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J Biol Chem. 2012 Oct 12;287(42):35768-78. doi: 10.1074/jbc.M112.396911. Epub 2012 Aug 22.

Mechanistic studies of semicarbazone triapine targeting human ribonucleotide reductase in vitro and in mammalian cells: tyrosyl radical quenching not involving reactive oxygen species.

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1
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

Abstract

Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β(2) subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(Fe(III)(2)-Y·)(Fe(III)(2))] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of β(2) have been proposed; one involves direct iron chelation from β(2), whereas a second involves Y· destruction by reactive oxygen species formed in situ in the presence of O(2) and reductant by Fe(II)-(3-AP). Inactivation of β(2) can thus arise from cofactor destruction by loss of iron or Y·. In vitro kinetic data on the rates of (55)Fe and Y· loss from [((55)Fe(III)(2)-Y·)((55)Fe(III)(2))]-β(2) under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid β(2) inactivation. Oxyblot(TM) and mass spectrometric analyses of trypsin-digested inhibited β(2), and lack of Y· loss from H(2)O(2) and O(2)(•) treatment together preclude reactive oxygen species involvement in Y· loss. Three mammalian cell lines treated with 5 μm 3-AP reveal Y· loss and β(2) inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(Fe(III)(2)-Y·)(Fe(III)(2))]-β(2) in lysates, similar iron-content in β(2) immunoprecipitated from 3-AP-treated and untreated [(55)Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β(2) are most consistent with Y· loss being the predominant mode of inhibition, with β(2) remaining iron-loaded and stable. A model consistent with in vitro and cell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y· of the [(Fe(III)(2)-Y·)(Fe(III)(2))] cofactor of β(2).

PMID:
22915594
PMCID:
PMC3471726
DOI:
10.1074/jbc.M112.396911
[Indexed for MEDLINE]
Free PMC Article

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