Aisha A. Alsfouk ^1* Abdelmoujoud Faris ^2,3* Ivana Cacciatore ^4,5 Radwan Alnajjar ⁶

• ¹ Department of Rehabilitation Sciences, College of Health and Rehabilitation Sciences, Princess Nourah bint Abdulrahman University, Riyadh, Riyadh, Saudi Arabia
• ² Faculty of Science and Technology, Sidi Mohamed Ben Abdellah University, Fez, Morocco
• ³ Sidi Mohamed Ben Abdellah University, Fes, Morocco
• ⁴ Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Tuscany, Italy
• ⁵ Department of Pharmacy, University 'G. d'Annunzio' of Chieti-Pescara, Chieti, Italy
• ⁶ School of Computer Science, Faculty of Science, University of Nottingham, Nottingham, England, United Kingdom

Cyclin-dependent kinase 9 (CDK9) and cytochrome P450 3A4 (CYP3A4) have emerged as promising targets in the development of anticancer drugs, presenting a consistent challenge in the quest for potent inhibitors.Employing a range of computational methodologies, this study systematically investigated the binding mechanisms of pyrimidine derivatives against CDK9 and CYP3A4. Techniques such as 3D-QSAR, molecular docking, molecular dynamics (MD) simulations with subsequent binding free energy MM/PBSA, and ADMET studies were applied. The field-based model demonstrated high Q2 (0.66) and R2 values (0.99), proving its accuracy in predicting in silico inhibitory activity against the target of this study. The screening process followed in this work led to the discovery of powerful new inhibitor compounds. Of the 15 new compounds designed, three have a high affinity with the target (ranging from -8 to -9 kcal/mol kcal/mol) were singled out through docking filtration for more detailed investigation. As well as, a reference compound with a substantial pIC50 value of 8.4, serving as the foundation for the development of the new compounds, was included for comparative analysis. To elucidate the essential features of CDK9 and CYP3A4 inhibitor design, a comparative analysis was conducted between 3D-QSAR-generated contours and molecular docking conformations of ligands. Molecular dynamics simulations were carried out for a duration of 100 ns on selected docked complexes, specifically those involving novel compounds with CDK9 and CYP3A4 enzymes. Additionally, the binding free energy for these complexes was assessed using the MM/PBSA method, which evaluates the free energy landscape of protein-ligand interactions.. The results of MM/PBSA highlighted the strength of the new compounds in enhancing interactions with the target protein, which favors the results of molecular docking and MD simulation. These insights contribute to a deeper understanding of the mechanisms underlying CDK9 and CYP3A4 inhibition, offering potential avenues for the development of innovative and effective CDK9 inhibitors.