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Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs, inhibitors and implications for
Cristina Mazzon1, Chiara Rampazzo, Maria Chiara Scaini
1Department of Biology, University of Padova, 35131 Padova, Italy.
Abstract:
Nucleoside analogs act as prodrugs that must be converted to 5'-phosphates by intracellular kinases to become active in the treatment of viral and oncological diseases. Activation may be reversed by dephosphorylation if the 5'-phosphates are substrates for 5'-nucleotidases. Dephosphorylation by cytosolic enzymes decreases the efficacy of the analogs, whereas dephosphorylation by mitochondrial enzymes may decrease mitochondrial toxicity. Both effects may influence the outcome of therapy. We investigated the dephosphorylation of the 5'-phosphates of commonly used nucleoside analogs by two cytosolic (cN-II and dNT-1) and one mitochondrial (dNT-2) nucleotidase. Most uracil/thymine nucleotide analogs were dephosphorylated by all three human enzymes but cytosine-containing nucleotide analogs were inactive. Only cN-II showed some activity with the monophosphates of the two purine analogs 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosylguanine. We conclude that overproduction of any of the three 5'-nucleotidases cannot explain development of resistance against cytosine analogs but that overproduction of cN-II could lead to resistance against purine analogs. Of the tested analogs, only (E)-5-(2-bromovinyl)-2'-deoxyuridine was preferentially dephosphorylated by mitochondrial dNT-2. We propose that in future developments of analogs this aspect be considered in order to reduce mitochondrial toxicity. We tested inhibition of dNT-1 and dNT-2 by a large variety of synthetic metabolically stable nucleoside phosphonate analogs and found one (PMcP-U) that inhibited dNT-1 and dNT-2 competitively and a second (DPB-T) that inhibited dNT-2 by mixed inhibition. Both inhibitors are useful for specific 5'-nucleotidase assays and structural studies and may open up possibilities for therapy.
Insights
Nucleoside analog drugs can be deactivated by dephosphorylation. This study shows how different nucleotidases affect drug efficacy and toxicity, identifying potential resistance mechanisms and therapeutic strategies.
Area of Science:
- Biochemistry
- Pharmacology
- Molecular Biology
Background:
- Nucleoside analogs are prodrugs requiring intracellular phosphorylation for activity against viral and oncological diseases.
- Dephosphorylation of active 5'-phosphates by 5'-nucleotidases can reduce drug efficacy and alter toxicity.
- Understanding nucleotidase activity is crucial for optimizing nucleoside analog therapy.
Purpose of the Study:
- To investigate the dephosphorylation of nucleoside analog 5'-phosphates by specific human cytosolic (cN-II, dNT-1) and mitochondrial (dNT-2) nucleotidases.
- To determine the impact of nucleotidase activity on the efficacy and toxicity of common nucleoside analogs.
- To identify potential mechanisms of drug resistance and explore strategies for reducing mitochondrial toxicity.
Main Methods:
- Enzymatic assays using purified human cytosolic (cN-II, dNT-1) and mitochondrial (dNT-2) nucleotidases.
- Testing dephosphorylation of various uracil, thymine, cytosine, and purine nucleoside analog 5'-phosphates.
- Evaluating inhibition of dNT-1 and dNT-2 by synthetic nucleoside phosphonate analogs.
Main Results:
- Most uracil/thymine analogs were dephosphorylated by all tested nucleotidases; cytosine analogs were inactive.
- Cytosolic enzyme cN-II showed activity against monophosphates of 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosylguanine.
- (E)-5-(2-bromovinyl)-2'-deoxyuridine was preferentially dephosphorylated by mitochondrial dNT-2.
- Overproduction of cN-II may cause resistance to purine analogs.
- PMcP-U and DPB-T were identified as competitive and mixed inhibitors of dNT-1/dNT-2 and dNT-2, respectively.
Conclusions:
- Dephosphorylation patterns vary significantly among nucleoside analogs and nucleotidases.
- Overproduction of cN-II could be a resistance mechanism for purine analogs.
- Targeting mitochondrial dNT-2 may reduce toxicity of specific analogs like (E)-5-(2-bromovinyl)-2'-deoxyuridine.
- Developed inhibitors (PMcP-U, DPB-T) are valuable tools for nucleotidase research and potential therapeutic development.