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Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide
W B Parker1, A R Bapat, J X Shen
1Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill 27599.
Abstract:
Recently, 2-halogenated deoxyadenosine analogs (F, Cl, and Br) have been shown to have antitumor activity. These analogs are phosphorylated by cells and are believed to exert their cytotoxic action at the nucleoside triphosphate level. In this work the interaction of these nucleoside triphosphate analogs with potential targets, such as DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase was examined in detail. All of these compounds competitively inhibited the incorporation of dAMP into DNA by DNA polymerase alpha, beta, or gamma. F-dATP was able to completely substitute for dATP using DNA polymerase alpha and gamma, but not with DNA polymerase beta. Cl-dATP and Br-dATP substituted poorly for dATP using DNA polymerase alpha and beta. Extension of a 32P-labeled primer by DNA polymerase alpha, beta, or gamma on a single-stranded M13 template showed that these compounds were incorporated into the 3' end of the growing DNA chain and that elongation beyond the incorporated analogs was significantly retarded for Cl-dATP and Br-dATP using either DNA polymerase alpha or beta. DNA primase using poly(dC) as template was inhibited by these compounds at a concentration 4 to 5 times greater than that required for 2-F-araATP. The 2-halogenated dATP analogs were potent inhibitors of ADP reduction by ribonucleotide reductase. In conclusion, the cytotoxic action of 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine may partially be mediated through the mechanism of "self-potentiation," by depression of the deoxynucleoside triphosphate pools due to inhibition of ribonucleotide reductase, which would facilitate their incorporation into DNA and result in the inhibition of DNA synthesis.
Insights
2-halogenated deoxyadenosine analogs show antitumor activity by inhibiting DNA synthesis. These compounds, particularly chloro- and bromo-deoxyadenosine, may self-potentiate their cytotoxic effects by depleting deoxynucleoside triphosphate pools.
Area of Science:
- Biochemistry
- Molecular Biology
- Anticancer Drug Development
Background:
- 2-halogenated deoxyadenosine analogs (F, Cl, Br) exhibit antitumor properties.
- Cellular phosphorylation leads to nucleoside triphosphate analogs believed to cause cytotoxicity.
Purpose of the Study:
- To investigate the interaction of 2-halogenated deoxyadenosine triphosphate analogs with key enzymes involved in DNA synthesis.
- To elucidate the mechanisms underlying the cytotoxic action of these analogs.
Main Methods:
- Enzyme inhibition assays using DNA polymerase alpha, beta, and gamma, and DNA primase.
- Assessment of analog incorporation into DNA and subsequent chain elongation.
- Evaluation of inhibition of ribonucleotide reductase activity.
Main Results:
- All analogs competitively inhibited DNA polymerase activity.
- F-dATP fully substituted for dATP with DNA polymerase alpha and gamma, but not beta.
- Cl-dATP and Br-dATP incorporation significantly retarded DNA chain elongation and potently inhibited ribonucleotide reductase.
Conclusions:
- 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine cytotoxicity may involve self-potentiation.
- Inhibition of ribonucleotide reductase depletes deoxynucleoside triphosphate pools, enhancing analog incorporation and DNA synthesis inhibition.