Searching for the specific inhibitor of S1-P1 nuclease using fragment screening

K. Špeldová^1,2, T. Kovaľ², K. Adámková^2,3, B. Husťáková^2,3, M. Trundová², J. Dušková², P. Kolenko^1,2 and J. Dohnálek²

¹Czech Technical University in Prague, Břehová 7, 115 19 Prague, Czech Republic

²Institute of Biotechnology of the Czech Academy of Sciences, Biocev, Průmyslová 595, Vestec, Czech Republic

³University of Chemistry and Technology Prague, Department of Biochemistry and Microbiology, Technická 5, 166 28 Prague 6, Czech Republic

speldkar@fjfi.cvut.cz

S1-P1 nucleases, hydrolases that cleave phosphodiester bonds of nucleic acids, are found in plants, fungi, bacteria and trypanosomatid [1]. They are dependent on divalent metals such as zinc, calcium or magnesium. These metals are usually found in the active site and form the trinuclear cluster. Another important part of the active site (NSB1) is responsible for binding the nucleobase and sugar group of the substrates [2]. It is theoretically possible to use structure-based inhibitor design in order to synthetize highly specific inhibitors of S1-P1 nucleases.

In order to study the binding of ligands to the S1-P1 nuclease active site the crystallographic fragment screening method was used as it has abundant applications in the discovery of suitable inhibitors and subsequent drug development. S1-P1 nuclease crystals were soaked in ligand solutions prepared using Frag Xtal Screen (Jena Bioscience) and then vitrified. These crystals were then subjected to X-ray structural analysis at the BESSY II synchrotron radiation source in Berlin [3]. The diffraction data were subsequently processed and the structures of the complexes were determined by molecular replacement. Binding of different fragments in the active site of the nuclease was analysed and compared to the known binding of native products.

1. M. Trundova, T. Koval, R.J. Owens, K. Fejfarova, J. Duskova, P. Kolenko, J. Dohnalek, Int J Biol Macromol. 114, (2018), 776-787.

2. T. Koval, J. Dohnálek, Biotechnol Adv. 36, (2018), 603-612.

3. U. Mueller, R. Förster, M. Hellmig, F. U. Huschmann, A. Kastner, P. Malecki, S. Pühringer, M. Röwer, K. Sparta, M. Steffien, M. Ühlein, P. Wilk, M. S. Weiss, European Physical Journal Plus, 130, (2015), 141.

This work was supported by the institutional support of IBT CAS, v. v. i. (RVO: 86652036), from the grant of Specific university research – grant No A1_FPBT_2021_003, Grant Agency of the Czech Technical University in Prague (grant No. SGS19/189/OHK4/3T/14), European Regional Development Fund grants CZ.02.1.01/0.0/0.0/15_003/0000447, and by the Ministry of Education, Youth and Sports of the Czech Republic (LM2015043 and LM2018127, support of Biocev CMS – core facilities Crystallization of Proteins and Nucleic Acids, and Diffraction Techniques of CIISB, part of Instruct-ERIC).