain the top hits based on the binding energy and pharmacokinetic profile. Based on the virtual screening results, the top 5 compounds were selected for further studies including the MD simulation and binding free energy calculations using MM-PBSA and MM-GBSA. The MD simulation analysis was conducted to assess the dynamic stability of the complexes following virtual screening. The results showed that compounds 2231 (Z465849926), 2267 (Z56774766), 2760 (Z19047271), 3765 (Z1203029456), and 4612 (Z26475533) exhibited stable RMSD and no marked fluctuations were observed for 50 ns simulation. Similarly, the RMSF analysis and RoG were in the acceptable range following binding of the ligands with the protein. Similarly, the compounds formed a variable number of hydrogen bonds with the target protein during the simulation. The binding free energies calculation suing MM-PBSA and MM-GBSA revealed that compounds negative and favorable binding energies and validated the results of the MD simulation. The DFT calculations for the selected compounds to assess the stability and reactivity of the compounds using HOMO and LUMO energy gaps. Furthermore, pharmacokinetic studies were also conducted for the selected compounds. In conclusion, the selected compounds exhibited marked activity against the prostate cancer target and can be employed to deal with the prostate following comprehensive in vivo studies.</p>