Projects

Project J1-4402 is financially supported by the Slovenian Research Agency.

Cancer is one of the leading causes of death. The complex nature of cancer depends on genetic predispositions and environmental influences and poses a major challenge for treatment, as the efficiency of treatment depends on the specific response of each cancer tissue to a particular drug. The exploration of novel molecules that provide an efficient means to combat cancer based on deep understanding of the underlying molecular mechanisms is one of the priorities of the knowledge-based society, and the discovery of anticancer drugs goes down in history as one of the triumphs of human scientific endeavors. In this project, we will investigate type II DNA topoisomerases, biological nanomachines capable of altering the topology of the DNA molecule by creating double-stranded breaks in the first bound DNA molecule thus allowing the second DNA molecule to pass through the break. In this process, they use the energy of ATP hydrolysis, which is converted into mechanical work. A key member of the family is the human DNA topoisomerase IIα, an established anticancer target whose inhibitors have been successfully introduced into clinical practice as part of standard chemotherapy. In revisiting this established anticancer target, we will address two major challenges associated with it. The first challenge is that, despite intensive research, current structural and biochemical data provide only limited atomistic insight into the catalytic and conformational mode of action of type II topoisomerases. The open questions concern the conformational changes coupled with passage of the T segment, movements of the N- and C-terminal domains in the different steps of the catalytic cycle, and the mechanism of ATP hydrolysis. The second challenge is associated with its inhibitors, topo II poisons, which are included in many chemotherapies. The mode of action of these drugs, the formation of a tertiary complex between the DNA, topo IIα and topo II poison resulting in DNA damage, is directly responsible for severe cardiotoxicity and the induction of secondary tumors. Therefore, there is a need for new molecules to improve the safety profile of this approach in cancer treatment. To address the challenges described above, research activities will proceed in two directions. First, based on structural and biochemical information, we will use molecular simulations and multiscale QM /MM calculations to derive a dynamical model of the type II topoisomerase . This will include the study of the catalytic mechanism of ATP hydrolysis and molecular simulation studies of different conformational states/steps in the catalytic cycle. We will also perform some biochemical experiments, for example point mutations in the ATP binding site. The models created will improve the mechanistic understanding of how topo II molecular machine is able to alter DNA topology and provide guidelines for new subsequent experiments. At the same time, we will investigate a therapeutically unexploited paradigm of human topoisomerase IIα inhibition that can circumvent the drawbacks associated with the mechanism of action of topo II poisons. This research involves the development of an integrated computational platform to design and identify catalytic inhibitors that bind to the ATP binding site of the ATPase domain of topoisomerase IIα, thereby stopping the catalytic cycle. Compound screening will utilize a broad chemical space of available and newly synthesized compounds as well as natural products. Selected chemical classes will then be characterized in enzymatic, functional, biophysical, and cell-based in vitro assays to identify and optimize compounds suitable for later development in the drug discovery pipeline.