A Ferrous-triapine complex mediates formation of reactive oxygen species that inactivate human ribonucleotide reductase

Abstract

Ribonucleotide reductase plays a central role in cell proliferation by supplying deoxyribonucleotide precursors for DNA synthesis and repair. The holoenzyme is a protein tetramer that features two large (hRRM1) and two small (hRRM2 or p53R2) subunits. The small subunit contains a di-iron cluster/tyrosyl radical cofactor that is essential for enzyme activity. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a new, potent ribonucleotide reductase inhibitor currently in phase II clinical trials for cancer chemotherapy. Ferric chloride readily reacts with Triapine to form an Fe(III)-(3-AP) complex, which is reduced to Fe(II)-(3-AP) by DTT. Spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide prove that Fe(II)-(3-AP) reduces O 2 to give oxygen reactive species (ROS). In vitro activity assays show that Fe(II)-(3-AP) is a much more potent inhibitor of hRRM2/hRRM1 and p53R2/hRRM1 than Triapine. Electron paramagnetic resonance measurements on frozen solutions of hRRM2 and p53R2 show that their tyrosyl radicals are completely quenched by incubation with Fe(II)-(3-AP). However, the enzyme activity is maintained in protein samples supplemented with catalase alone or in combination with superoxide dismutase. Furthermore, catalase alone or in combination with superoxide dismutase markedly decreases the antiproliferative effect of Triapine in cytotoxicity assays. These results indicate that Triapineinduced inhibition of ribonucleotide reductase is caused by ROS. We suggest that ROS may ultimately be responsible for the pharmacologic effects of Triapine in vivo. [Mol Cancer Ther 2006;5(3):586 -92]

Phase I study of the ribonucleotide reductase inhibitor 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) in combination with high dose cytarabine in patients with advanced myeloid leukemia

“…Methemoglobinemia is a recognized complication of 3-AP administration, and has been linked to the generation of reactive oxygen species [16,17]. The development of clinically significant met-Hg with 3-AP use was initially reported in patients with G6PD deficiency [13].…”

Chelators at the Cancer Coalface: Desferrioxamine to Triapine and Beyond

“…A recent study reported that the Triapine-Fe(II) complex was significantly more active at inhibiting ribonucleotide reductase than free Triapine (20). Triapine did not remove Fe from the active site of R2 or p53R2.…”

“…Triapine did not remove Fe from the active site of R2 or p53R2. Instead, this chelator formed a complex with Fe(III), which was reduced to Fe(II) that generated reactive oxygen species and quenched the ribonucleotide reductase tyrosyl radical (20). This inactivated the enzyme and prevented DNA repair and synthesis (20).…”

Rákellenes tioszemikarbazonok és fémkomplexeik: a stabilitás és a biológiai aktivitás kapcsolata

“…A képzõdõ vas(II)-TSK komplex közvetlenül vagy még inkább az oxigénnel való reakciója során képzõdõ reaktív oxigén származékok (ROS) által képes a katalitikus centrumban lévõ Tyr gyököt kioltani, ami az enzim inaktiválásához vezet. 11 Ennek következtében a TSK-k vas(II/III)ionokkal képzett komplexeinek oldatbeli stabilitása és redoxi tulajdonsága egyértelmûen befolyásol(hat)ja a TSK-k biológiai hatását.…”