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 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. 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 tha activity of 5'-nucleotidases that dephosphorylate ribo- and deoxyribonucleoside monophosphates. 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.

The main goal of this project is structure-assisted design of a potent and selective inhibitors of mammalian 5'‑nucleotidases based on nucleoside phosphonic acid derivative scaffold. In general, compounds of a strong and selective inhibitory potency are of high medicinal interest as antimetabolites for anticancer and antiviral therapy.

We have used the structure of human mitochondrial 5'-nucleotidase [6] and 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).

Using this approach we have identified compounds specifically inhibiting mdN and cdN with inhibitory potency being 100 and 50 times better compared to the reported mdN and cdN inhibitors, respectively. Structural details of interactions of newly identified compounds with mdN were investigated through determination of high-resolution crystal structures.

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