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

ABSTRACT: Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the ?(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 ?(2) have been proposed; one involves direct iron chelation from ?(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 ?(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))]-?(2) under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid ?(2) inactivation. Oxyblot(TM) and mass spectrometric analyses of trypsin-digested inhibited ?(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 ?m 3-AP reveal Y· loss and ?(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))]-?(2) in lysates, similar iron-content in ?(2) immunoprecipitated from 3-AP-treated and untreated [(55)Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited ?(2) are most consistent with Y· loss being the predominant mode of inhibition, with ?(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 ?(2).

SUBMITTER: Aye Y

PROVIDER: S-EPMC3471726 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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

Aye Yimon Y Long Marcus J C MJC Stubbe JoAnne J

The Journal of biological chemistry 20120822 42

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 ...[more]

PMID: 22915594

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Project description:Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-sensitive Fe-O-Fe symmetric stretching (483 cm(-1)) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g(1)-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053-33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.

Dataset Information

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

Publications

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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