Approaching the mechanism of anticancer activity of a copper(II) complex through molecular modelling, docking and dynamic studies. I.N. Zoi1 , A.X. Lygeros1 , G.D. Geromichalos2 and G.A. Katsoulos1 1 Department of Chemistry, Aristotle University, Thessaloniki, Greece 2 Symeonidion Research Center, Theagenion Cancer Hospital, Thessaloniki, Greece * Corresponding author: izoi@chem.auth.gr INTRODUCTION METHODS In an attempt to understand the molecular basis of the drug action of several copper(II) chelates, we studied theoretically their chemical interactions involved in the complex processes of drug delivery and their reactions with a variety of biological molecules. We have focused in the theoretical study of copper(II) complexes of the formula Cu(SalNEt2)-X, where SalNEt2 stands for the anion of the N-(2-(diethylamino)ethyl)salicylidenaminato and X = citrate, tartrate, and salicylate anion, as well as their adducts with various biological molecules. The complexes have shown a potent cytotoxic activity against a panel of human cancer cell lines in vitro 1,2,3. DFT calculations demonstrated that the mechanism of the cellular damage can be explained, at least in part, by the ability of the nucleobases and nucleotides to nucleophilic attack the complexes. It was also verified through molecular docking studies the hypothesis that the mechanism of action of complexes involves the binding to DNA and RNA and also the inhibition of DNA topoisomerases I and II. In order to explain the inhibition profile, we explored the interactions of each complex within the binding cavity of the aforementioned proteins’ active site. Moreover, through molecular modelling we stepped forward to build new molecules with advanced properties. These findings represent a prompting to search systematically for the possible interaction of the complexes with other cellular elements, which is one of the most important goals in drug design. The aim of this study was to investigate the in vitro activity of copper chelates, against a panel of 2 human cancer cell lines: HT29 (colon), A-549 (lung). Experiments were performed on cells grown in adherence in 96-well microplates and cytotoxicity was evaluated by means of SRB, XTT, WST-1 and BrdU metabolic assays. The drug concentrations that induce cytotoxicity on 50% of cells (IC50) were calculated for each cell line . Beyond the in vitro activity assessment of the compounds, we aimed at the elucidation of the underlying mechanism of action carrying out molecular docking calculations via BioMedCaChe program, which is part of the CaChe package. The molecule was built in 3D coordinates and its best (lower energy) conformation was detected, by geometrical optimization of its structure as implemented in the same molecular modeling program suite. Geometry optimization was accomplished via DFT calculations. (Functional: B88PW91, Basis set: DGDZVP). To identify molecular determinants responsible for the binding mode of the molecules, we have used in our calculations the determined X-ray crystal structures of human DNA topoisomerases I (2.1 Å resolution, PDB entry code: 1K4T) & II (2.51 Å resolution, PDB entry code: 1ZXN), obtained from the Brookhaven Protein Data Bank (RCSB). CONCLUSION RESULTS In vitro results revealed that Cu(SalNEt2)citrate, - tartrate, - salicylate complexes, caused a dose-dependent inhibition of cell proliferation. The found IC50 values (microg/ml) are: 52.5 (HT29), 156.0 (A-549) for Cu(SalNEt2)citrate, 100.1 (HT29), 82.6 (A-549) for Cu(SalNEt2)tartrate and 99.8 (HT29) , 103.5 (A-549) for Cu(SalNEt2)salicylate. The elucidation of copper complexes’ molecular mechanism of action regarding DNA topoisomerase pathway was attempted via molecular docking studies with the crystallographic structures of the proteins. All copper chelates were found to be positioned inside the binding cavity of both proteins. The best possible binding modes of copper (II) complexes in DNA topo I & II are displayed in Figures 1, 2. The interactions between Cu(SalNEt2)Citrate complex and DNA topo I are mainly governed by hydrogen bonds and polar contacts with residues Asn722 and Glu332. Furthermore, the Cu-citrate complex interacted with nucleobases G12, T10 , C112.Binding with DNA topo II, are mediated through residues Glu31 ,Glu185 , Tyr186 , Lys131 , Val30. Cu(SalNEt2)tartrate complex interacts with the residues Asp 533, Arg 364, Thr 718 of DNA top I and with Lys 131, Tyr 186, Ser 29 of DNA top II. Within the binding cavity of DNA topo I Cu(SalNEt2)salicylate complex interacts with nucleobase C112 and amino acid Asp 533 and with Lys 131 in the active side of DNA topoisomerase II. Undertaken docking approach could be informative in pointing out the mechanism of action of the complex in a molecular level. Docking studies revealed the functional role of amino acids and nucleobases, for the copper(II) chelates complexation with the proteins. These findings represent a prompting to search systematically for the possible interaction of the complex with other cellular elements in signal transduction which are of fundamental importance in cell proliferation. New insights in the mechanism of interaction may come into light through further molecular modeling experiments which are underway. Figure 3: (a)LUMO of Cu(SalNEt2)citrate (b) HOMO of guanine (c) adduct a b c c a b Figure 4: (a)LUMO of Cu(SalNEt2)citrate (b) HOMO of glutamic acid (c) adduct a b c Figure 1: Cu(SalNEt2)citrate, (a)- tartrate, (b) – salicylate (c) docked into DNA topoisomerase I Figure 6:DNA topoisomerase II c a b 6 5 Figure 2: Cu(SalNEt2)citrate, (a)- tartrate, (b) – salicylate (c) docked into DNA topoisomerase II Figure 5:DNA topoisomerase I