The novel selective inhibitors of cyclin-dependent kinase 4/6: in vitro and in silico study

www.nature.com/scientificreports OPEN The novel selective inhibitors of cyclin‑dependent kinase 4/6: in vitro and in silico study Ni Made Pitri Susanti 1,2, Fransiska Kurniawan 1, Sophi Damayanti 1, Rahmana Emran Kartasasmita 1 & Daryono Hadi Tjahjono 1* One of the main regulators in the cell cycle is cyclin-dependent kinase 4 and 6 (CDK4/6). FDA has approved CDK4/6 inhibitors for the treatment of patients with metastatic breast cancer. However, the development of selective agents remains problematic due to the conservation of their ATP binding sites. In the previous in silico study, ZINC585292724, ZINC585292587, ZINC585291674, and ZINC585291474 have been identified as potential inhibitors. Therefore, the present study aimed to analyze the selectivity and inhibitory activity of the four compounds against CDK4/6 in vitro as well as determine the potential for their further development in silico. The in vitro results showed that the four compounds had good selectivity towards both kinases, due to their similar structure. In agreement with the in silico results, ZINC585291674 produced the best inhibitory activity against CDK4 and CDK6, with IC50 of 184.14 nM and 111.78 nM, respectively. Their ADMET profile were also similar to reference compound of Palbociclib. Based on this, ZINC585291674 can be used as a lead compound for further inhibitor development. One of the fundamental biological processes is cell proliferation which occurs in a series of stages constituting the cell cycle. This cycle includes interphase (preparation of DNA replication in the G1 phase, DNA replication in the S phase, and preparation of mitosis in G2) and metaphase, characterized by sister chromatids’ separation. Besides, there is an inactive or G0 phase, where most of the non-proliferative cells are l ocated1,2. The cycle in normal cells is monitored by checkpoint as a control system, which induces cell cycle arrest during abnormal division, and enables cell repair, thereby ensuring a proper DNA synthesis and chromosomal s eparation3,4. Cyclin-dependent kinases (CDKs), the serine/threonine family of protein kinases, are one of the most important proteins regulating cell cycle transitions. CDK forms heterodimers with cyclin, the regulatory subunits for its a ctivity5,6. Among the CDK family, CDK4 and CDK6 (CDK4/6) are particularly important as they play a fundamental role in the G1/S transition through phosphorylation of retinoblastoma protein (Rb) after activation by cyclin D when cells sense mitogenic signals6. The cell cycle is deregulated in malignant cells, characterized by abnormal and uncontrolled proliferation due to dysregulation and mutations of various regulators, such as CDK, CAK (CDK-activating kinase), CKI (CDK-inhibitors), CDK substrates, cyclins, and checkpoint p roteins5–7. Several oncogenes can be activated in cancer, including those in the RAS/RAF/MEK/ERK, PI3K/Akt/mTOR, JAK/STAT, Wnt/ β-catenin, and BTK/NF-κB pathways, all of which converge on CDK4/6-cyclin D complex. Additionally, mutations of tumor suppressor genes e.g. p53, tend to increase CDK4/6 activity through the release of inhibitory P 21CIP18,9. Therefore, CDK4/6 serves as a center for tumorigenesis. In 2020, breast cancer is the leading cause of women’s death in the world with an estimated 9.2 million new c ases10. The hormone receptorpositive/human epidermal growth factor receptor 2 negative (HR+/HER2-) is the commonest breast cancer type, where CCND1 as an encoding of cyclin D1, is profoundly e xpressed11,12. These features make CDK4/6 an attractive target for anticancer therapy. Heterocyclic ring structures with secondary amine bridges have been widely used in the development of CDK inhibitors, particularly those selective for CDK4/6, including the three FDA-approved compounds, palbociclib, ribociclib, and abemaci-clib13–18. They prevent substrate phosphorylation by competing with ATP at the ATPbinding pocket of CDK4/619. Besides being able to form hydrogen bonds, the heterocyclic ring structure is also known to occupy the adenine binding site of ATP in the CDK hinge r egion20. Previous in silico studies identified ZINC585292724, ZINC585292587, ZINC585291674, and ZINC585291474 as potential CDK4/6 inhibitors as presented in Fig. 1. These four compounds contain a heterocyclic ring with a secondary amine chain in their 1 School of Pharmacy, Bandung Institute of Technology, Bandung 40132, Indonesia. 2Study Program of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Udayana, Badung 80361, Indonesia. *email: Scientific Reports | (2024) 14:23505 | https://doi.org/10.1038/s41598-024-71865-7 1 Vol.:(0123456789) www.nature.com/scientificreports/ H N N N N O N N HN O (a) N N O O N N N N NH N N N N N HN N (b) (c) N N O S N N O NH N N N N N N N HN S (e) (d) Fig. 1.  The chemical structures of (a) Palbociclib, (b) ZINC585291674, (c) ZINC585292724, (d) ZINC585291474, and (e) ZINC585292587. structure21. Therefore, this current study aimed to analyze the selectivity and inhibitory activity of the four compounds against CDK4/6 in vitro as well as determine the potential for their further development in silico. Results and discussion In vitro studies In vitro studies were conducted by testing a single concentration of 1 µM compound to determine their selectivity using CDK6 and six other targets, including CDK1, CDK2, CDK3, CDK5/p25, CDK5/p35, and CDK9. According to binding free energy (∆Gbind) calculation during the previous in silico s tudy21, ZINC585291674 had the lowest ∆Gbind, followed by ZINC585292724, ZINC585291474, and ZINC585292587. The results in Table 1 showed the six enzymes other than CDK6 possessed full enzymatic activity > 90%, implying the four compounds lacked inhibitory activity against them. These indicated that the four compounds had good selectivity towards CDK6, and possibly for CDK4 due to the high homology of both kinases with up to 70% structural similarity22. The inhibitory activity of ZINC585291674 was the best, followed by ZINC585292724, ZINC585291474, and ZINC585292587 with a decrease in enzyme activity to 12%, 18%, 23%, and 35%, respectively. This was directly proportional to the in silico result, consequently, ZINC585291674 and ZINC585292724 were classified with strong inhibitory activity, while ZINC585291474 and ZINC585292587 had moderate inhibitory activity. CDK4/6 inhibitory activity was assessed by determining the IC50, and the results are presented in Fig. 2 and Table 2. ZINC585291674 had the lowest IC50 value against CDK4/6, followed by ZINC585292724, % Activity* Compound CDK1 CDK2 CDK3 CDK5/p25 CDK5/p35 CDK6 CDK9 Palbociclib 82 79 90 97 103 2 106 ZINC585291674 93 91 91 98 102 12 101 ZINC585292724 99 104 102 99 101 18 107 ZINC585291474 98 95 101 92 105 23 117 ZINC585292587 101 107 97 115 102 35 104 Table 1.  The kinase activity of 1 µM concentration of the hit compounds. * > 80%: no activ (...truncated)