Mechanistic Studies of Semicarbazone Triapine Targeting Human Ribonucleotide Reductase in Vitro and in Mammalian Cells

Abstract

Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β2 subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(FeIII2-Y·)(FeIII2)] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of β2 have been proposed; one involves direct iron chelation from β2, whereas a second involves Y· destruction by reactive oxygen species formed in situ in the presence of O2 and reductant by Fe(II)-(3-AP). Inactivation of β2 can thus arise from cofactor destruction by loss of iron or Y·. In vitro kinetic data on the rates of 55Fe and Y· loss from [(55FeIII2-Y·)(55FeIII2)]-β2 under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid β2 inactivation. OxyblotTM and mass spectrometric analyses of trypsin-digested inhibited β2, and lack of Y· loss from H2O2 and O2̇̄ treatment together preclude reactive oxygen species involvement in Y· loss. Three mammalian cell lines treated with 5 μm 3-AP reveal Y· loss and β2 inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(FeIII2-Y·)(FeIII2)]-β2 in lysates, similar iron-content in β2 immunoprecipitated from 3-AP-treated and untreated [55Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β2 are most consistent with Y· loss being the predominant mode of inhibition, with β2 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 [(FeIII2-Y·)(FeIII2)] cofactor of β2.

Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes

“…Unlike HU, however, neither metal-free triapine nor its metal complexes are potent reductants [22]. Studies on the mechanism of inhibition of RNR by triapine using recombinant RNR proteins in the presence or absence of a reducing agent, generated several models, including inactivation of the enzyme by ROS generated from redox cycling of the triapine/Fe complex, direct reduction of the tyrosyl radical by triapine/Fe 2+ , and mobilization of Fe from the di-iron center by metal-free triapine [22,41,42]. In our study, FeCl 3 at 100 µM in the medium exerted no effect on the cytotoxic activity of triapine (Fig.…”

Effects of Terminal Dimethylation and Metal Coordination of Proline-2-formylpyridine Thiosemicarbazone Hybrids on Lipophilicity, Antiproliferative Activity, and hR2 RNR Inhibition

“…22,23,24 However, quite recently it was found that quenching of the tyrosyl radical is not oxygen dependent, suggesting that it might be reduced directly by the Fe(II)-Triapine complex without involvement of ROS. 25 A second known target for HCTs is topoisomerase IIα (Topo IIα) an enzyme which controls the DNA topology during cell division by inducing temporary double strand breaks. 26,27,28,29 A series of Topo IIα inhibiting HCTs showed high affinity for the enzymes ATP binding pocket, thus acting as catalytic inhibitor of Topo IIα without the generation of DNA double strand breaks.…”

Conserved electron donor complex Dre2–Tah18 is required for ribonucleotide reductase metallocofactor assembly and DNA synthesis

“…However, the self-assembly process is inefficient in general, pointing to the importance of a biosynthesis pathway for controlled cofactor assembly (28). The Y• in cells also can be destroyed rapidly by endogenous reductants or exogenous reducing agents such as hydroxyurea (HU) and triapine (29,30, and thus must be repaired to restore RNR activity (Fig. 1A).…”