SELECTIVE INHIBITORS OF 5´-NUCLEOTIDASES
Jiøí Brynda1,2, Petr Pachl1,
Ivan Rosenberg 2, Milan Fábry1,
Pavlína Øezáèová1,2
1Institute
of Molecular Genetics, and 2Institute of
Organic Chemistry and Biochemistry AS CR, Flemingovo nám. 2, Praha 6, 16610, Czech
Republic, brynda@img.cas.cz
The monophosphate
5′-nucleotidases are ubiquitous
enzymes that catalyze the dephosphorylation of
nucleoside monophosphates and regulate thus
nucleotide and nucleoside pools in cell [1-6]. The ribonucleotides
and deoxyribonucleotides could be synthesized de novo from low molecular weight
precursors or by a salvage pathway from nucleosides or nucleobases
originating from 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 nucleic acid synthesis. The phosphorylation
by cellular nucleoside kinases is opposed by the activity
of 5'-nucleotidases that dephosphorylate ribo- and deoxyribonucleoside monophosphates [2-4]. 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 aim of this project is
structure-assisted design of highly selective inhibitors of mammalian 5'‑nucleotidases
based on nucleoside phosphonic acid derivative
scaffold. In general, the compounds with a strong and selective inhibitory
potency are of high medicinal interest as antimetabolites
for anticancer and antiviral therapy.
Using the structure of human mitochondrial
5'-nucleotidase [6] and our structure of cytosolic 5'-nucleotidase we
performed in silico
screening of a virtual library containing 29 thousand compounds. A set of
compounds with highest scores was synthesized and screened for their inhibitory
effect toward two isoforms of human 5'-nucleotidase: cytosolic (cdN) and mitochondrial
(mdN).
By this approach we have discover compounds
specifically inhibiting the mdN and cdN with inhibitory potency two orders of magnitude better
compared to the formerly reported mdN and cdN inhibitors, respectively. Structural details of
interactions of newly identified compounds with both nucleotidases
were investigated through determination of high-resolution crystal structures.
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Interactions between deoxyribonucleotide and DNA
synthesis. Annu Rev Biochem., 57 (1988), 349-74.
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.
Acknowledgements. This work was supported by the Grant agency of the Czech Republic
(research project No. GA 203/09/0820). Diffraction data have been collected on
BL14.1 and BL14.2 operated by the Helmholtz-Zentrum
Berlin (HZB) at the BESSY II electron storage ring.