Mechanistic studies of semicarbazone triapine targeting human ribonucleotide reductase in vitro and in mammalian cells: tyrosyl radical quenching not involving reactive oxygen species.
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).
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Yimon
Y
Aye
Graduate Program in Biochemistry and Biophysics, Brandeis University, Waltham, Massachusetts 02454.
Marcus J C
MJC
Long
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. Electronic address: stubbe@mit.edu.
JoAnne
J
Stubbe
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004795:Enzyme Stability; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D020014:K562 Cells; D051379:Mice; D041681:NIH 3T3 Cells; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology*; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D013882:Thiosemicarbazones / Q000494:pharmacology*
D021122:Protein Subunits; D011725:Pyridines; D013882:Thiosemicarbazones; D013481:Superoxides; C078157:3-aminopyridine-2-carboxaldehyde thiosemicarbazone; D006861:Hydrogen Peroxide; D007501:Iron; D012264:Ribonucleotide Reductases