Structure-based drug design of selective 5'-nucleotidases inhibitors

Petr Pachl¹, Jiří Brynda^1,2, Ivan Rosenberg ², Milan Fábry¹, Pavlína Řezáčová^1,2

¹Institute of Molecular Genetics, Flemingovo nam. 2, Prague 6, Prague, 16610, Czech Republic, ²Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo nam. 2, Prague 6, Prague, 16610, Czech Republic

The monophosphate 5'-nucleotidases, including 5'(3')-deoxyribonucleotidase, belong to a family of enzymes that catalyze the dephosphorylation of nucleoside monophosphates. The ribonucleotides and deoxyribonucleotides can be synthesized de novo from low-molecular-weight precursors or by salvage from nucleosides or nucleobases produced in catabolism of nucleic acids[1]. In this salvage pathway, ribonucleotides and deoxyribonucleotides are phosphorylated by nucleoside and nucleotide kinases to maintain sufficient pools of dNTP's and NTP's for synthesis of DNA and RNA, respectively. The phosphorylation by cellular nucleoside kinases is opposed by 5'-nucleotidases that dephosphorylate ribo- and deoxyribonucleoside monophosphates[2,3,4]. Besides their role in the regulation of physiological dNTP pools, substrate cycles between ribonucleotidases and kinases may affect the therapeutic action of pyrimidine nucleoside analogs used as anticancer and antiviral agents. Such compounds require the nucleoside kinases activity for phosphorylation to their active forms. Results of clinical and in vitro studies propose that an increase in nucleotidase activity can interfere with nucleoside analogue activation resulting in drug resistance[5].

The main goal of this project is the search for potent and selective inhibitors of mammalian 5'-nucleotidases based on nucleoside phosphonic acids and their derivatives and comparison of sensitivity of 5'-nucleotidases isolated from various sources toward individual inhibitors.

We have prepared 2 types of human 5'-nucleotidase: cytosolic and mitochondrial by recombinant expression in E. coli. Two strategies of finding potential inhibitors are used. First, a random series of nucleoside phosphonic acids derivatives are tested. Second, testing of rationally designed compounds based on a published structure of known inhibitor-enzyme complex[6]. In this structure-based design we have already found 100x better inhibitor of mitochondrial nucleotidase and some other promising compounds.

In general, compounds of strong and selective inhibitory potency are of high medicinal interest as antimetabolites for anticancer and antiviral therapy.

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[2] S.A. Hunsucker, B.S. Mitchell, J. Spychala: The 5'-nucleotidases as regulators of nucleotide and drug metabolism. Pharmacol Ther., 107 (2005), 1-30.

[3] V. Bianchi, E. Pontis, P. Reichard: Interrelations between substrate cycles and de novo synthesis of pyrimidine deoxyribonucleoside triphosphates in 3T6 cells. Proc Natl Acad Sci U S A, 83 (1986), 986-90.

[4] P. Bianchi, E. Fermo, F. Alfinito, C. Vercellati, M. Baserga, F. Ferraro, I. Guzzo, B. Rotoli, A. Zanella: Molecular characterization of six unrelated Italian patients affected by pyrimidine 5'-nucleotidase deficiency. Br J Haematol, 122 (2003), 847-51.

[5] C. Mazzon, C. Rampazzo, M.C. Scaini, L. Gallinaro, A. Karlsson, C. Meier, J. Balzarini, P. Reichard, V. Bianchi: Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs, inhibitors and implications for therapy. Biochem Pharmacol, 66 (2003), 471-9.

[6] A. Rinaldo-Matthis, C. Rampazzo, J. Balzarini, P. Reichard, V. Bianchi, P. Nordlund: Crystal Structures of the Mitochondrial Deoxyribonucleotidase in Complex with Two Specific inhibitors. Mol Pharmacol, 65 (2004), 860-867