Experimental and computational investigation of topoisomerase as a possible target of titanium(IV) anticancer complexes

Titanium(IV) complexes, due to their promising activity coupled with lower toxicity and unique chemical versatility, have emerged as alternatives to conventional platinum-based anticancer therapeutics. Several titanium complexes have been found effective against tumors, for instance, titanocene dichloride (TDC) and budotitane. These two titanium(IV) complexes went to clinical trials as non-platinum anticancer candidates, motivated by the clinical success of cisplatin. The main target and focus for the mechanism of action for TDC and budotitane, due to their structural similarities to cisplatin, has been DNA. However, the exact mechanism of action of titanium(IV) anticancer complexes on cancer cells remains largely unknown. DNA topoisomerases have been postulated as one of the possible targets for titanium(IV) anticancer complexes. These enzymes, namely topoisomerase I and II (topo I and II), play crucial roles in controlling and relaxing DNA conformation and topology during the replication process. The main objective of this study is to interrogate the molecular mechanisms of action of selected titanium(IV) complexes, specifically titanium(IV) citrate, titanium(IV) ascorbate, titanium(IV) catecholate, and titanium(IV) desferrioxamine B (DFOB), with a focus on their interaction with DNA and topoisomerase enzymes.To explore the biological interactions of titanium(IV) complexes, a combination of experimental and computational approaches were employed in this study. Human topoisomerase I inhibition assays were performed to evaluate the inhibition ability of titanium(IV) complexes. The magnitude of inhibition activity of five titanium(IV) complexes was quantified as the relaxation of supercoiled plasmid DNA pBR322 using agarose electrophoresis. The assay results revealed that among the tested compounds, titanium (IV) citrate and titanium (IV) catecholate bearing moderately labile oxygen-donor ligands exhibited strong inhibition potential, surpassing other titanium(IV) complexes. The results demonstrate that the inhibition of enzyme activity of titanium(IV) complexes is influenced by ligand type and overall ligand-metal stability. To evaluate the binding strength, mode, and thermodynamic parameters of binding between titanium(IV) complexes and calf thymus DNA (ctDNA), spectroscopic characterization techniques, including fluorescence spectroscopy, circular dichroism (CD), and isothermal titration calorimetry (ITC) were employed. Fluorescence quenching and Stern-Volmer analyses confirmed the tested titanium(IV) complexes partially displaced the intercalated ethidium bromide, and the weak quenching constant suggests these titanium(IV) complexes preferentially associate with ctDNA via outer-sphere coordination rather than - stacking. Circular dichroism (CD) spectra showed minor spectral shifts without loss of the characteristic B-DNA signal, suggesting that titanium(IV)-ctDNA interaction occurs mainly through groove or backbone association rather than intercalation. Isothermal titration calorimetry (ITC) demonstrated strong exothermic binding profiles, indicative of favorable interactions between tested titanium(IV) complexes and ctDNA. Titanium(IV) citrate showed the highest affinity with a strong exothermic enthalpy change. Molecular docking simulations were carried out using AutoDock 4.2 and ORCA to model the interactions between titanium(IV) complexes and biological targets such as topoisomerase I, topoisomerase II, human serum albumin (HSA), and transferrin (sTF). Titanium (IV) citrate ranked strongest by displaying the lowest binding energy and strongest theoretical inhibition constant across all targets. In docking with the topoisomerase I–DNA covalent complex, titanium(IV) citrate was positioned near the catalytic tyrosine–phosphate interface, consistent with its observed enzyme inhibition. Docking with human serum albumin (HSA) and transferrin (sTF) revealed that these proteins can coordinate and stabilize titanium(IV) species, suggesting their possible roles in systemic transport and cellular delivery. Overall, this thesis combines enzymatic, spectroscopic, thermodynamic, and computational evidence to provide a comprehensive view of how titanium(IV) complexes enter cells, bind to biomolecules, and inhibit key enzymes involved in DNA topology. Titanium(IV) citrate emerges as the most biologically active complex, exhibiting strong DNA binding, significant topoisomerase inhibition, and favorable docking interactions.