JBIC Journal of Biological Inorganic Chemistry
October 2015, Volume 20, Issue 7, pp 1205-1217
Inhibition of cyclin-dependent kinase CDK1 by oxindolimine ligands and corresponding copper and zinc complexes.
- Rodrigo Bernardi Miguel1 • Philippe Alexandre Divina Petersen2 • Fernando A. Gonzales-Zubiate3 • Carla Columbano Oliveira3 • Naresh Kumar1 • Rafael Rodrigues do Nascimento2 • Helena Maria Petrilli2 • Ana Maria da Costa Ferreira1*
1 Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, São Paulo 05508-000, SP, Brazil;
2 Departamento de Física dos Materiais e Mecânica, Instituto de Física, Universidade de São Paulo, Rua do Matão, Travessa R 187, São Paulo 05508-090, SP, Brazil; 3 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Av. Prof. Lineu Prestes 748, São Paulo 05508-000, SP, Brazil
Oxindolimine-copper(II) and zinc(II) complexes that previously have shown to induce apoptosis, with DNA and mitochondria as main targets, exhibit here significant inhibition of kinase CDK1/cyclin B protein. Copper species are more active than the corresponding zinc, and the free ligand shows to be less active, indicating a major influence of coordination in the process, and a further modulation by the coordinated ligand. Molecular docking and classical molecular dynamics provide a better understanding of the effectiveness and kinase inhibition mechanism by these compounds, showing that the metal complex provides a stronger interaction than the free ligand with the kinase ATP-binding site. The metal ion introduces charge in the oxindole species, giving it a more rigid conformation that then becomes more effective in its interactions with the protein kinase active site. Analogous experiments resulted in no significant effect regarding phosphatase inhibition. These results can explain the cytotoxicity of these metal complexes towards different tumor cells, in addition to its capability of binding to DNA, and decreasing membrane potential of mitochondria.
Essential metal complexes of oxindolimine ligands (figure1, as an example) were studied  as alternative antitumor agents to cisplatin and its analogs, the single class of metallodrugs already approved to cancer therapy by the Food and Drug Administration (FDA). These ligands were designed based on oxindole derivatives that have already entered some phase of clinical trials (phase II and III) as antitumor agents, or that have been approved by FDA as Sunitinib [2-4 ]. Binding and consequent oxidative damage to DNA caused by these oxindolimine complexes have been previously detected , indicating a significant nuclease activity of this class of compounds. In addition, they can act as decoupling agent toward mitochondria . These focused copper(II) and zinc(II) complexes can also efficiently inhibit specific proteins. Topoisomerase IB has already been tested through enzymatic kinetic assays and molecular docking simulations, being inhibited by such complexes remarkably at the cleavage reaction, but only partially at the religation step .
Figure 1 – Oxindolimine-copper(II) complex, [Cu(isapn)], DFT (quantum mechanics) optimized structure.
Since protein kinases play crucial roles in cellular signal transduction and regulation, the activity of these metal complexes on CDK1 protein, activated by cyclin B, was additionally investigated using 32P-g-ATP, and histone H1 as a phosphorylation substrate. Further, the effect on alkaline phosphatase, responsible for dephosphorylation, was observed. Possible mechanisms of action of such metal complexes were then proposed, based on theoretical simulations that rationalize and give support to the observed experimental results.
In the concentration range (5 to 50 µM), a series of complexes containing copper(II) or zinc(II) exhibited remarkable inhibition of histone phosphorylation. The free ligands were much less active than the corresponding metal complexes, and copper derivatives were more efficient than the analogous zinc compounds (see figure 2). Therefore, the process is modulated by both the metal ion, and the ligand structure. Its mode of action includes not only generation of reactive species (mainly hydroxyl radicals, in the case of copper), but also interactions with the protein via hydrogen bonding, electrostatic forces, and coordinative bonding. Coordination of the ligand to the metal ion provides charge and a more rigid structure to the ligands, causing a significant enhancement in its inhibition effect. In other words, metal ions provide peculiar three-dimensional shapes for organic compounds upon coordination, interfering in its electronic properties, increasing their possibility of molecular recognition by vital biomolecules and consequently their biological properties.
Figure 2 – Inhibition of CDK1/cyclin B kinase activity in the presence of metal complexes
Theoretical simulations indicated that close interactions with different amino acid residues, and different numbers of hydrogen bonds occur between the free or the coordinated ligand to copper ion and the protein, at the protein active site (figure 3). Molecular dynamics results revealed good stability of both species at the ATP-binding site of CDK1, corroborating initial positions, obtained by molecular docking. Similar theoretical results were obtained for the ligands isapn and isaepy inserted at the active site of CDK2 , attesting that more interactions occur when the ligand is coordinated to the copper ion.
Figure 3 – Theoretical simulation of [Cu(isapn)] species inside the ATP-binding site of cyclin-dependent kinase (CDK1).
In recent studies in the literature, some octahedral rhodium, ruthenium or iridium complexes with sophisticated pyridocarbazoles were reported as efficient kinase inhibitors [9, 10]. Some of such complexes, in a series that were inspired in staurosporine , a natural high-affinity inhibitor of nearly all mammalian kinases, showed IC50 values in the nM range toward different kinases. However, in those metal complexes the ligands show structurally a high stereochemical complexity, leading to many stereoisomers, and therefore carefully tailored complexes must be obtained in order to achieve binding selectivity . Some cyclam fused naphthalimides metal complexes were also described in analogous studies as multi-target receptor tyrosine kinase (RTK) inhibitors , attesting that different scaffolds can efficiently act in the inhibition of kinases.
Our reported results showed that besides DNA and mitochondria, the oxindolimine-metal complexes have kinases as important intracellular targets, acting as multifunctional agents in a synergistic process involving the metal center, and the coordinated ligand. These ligands are easily prepared from isatin, a tryptophan metabolite, and diverse amines, and formed metal complexes kinetic and thermodynamically very stable . The main importance of these results consists in providing a possibility of rational design of new oxindolimine ligands and corresponding metal complexes that could act as even more efficient kinase inhibitors.
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This work was supported by FAPESP (Grants 2010/51842-3, 2011/50318-1, and 2013/07937-8), CNPq (Grant 573530/2008-4), CAPES, and PRPUSP (Grant 2011.1.9352.1.8). The authors are also grateful to the networks INCT INEO, INCT Redoxoma (FAPESP/CNPq/CAPES), NAP Redoxoma (PRPUSP), CEPID Redoxoma (FAPESP) and BioMol/CAPES (Computational Biology Project).
Ana Maria da Costa Ferreira, Ph.D.
Professor of Inorganic Chemistry
Dept. of Chemistry, Institute of Chemistry
University of São Paulo
Av. Prof. Lineu Prestes, 748
05508-000 São Paulo, SP – BRAZIL