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DYNAMICAL MODEL OF A TYPE II DNA TOPOISOMERASE BIOLOGICAL NANOMACHINE AND DESIGN OF CATALYTIC INHIBITORS

ARRS project code: J1-4402

Principal investigator:: Assoc. Prof. Andrej Perdih

PROJECT DESCIPTION

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.

RESEARCH CHALLENGE 1: Deciphering the mechanism of a type II topoisomerase nanomachine

Type II topoisomerases are complex biological nano machines that have been investigated before, yet available structural and biochemical data about the topoisomerases II currently provide only a limited insight into the catalytic and conformational aspects of its mechanism such as conformational changes occurring during its catalytic cycle and the mechanism of ATP hydrolysis to name a few.

RESEARCH CHALLENGE 2: Development of safer topoisomerase IIα inhibitors for chemotherapy

Topoisomerase IIα inhibitors-topo II poisons comprise a vital core part of many chemotherapy regimens. However, safer, and comparably efficient molecules with fewer side effects acting via an alternative inhibition mechanism must be discovered. Exactly the mode of action of topo II poisons, the formation of a tertiary complex between DNA, topo IIα and topo II poison drug, resulting in DNA damage is responsible for the severe cardiotoxicity side effects and induction of secondary tumors. In addition, observed resistance of cancer cell topo II poisons also calls for the development of topo II acting molecules.

In this project two PROJECT OBJECTIVES to address these challenges are envisioned:

PROJECT OBJECTIVE 1: Construction of a dynamical model of a type II topoisomerase

Based on experimental information we will utilize molecular simulations, to construct a dynamical model of a type II topoisomerase. First, we will with MD and multiscale QM/MM calculations provide insights into the dynamics of ATPase domain and study the catalytic mechanism of the ATP hydrolysis. Next, we will study the conformational behavior of the complete topo II enzyme with DNA segments at several proposed steps/conformational states of its catalytic cycle. This will generate the atomistic picture of this complex event of DNA topology simplification. We will also perform biochemical experiments to provide some experimental validation of the created molecular models.

PROJECT OBJECTIVE 2: Development of catalytic topo IIα inhibitors

We will explore and validate a new, clinically not yet utilized paradigm of topo IIα inhibition by developing and characterizing novel catalytic inhibitors of topo IIα that target the ATP binding site at its ATPase domain and would be suitable for subsequent preclinical development as anticancer drugs. Probing this alternative topo-IIα inhibition paradigm could potentially lead to safer and comparably effective molecules for this established target. Indeed, the main side effects are directly related to the mechanism of action of existing topo IIα drugs that induce DNA damage, and this inhibition approach can efficiently circumvent this weakness.

Participating organizations in the project:

National Institute of Chemistry, Ljubljana

   

University of Ljubljana, Faculty of Pharmacy  

         

National Institute of Biology, Ljubljana

                   

PROJECT TEAM

I. National institute of Chemistry, Ljubljana

Dr. Andrej Perdih (Principal investigator) SICRIS

Dr. Janez Mavri SICRIS

Dr. Simona Golič Grdadolnik SICRIS

Dr. Jure Borišek SICRIS

Dr. Jernej Stare SICRIS

Dr. Alja Prah SICRIS

Barbara Herlah SICRIS

Iza Ogris SICRIS

Eva Prašnikar SICRIS

II. University of Ljubljana, Faculty of Pharmacy  

Dr. Marija Sollner Dolenc SICRIS

Dr. Izidor Sosič SICRIS

Alja Kodila SICRIS

III. National institute of Biology, Ljubljana

Dr.  Bojana Žegura SICRIS

Dr. Metka Filipič SICRIS

Dr. Katja Kološa SICRIS

Dr. Alja Štern SICRIS

PROJECT WORKFLOW

Project research activities will be conducted in five synergistically linked WORK PACKAGES (WP) as depicted in the scheme bellow. WP1 and WP2 focus primarily on computational work while WP3, WP4, WP5 cover the experimental activities. All this will enable the most efficient implementation of PROJECT OBJECTIVES 1 and 2.

In WP1 and WP2, we will primarily use computational chemistry methods and molecular simulations to derive a dynamic model of the type II topoisomerase. In addition, we will also perform some biochemical experiments to provide experimental support for the derived model results. Then, the simulation-based design of topo IIα catalytic inhibitors will be initiated using static and dynamic drug design methods. Following the design and molecular simulation phases, synthesis and selection of molecules either from the investigated compound libraries or from the proposed optimized analogues of the existing lead compounds will be performed in WP3. All compounds will then be experimentally evaluated at in vitro and cell-based levels in WP4 and WP5. The obtained results will be incorporated into the next compounds optimization step.

PROJECT RESULTS

5. HERLAH Barbara, JANEŽIČ Matej, OGRIS Iza, GOLIČ GRDADOLNIK Simona, KOLOŠA Katja, ŽABKAR Sonja, ŽEGURA Bojana, PERDIH Andrej. Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα: Dynophore-derived discovery. Biomedicine & Pharmacotherapy (175),  2024, 116676. Full text

4. HERLAH, Barbara, PAVLIN, Matic, PERDIH Andrej. Molecular choreography: Unveiling the dynamic landscape of type IIA DNA topoisomerases before T-segment passage through all-atom simulations. International Journal of Biological Macromolecules, 2024, 269, 131991 Full text

3. PAVLIN, Matic, HERLAH, Barbara, VALJAVEC, Katja, PERDIH, Andrej. Unveiling the interdomain dynamics of type II DNA topoisomerase through all-atom simulations : implications for understanding its catalytic cycle. Computational and Structural Biotechnology Journal. 2023, vol. 21, str. 3746-3759. Full text

2.  CVIJETIĆ, Ilija N., HERLAH, Barbara, MARINKOVIC, Aleksandar, PERDIH, Andrej, BJELOGRLIC, Snezana K. Phenotypic discovery of thiocarbohydrazone with anticancer properties and catalytic inhibition of human DNA topoisomerase IIα.Pharmaceuticals. 2023, vol. 16, iss. 3, [article no.] 341. Full text

1. OGRIZEK, Mitja, JANEŽIČ, Matej, VALJAVEC, Katja, PERDIH, Andrej. Catalytic mechanism of ATP hydrolysis in the ATPase domain of human DNA topoisomerase IIα. Journal of chemical information and modeling. 2022, vol. 62, iss. 16, str. 3896–3909. Full text

FUNDING

The reserach project is funded by the Slovenian Reserach Agency (ARRS) with 2187 annual hours of price class C for a period of 3 years.

Funding period: 1. 10. 2022 - 30. 9. 2025

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