Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

Abstract

DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity.

Thiopurine Derivative-Induced Fpg/Nei DNA Glycosylase Inhibition: Structural, Dynamic and Functional Insights

“…The compound 2TX was identified in a DNA base analog screening as an uncompetitive inhibitor of the E. coli formamidopyrimidine-DNA glycosylase (EcFpg) [30]. We confirmed the inhibitory effect of 2TX on ZnF-containing enzymes from the Fpg/Nei DNA glycosylase structural superfamily (including LlFpg, EcNei and hNeil2) [18]. Although the precise mode of action of 2TX remains to be clarified, we established in solution and by X-ray analysis that-unexpectedly-2TX chemically and selectively targets the two most exposed cysteine residues of the ZnF in these enzymes.…”

“…While recent studies have demonstrated the relevance of the research to design innovative anticancer strategies, only a few reported the search for hOgg1 and hNeil1 inhibitors [18][19][20][21]. In previous work, we initiated this study on DNA glycosylases from the structural Fpg/Nei superfamily [18,22,23]. These enzymes recognize and excise oxidized bases in DNA by catalyzing the cleavage of the N-glycosydic bond between the damaged base and its associated sugar.…”

“…In an effort to exploit the extrahelical base lesion recognition mechanism used by these enzymes for removing oxidized bases, 2-thioxanthine (2TX) was identified among other purine and pyrimidine derivatives as an inhibitor of the bacterial Fpg and Nei enzymes and human hNeil2 [18,30]. Surprisingly, enzyme kinetic experiments with the Escherichia coli Fpg protein proposed an uncompetitive inhibition mode.…”

“…Other 2TX enzyme target sites, however, are not excluded, but the irreversible character of the inhibition at a high 2TX concentration compromises the correct interpretation of enzymatic kinetics data. Although the ZnF oxidation mechanism mediated by 2TX remains unclear, it does explain why hNei1, which lacks a ZnF, is resistant to 2TX and why a strong disulfide reducer, such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP), protects the ZnF-containing enzymes from the 2TX inhibitory effect [18].…”

Small Molecule Inhibitors of 8-Oxoguanine DNA Glycosylase-1 (OGG1)

“…40 Similarly, a recently identified Fpg inhibitor also decreased the activity of other closely related DNA glycosylases. 50 Ongoing experiments of OGG1 cocrystallization with these compounds are anticipated to uncover how these molecules display such high specificity.…”