A yeast-based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis.

We introduce an approach for detection of drug-protein interactions that combines a new yeast three-hybrid screening for identification of interactions with affinity chromatography for their unambiguous validation. We applied the methodology to the profiling of clinically approved drugs, resulting in the identification of previously known and unknown drug-protein interactions. In particular, we were able to identify off-targets for erlotinib and atorvastatin, as well as an enzyme target for the anti-inflammatory drug sulfasalazine. We demonstrate that sulfasalazine and its metabolites, sulfapyridine and mesalamine, are inhibitors of the enzyme catalyzing the final step in the biosynthesis of the cofactor tetrahydrobiopterin. The interference with tetrahydrobiopterin metabolism provides an explanation for some of the beneficial and deleterious properties of sulfasalazine and furthermore suggests new and improved therapies for the drug. This work thus establishes a powerful approach for drug profiling and provides new insights in the mechanism of action of clinically approved drugs.

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases.

BACKGROUND: (5R) and (5S) diastereomers of 1-[2-deoxy-beta-D-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-beta-D-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2'-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes. METHODS: Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements. RESULTS: In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the kcat/Km ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase. CONCLUSIONS: The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates. GENERAL SIGNIFICANCE: The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.

Radiation-induced DNA damage: formation, measurement, and biochemical features.

Most of the reactions induced by *OH radicals (indirect effects) and by one-electron oxidation (direct effects) as the result of exposure to ionizing radiation may be described for the four main DNA nucleobases. Relevant information is now available on the formation of single and tandem base lesions implicating guanine as the most susceptible DNA component to the deleterious effects of ionizing radiation. In contrast, there is still a paucity of information on the radiation-induced formation of base damage within cellular DNA. This is mostly a result of difficulties associated with the measurement of oxidized purine and pyrimidine bases that appear to be generated in very low yields. This is illustrated by the measurement of low amounts of E. coli formamidopyrimidine glycosylase- and endonuclease-III-sensitive sites in the DNA of neoplastic monocytes upon exposure to gamma rays (48 and 53 per 10(9) bases and per Gy, respectively) using a modified comet assay (the overall number of strand breaks and alkali-labile sites was estimated to be 130 per 10(9) bases and per Gy). More specifically, the level of several radiation-induced modified bases, including thymine glycols, 5-formyluracil, 5-(hydroxymethyl)uracil, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroadenine, together with related formamidopyrimidine derivatives was assessed using the suitable HPLC-MS/MS method. Information is also provided on the substrate specificity of DNA repair enzymes and the mutagenic potential of base lesions using site-specific modified oligonucleotides as the probes.

Recognition of cyclonucleoside lesions by the Lactococcus lactis FPG protein.

Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.

Recent aspects of oxidative DNA damage: guanine lesions, measurement and substrate specificity of DNA repair glycosylases.

This review discusses recent aspects of oxidation reactions of DNA and model compounds involving mostly OH radicals, one-electron transfer process and singlet oxygen (1O2). Emphasis is placed on the formation of double DNA lesions involving a purine base on one hand and either a pyrimidine base or a 2-deoxyribose moiety on the other hand. Structural and mechanistic information is also provided on secondary oxidation reactions of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a major DNA marker of oxidative stress. Another major topic which is addressed here deals with recent developments in the measurement of oxidative base damage to cellular DNA. This has been mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents, including UVA and ionizing radiations. In addition, the modified comet assay, which involves the use of bacterial DNA N-glycosylases to reveal two main classes of oxidative base damage, is applicable to isolated cells and is particularly suitable when only small amounts of biological material are available. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision pathways are briefly reviewed.

Chemical synthesis and biochemical properties of oligonucleotides that contain the (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-deoxyuridine DNA lesion.

The first chemical synthesis of (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-deoxyuridine [(5'S,5S,6S)-cyclo-5-OH-dHdU], a radiation-induced decomposition product of 2'-deoxycytidine in aerated solution, is reported. Subsequently, 2'-deoxycytidine was incorporated into oligodeoxyribonucleotides with defined sequences by using an optimized system of protection that takes into account the reactivity and stability of the modified building blocks. After deprotection and purification, the chemical composition of the modified DNA fragments was assessed by enzymatic digestions and mass spectrometry measurements. The MS analyses confirmed the presence and integrity of the lesion within the synthesized DNA fragments. In vitro replication and repair studies showed that (5'S,5S,6S)-cyclo-5-OH-dHdU acts as a block for DNA polymerases when inserted into DNA oligomers and is not excised by any of the tested DNA N-glycosylases. Therefore, (5'S,5S,6S)-cyclo-5-OH-dHdU may represent a potential lethal lesion within the cell if it is not removed by the nucleotide excision repair machinery.