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

Background: Diferric-tyrosyl radical [(Fe^III₂-Y·)(Fe^III₂)] cofactor-bearing subunit (β₂) of ribonucleotide reductase is targeted by a Phase-II cancer drug, Triapine (3-AP).

Results: Y· loss precedes iron loss without reactive oxygen species formation.

Conclusion: Fe(II)-(3-AP) inhibits β₂ catalytically resulting in iron-loaded β₂ with a reduced Y·.

Significance: Susceptibility of β₂ to inhibition via Y· reduction by metal complexes implicates a new avenue to develop RNR inhibitors.

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 [(Fe^III₂-Y·)(Fe^III₂)] 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 [(⁵⁵Fe^III₂-Y·)(⁵⁵Fe^III₂)]-β₂ under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid β₂ inactivation. Oxyblot^TM and mass spectrometric analyses of trypsin-digested inhibited β₂, and lack of Y· loss from H₂O₂ and O₂^˙̄ 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 [(Fe^III₂-Y·)(Fe^III₂)]-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 [(Fe^III₂-Y·)(Fe^III₂)] cofactor of β₂.

Introduction

Ribonucleotide reductases (RNRs)² supply the monomeric precursors required for DNA replication and repair (1). Two subunits, (α₂)_m(β₂)_n (m, n = 1–3) constitute active human (h) RNR (1, 2). α₂ contains the site of nucleotide reduction and binds allosteric effectors that control specificity and reduction rate (1–3). β₂ houses a diferric-tyrosyl radical cofactor [(Fe^III₂-Y·)(Fe^III₂)] (Fig. 1) essential for initiating thiyl radical formation in α₂, which initiates nucleotide reduction (3, 4). RNR plays a central role in nucleic acid metabolism (1, 2, 5, 6) and is the target of three cancer drugs used clinically (7), each targeting a different aspect of the RNR complex mechanism of catalysis and regulation (8–12). This paper focuses on understanding how 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine® or 3-AP, Fig. 1) specifically inactivates β₂ of hRNR in vitro and in cultured mammalian cells.

FIGURE 1.

Role of β₂ in hRNR catalysis and impact of β₂-specific inhibition by 3-AP. Holo-β₂ upon association with (α₂)_m constitutes hRNR that catalyzes nucleoside diphosphate (NDP) reduction to dNDPs, one of the key upstream processes that dictates dNTP pool homeostasis. Direct inhibition of β₂ can arise from either loss of Y· (Met-β₂ formation) or Fe^III₂-Y· (apoβ2 formation). Iron-chelating small molecules such as Triapine (3-AP) may also cause indirect inhibition by depletion of intracellular labile iron pool or by Fe(II)-(3-AP)-catalyzed generation of ROS in the presence of O₂. Blue inset, this study in vitro and in live mammalian cells suggests that β₂-specific inhibition involves reduction of Y· elicited by the active reductant, Fe(II)-(3-AP), catalytically recyclable by intracellular reductants, e.g. GSH. This inhibition results in iron-loaded-β₂ with Y· reduced, inferred in our model as Met-β₂ without the need to invoke ROS.

3-AP has efficacy in a variety of cell lines and animal cancer models (13–15). It is currently in phase II clinical trials (16, 17) resulting in renewed interest in its mechanism of cytotoxicity (18–24). Studies to date suggest that multiple mechanisms of 3-AP are involved in its cytotoxicity (13–15, 18–24). 3-AP is a chelator and readily forms both Fe(II) and Fe(III) complexes (19, 25, 26). The Fe(II)-(3-AP) in the presence of O₂ is able to catalyze generation of ROS (19, 27). The Fe^(III)/(II)-(3-AP) reduction potential is accessible in vivo by endogenous reductants (18, 19, 25, 26).

The diverse properties of 3-AP have resulted in a number of models by which it inhibits RNR. In one model free 3-AP is proposed to chelate the Fe(III) directly from the [(Fe^III₂-Y·)(Fe^III₂)] within β₂ (14, 28, 29), resulting in RNR inactivation. In a second model 3-AP is proposed to chelate iron from the intracellular iron pool(s) (18, 20) that could interfere with the essential [(Fe^III₂-Y·)(Fe^III₂)] assembly on β₂ (30). A third model, which is the one currently favored in the literature, is that Fe(III)-(3-AP) is reduced to Fe(II)-(3-AP) by endogenous reductants, which in turn reacts with O₂ and produces ROS that inactivate RNR (21, 27–29). A direct interaction of Y· with O₂^˙̄ has also been proposed to lead to β₂ inhibition observed in vitro (27–29). Although depletion of the labile iron pools and ROS generation are likely involved in late-stage cytotoxic pathways such as induction of apoptosis (21, 23, 24), we will argue that neither mechanism is likely to be important in RNR-specific inhibition. Previous pharmacological studies by Sartorelli and co-workers (13) and Keppler and co-workers (22) that examined the 3-AP-induced late-stage cytotoxicity in a number of cell lines over 2–4 days noted that blockage of DNA synthesis is induced within the initial hours of 3-AP incubation. Studies by Richardson and co-workers (18) have indicated that cellular iron uptake and efflux are minimally perturbed in 3-AP-treated cultured cells in the first hours of exposure. These interesting findings prompted us to undertake a detailed investigation to understand the mechanism of 3-AP-promoted inhibition of β₂ in vitro and in cell culture in the early stages of inhibitor treatment where cell viability remains high, cell cycle is not perturbed, and downstream cytotoxicity is not yet apparent.

Our studies in vitro have investigated the effect of 3-AP and its iron complexes on RNR activity, depletion of its essential Y·, and iron loss from the [(Fe^III₂-Y·)(Fe^III₂)] cluster of β₂ alone and in the active holo-complex [(α₂)_m(β₂] in a non-cycling or cycling state. The relative rates of Y· loss and iron loss have been measured and vary dependent upon the states of β₂ (β₂ alone, cycling- or non-cycling holo-complex). In all cases, however, Y· loss precedes iron loss, suggesting that β₂-specific inhibition can be explained by the direct reduction of Y·. These data implicate Fe(II)-(3-AP) as the reductant that can rapidly reduce Y·. The observations that Y· loss occurs in the presence and, importantly, in the absence of O₂ and that Y· levels are unaffected by O₂^˙̄ or H₂O₂ and the failure to detect oxidative modifications by additional studies using Oxyblot^TM technology and mass spectrometry on inhibited β₂ rule out ROS as the basis for Y· reduction.

We have also investigated the effects of 3-AP in K562, COS-1, and hydroxyurea (HU)-resistant TA3 cells on β₂ activity in cell lysates, ⁵⁵Fe content of β₂ immunoprecipitated from ⁵⁵Fe-labeled cells, the stability of the inhibited β₂, and the Y· levels in intact cells. Furthermore, Oxyblot^TM technology of the whole protein pool failed to reveal differences between 3-AP-treated and untreated cells. Together with in vitro data, the rapid and potent RNR inhibition in cells within 30 min of 3-AP treatment is most consistent with the formation of Fe(II)-(3-AP) from free 3-AP with intracellular Fe, which then engages in the direct reduction of the essential Y· of β₂. The inhibition occurs in the period where cell viability remains high, and the activity of α₂ and other abundant iron-dependent enzymes such as aconitase, a sensitive indicator of oxidative stress, are maintained. These findings further underscore the specificity of 3-AP-promoted β₂-specific inhibition and downplay nonspecific indirect models discussed above. They provide initial insight into understanding how inhibition of β₂ is involved in the cytotoxic effects of 3-AP.

EXPERIMENTAL PROCEDURES

In Vitro Assays on hRNR

All in vitro studies were performed on hRNR subunits recombinantly expressed and purified from Escherichia coli (10, 11). Specific activities for CDP reduction were 700–890 nmol min⁻¹mg⁻¹ for α and 3000–4100 nmol min⁻¹mg⁻¹ for β containing 0.9–1.2 Y·/β₂ and 3.6 iron/β₂. The Y· in the [(Fe^III₂-Y·)(Fe^III₂)] cluster of β₂ has a t½ ∼ 25 min at 37 °C in 50 mm Hepes (pH 7.6), 100 mm NaCl. All inhibition data, unless otherwise noted, have thus been adjusted for this intrinsic instability. The presence of 5 mm DTT in the storage buffer is necessary to preserve α₂ activity; thus in all experiments with holo-complex, 85 μm DTT is present. All experiments were carried out at physiological concentrations of subunits (0.5–2.5 μm (monomer) in 3T6 (6, 31–33), COS-1, HeLa, and NIH-3T3 cells (12)), except the EPR experiments where sensitivity limits require 5 μm β₂.

[(⁵⁵Fe^III₂-Y·)(⁵⁵Fe^III₂)] Cluster Assembly in β₂

Labeling of the active-site diiron center with ⁵⁵Fe and subsequent isolation and characterization are detailed in supplemental Materials and Methods. This procedure typically afforded ⁵⁵Fe-labeled β₂ with 1.0–1.2 Y·/β₂ (quantitated by EPR). The ferrozine assay (34) gave 3.2–3.6 iron/β₂. Non-specifically surface-bound S = 5/2 Fe^III was quantitated by EPR analysis of the same samples at 77 K involving double integration of the g = 4.3 signal relative to Fe^III-EDTA standard as detailed previously (35). It was less than 5% (<0.18 iron/β₂) of the total iron. Specific activity of β₂ reconstituted using an identical procedure to that described above with unlabeled FeCl₃ was 3000–4100 nmol min⁻¹mg⁻¹ of β. Reconstitution and active-site labeling processes were carried out on untagged and His₆-tag β₂ (10) and showed that the tag does not affect activity or the amount of surface-bound iron. All the in vitro experiments thus used tagged β₂. All in vitro experimental procedures are described in detail in the supplemental Materials and Methods.

Studies in Cultured Cells

COS-1, K562, and HU-resistant TA3 cells were selected as representative mammalian cell lines for the following reasons. COS-1 cells typically yield a relatively large quantity of total protein, and the endogenous β₂ activity per mg of total protein is 1.5–3-fold higher than that typically obtained from HeLa and NIH-3T3. K562 was chosen, as previous studies reveal endogenous levels of Y· can be detected by whole cell EPR (36). TA3 cells further support the data from K562 due to enhanced levels of Y· and also allow study of 3-AP in HU-resistant cells. All in-cell experimental procedures are provided in the supplemental Materials and Methods.

RESULTS

Mechanism of hRNR Inhibition by 3-AP in Vitro

All in vitro studies to date on 3-AP-induced inhibition of mouse and hRNR have primarily focused on the analysis Y· loss as a measure of enzyme inactivation (27–29). Because β₂ inhibition can arise from loss of iron, loss of Y·, or both, we have measured independently the effects of 3-AP and its iron complexes on the rates of Y· and active site iron loss.

Time-dependent Inhibition Assays

To establish if RNR activity is depleted in a time-dependent manner and whether this activity loss is associated with β₂ and/or α₂, 0.6 μm β₂ (α₂) was incubated with 0.3, 1, or 5 eq of 3-AP [per β(α)] for the indicated times and diluted into an assay mixture with a 7-fold excess of α₂ (β₂) and incubated an additional 3 min. The results shown in Fig. 2A indicate that with 1 eq or a 5-fold excess of 3-AP per β, >90% of the activity is lost within 10 and 1 min, respectively (Fig. 2A). The α₂ subunit activity under the same conditions remains unchanged (Fig. 2A), validating that 3-AP is a β₂ subunit-specific inhibitor. Incubation of β₂ with 0.3 eq of 3-AP/β, however, resulted in 50% activity loss at 20 min (Fig. 2A), greater than expected if 3-AP itself is the inhibitor. This result suggests that 3-AP has access to iron either from the endogenous loss from β₂ as previously reported in mouse (37) or from nonspecific surface-bound iron associated with the in vitro cluster assembly (10, 11). 3-AP-accelerated iron loss has previously been proposed to play a key role in β₂ inhibition (14, 29). If Fe(II)/Fe(III)-(3-AP) is generated, then in the presence of DTT (85 μm) that always accompanies α₂ in the assays, the metal chelate can redox-cycle (19, 25, 26), potentially causing RNR inhibition. These observations are consistent with previous proposals (27–29) that an iron-complex(es) of 3-AP is(are) the true inhibiting species. The kinetics of iron loss from β₂ and whether it is accelerated by 3-AP were, therefore, examined.

FIGURE 2.

Shown are in vitro kinetic studies on the α₂ and β₂ activity depletion (A), rate of active-site ⁵⁵Fe loss (B–D), or Y· loss (E) in β₂. All error bars are S.D. over duplicate experiments. A, 3-AP-promoted loss of α₂(β₂)-activity (data have been corrected for intrinsic instability) is shown. Inhibition mixture contained [r-His₆-α(β)]₂ = 0.6 μm, [3-AP] = 0, 0.36 (○), 1.2 (♦), or 6.0 (■ for α₂; ▴, for β₂) μm, and 100 μm KCl in 50 μm Hepes (pH 7.6). The assay mixture contained 0.15 μm [r-His₆-α(β)]₂, 1.1 μm [r-His₆-β(α)]₂, 3 mm ATP, 15 mm MgCl₂, and 1.0 mm 5-[³H]CDP, 100 mm KCl, 100 μm Trx, 1 μm Trx reductase, 2 mm NADPH in 50 mm Hepes (pH 7.6). B–D, analysis of the rate of ⁵⁵Fe loss. In all cases, the red square and blue circle, respectively, designate data with and without 3-AP. B is on β₂ alone. The reaction employed 1 μm (r-His₆-β)₂, 10 μm 3-AP, or 5% of DMSO in double distilled H₂O, 100 mm KCl, and 50 mm Hepes (pH 7.6). C is on non-cycling holo-complex. The reaction contained 1 μm (r-His₆-α)₂, 3 mm ATP, and 15 mm MgCl₂ in addition to all the components in B. D is on the cycling holo-complex. The reaction contained 100 μm Trx, 1 μm Trx reductase, 2 mm NADPH, and 1 mm CDP in addition to all the components in C. E, shown is the rate of Y· destruction (corrected for intrinsic Y· decay) analyzed by in vitro EPR experiments: by 3-AP alone (green diamond), Fe(III)-(3-AP) (purple square), Fe(II)-(3-AP) (orange triangle), Fe(III)-(3-AP) in the presence of 5 mm GSH (black inverted triangle, orange star). The orange triangle, black inverted triangle, and orange star constitute data from strictly anaerobic experiments. (○) and (⊗) indicate the effects on β₂ alone by either 0.5 μm (○) or 50 μm (⊗) H₂O₂ and (△) and (□) by either 50 or 1250 molar excess of O₂^˙̄ per β, respectively (generated using xanthine/xanthine oxidase). Representative data shown contained 5 μm (r-His₆-β)₂ and 50 μm 3-AP or its metal-chelate, except in orange star, where 3 μm was used. Also see supplemental Fig. S1A.

Quantitative Assessment of the Rate of Active-site Iron Loss in the Presence and Absence of 3-AP

⁵⁵Fe-Labeled β₂ was assembled in vitro from apoβ2, resulting in specific radioactivity of 652 cpm/nmol of iron with 3.6 iron/β₂ and specific activity in nucleotide reduction and Y· content identical to our previous reports (10, 11). Importantly, EPR analysis showed that the presence of non-specifically surface-bound iron is less than 5% of total iron. Thus ⁵⁵Fe is predominantly associated with the active site, and our assays monitor the rate of ⁵⁵Fe loss from this site. A time course for the rate of iron release at 37 °C in the absence and presence of 3-AP was examined under three different sets of conditions: β₂ alone and β₂ with α₂ in a 1:1 molar ratio (1 μm) either in the presence of the allosteric effector ATP (non-cycling holo-complex) or in the presence of the ATP, CDP, thioredoxin (Trx)/thioredoxin reductase/NADPH reducing system (cycling holo-complex). In the absence of 3-AP, the data reveal that the [(Fe^III₂-Y·)(Fe^III₂)] exhibits very different intrinsic stabilities in the three cases (Fig. 2, B–D). When β₂ is alone (Fig. 2B) or with α₂ in a non-cycling state (Fig. 2C), 20% of the ⁵⁵Fe is lost over 20 min. Because non-specifically bound ⁵⁵Fe is <5% that of total iron, the loss detected is associated with loss from metallo-cofactor. The data additionally demonstrate that the intrinsic iron loss is not associated with DTT. DTT is absent in the β₂ alone experiment but present at 85 μm in the non-cycling holo-complex (compare, Fig. 2, B and C). DTT is required to preserve α₂ stability and is thus carried over from the α₂ storage solution in all in vitro studies on the holo-complex. In contrast, 90% of the iron is spontaneously lost in 20 min under cycling conditions (Fig. 2D). The observed intrinsic lability in the cycling holo-complex is consistent with previous reports on ⁵⁹Fe release from the cycling mouse β₂ in the presence of DTT (∼60% loss in 30 min) (37). Our data reveal that the intrinsic lability of the cofactor is DTT-independent (Fig. 2, B and C) and that the cycling state of holo-hRNR provides an additional mechanism for iron loss.

Identical experiments to those described above were then carried out in the presence of 3-AP. In the case of β₂ alone, a 5-eq excess of 3-AP over β (Fig. 2B) only elicits 20% iron loss in 20 min, similar to the results in its absence (Fig. 2B). This provides compelling evidence that the free ligand does not accelerate active site iron loss on β₂ alone. In contrast, with the addition of α₂, under conditions that result in non-cycling- or cycling-holo-complex, 3-AP greatly enhances the rate of iron release, leading to almost complete loss in 20 min with a t½ of 4.8 min in both cases (Fig. 2, C and D). The observed increase in loss of iron from the holo-complex in the presence of 3-AP likely implies that formation of the holo-complex may lead to a state(s) that allows the free 3-AP to access iron from the active site, a process not feasible in the case of β₂ alone (Fig. 2B). Alternatively, the presence of reductant DTT (carried over with α₂), although having no effect on the intrinsic lability of the cofactor, could redox-cycle the in situ-assembled Fe(III)-(3-AP) such that the diferric center in holo-complex is labilized by reduction. Future experiments should provide a mechanistic explanation of these results. Our focus at this time was to determine whether the varied rates and amounts of iron loss observed in the three states of β₂ studied, and whether Y· loss or both are responsible for β₂ inhibition.

Rate of Y· Loss from [(Fe^III₂-Y·)(Fe^III₂)]-β₂

EPR studies that monitor Y· were carried out first on β₂ alone under a variety of conditions: with 3-AP alone, with Fe(III)-(3-AP), with Fe(II)-(3-AP) ± O₂, and with Fe(III)-(3-AP) and GSH ± O₂ (Fig. 2E). The Y· in hRNR-β₂ is inherently unstable; t½ of 25 min at 37 °C (11). Control experiments measuring the effect of Fe(II), Fe(III), GSH, and combinations of these on the rate of Y· loss indicated minimal changes relative to inherent instability. The data shown in Fig. 2E have been adjusted for the respective intrinsic rates of Y· loss.

In the first experiment 5 eq 3-AP/β was incubated with β₂ alone, and as shown in Fig. 2E, 80% of Y· is lost within 20 min. The measured rate of Y· loss induced by the 3-AP alone is similar to that reported for mouse β₂ in the absence of DTT (29). However, comparison of the rate of Y· loss with the rate of ⁵⁵Fe loss for β₂ alone (Fig. 2B) provides the first quantitative evidence that 3-AP effectively targets Y· during a time frame in which its impact on the diferric center remains minimal (Fig. 2, B versus E). Our in vitro data on β₂ alone thus suggest that Y· quenching is the principle mechanism of enzyme inhibition and leads to iron-loaded β₂.

To further investigate if Y· quenching precedes iron loss in non-cycling holo-complex in the presence of 3-AP, similar EPR analyses were performed and compared with the rates of ⁵⁵Fe loss (Fig. 2, C and E). Y· loss was 80% complete in 1 min (supplemental Fig. S1B), whereas 40% of total iron was lost in the same timeframe (Fig. 2C). Whether the mechanism of iron loss is coupled to Y· loss in this short time frame remains to be established. However, if a single mechanism is at play, iron is lost at a rate 5× slower than Y· over the 20-min duration of the assay.

Rapid Y· Reduction Mediated by Fe(II)-(3-AP)

In an effort to understand the form of 3-AP responsible for inhibition of β₂, loss of Y· was also examined in the presence of Fe(III)-(3-AP), Fe(II)-(3-AP) ± O₂, and with Fe(III)-(3-AP) and GSH ± O₂ (Fig. 2E). With 5 eq of preassembled Fe(III)-(3-AP) (per β), the rate of Y· loss was similar to the case with 5 eq of free 3-AP/β (Fig. 2E). In contrast, when preassembled Fe(II)-(3-AP) was examined, Y· destruction was complete within the first time point. The same rapid Y· loss with a t½ of <0.5 min was observed when the experiment was repeated under strictly anaerobic conditions (Fig. 2E). These data suggest that Fe(II)-(3-AP) is the active species and that ROS are unlikely to be involved.

To provide additional support for proposed role of Fe(II)-(3-AP), the EPR experiment with preassembled Fe(III)-(3-AP) complex and β₂ alone was replicated in an anaerobic chamber in the presence of 5 mm GSH. GSH was chosen as a reductant due to its physiological role and abundance, and the reported range of redox potentials suggests its ability to reduce Fe(III)-(3-AP) (38). The rapid rate of Y· loss with GSH and in the absence of O₂ (Fig. 2E) is consistent with Fe(II)-(3-AP) being the active inhibitor. Importantly, rapid Y· loss is also achieved with substoichiometric amounts of Fe(III)-(3-AP) (0.3 eq/β) when GSH is present (Fig. 2E). These results together suggest that reduction of Y· can be elicited by Fe(II)-(3-AP) catalytically without ROS formation. The chemical mechanism and kinetics of Y· reduction will be the focus of future studies.

EPR Analysis of Y· in β₂ Alone in the Presence of H₂O₂ and O₂^˙̄

The effect of H₂O₂ and O₂^˙̄ was next evaluated. Earlier studies on E. coli β₂ at 25 °C reported H₂O₂-dependent Y· loss (∼30% loss by 5.5 mm H₂O₂ in 1 h) (39) as well as irreversible enzyme inactivation by enzymatically generated O₂^˙̄ (∼80% loss by 3 μm steady-state O₂^˙̄ in 0.5 h) (40). Corresponding experiments on eukaryotic β₂ have not been reported. Incubation of 5 μm hRNR β₂ with 50 or 0.5 mm H₂O₂ at 37 °C (Fig. 2E) showed no appreciable loss of Y· with respect to controls. Similar experiments in the presence of steady-state concentrations of 0.3 and 3 μm O₂^˙̄ generated using xanthine/xanthine oxidase (41–43) revealed that the Y· levels remained unaltered (Fig. 2E). Thus H₂O₂ and O₂^˙̄ are both incapable of inducing Y· loss at the rate observed in the presence of Fe(II)-(3-AP).

In Vitro Oxyblot^TM Analysis of Inhibited β₂ Alone and Cycling- and Non-cycling Holo-complexes

A hallmark of ROS action on proteins via metal-catalyzed oxidation is the formation of protein carbonyls (44). These chemotypes can be probed using Oxyblot^TM (Millipore) analysis in which the carbonyls are derivatized to 2,4-dinitrophenyl hydrazones, which are then detected by Western blotting (45). Thus Oxyblots were used to determine if nonspecific oxidative damage could be responsible for β₂ inhibition by 3-AP. β₂ alone and non-cycling- and cycling holo-complexes were incubated with and without 3-AP for 20 min. Proteins in controls and samples with 3-AP were derivatized with 2,4-dinitrophenylhydrazine and subjected to gel electrophoresis followed by Western blot analysis. Protein carbonyls were not detected within β₂ treated with 3-AP under any conditions examined (supplemental Fig. S2A). A positive control was carried out with β₂ treated with excess ascorbate and iron. SDS-PAGE/Western analysis revealed that β (47 kDa monomer) in these conditions underwent a gel shift to higher a molecular weight species, presumably associated with cross-linking during oxidative damage. Interestingly, Oxyblot analysis on the cycling holo-complex indicated that Trx reductase was oxidized, further indicating that the Oxyblot could readily detect oxidation; no oxidation, however, was associated with β₂ (supplemental Fig. S2A). Omission of 2,4-dinitrophenylhydrazide resulted in complete absence of all the bands. Given the reported sensitivity (fM) of the Oxyblot method, the data imply that Fe(II)-(3-AP)-catalyzed protein oxidation is not involved in β₂ inhibition.

Trypsin Digest LCMS Analysis of Inhibited β₂

To further determine if modification accompanies inhibition of β₂, LCMS analyses were performed on in-gel-digested samples of 5 μm β₂ treated in vitro with no 3-AP but 50 μm concentrations of either Fe(II)-(3-AP) or H₂O₂ for 20 min at 37 °C. The resulting peptides were examined for side-chain oxidation (supplemental Table S1A) and hydroxylation (supplemental Table S1B). Qualitatively, there was no significant difference between these three samples. The modifications detected are likely caused by sample handling during protein isolation, gel electrophoresis, or the in-gel digestion process. The Oxyblot and mass spectrometric data suggest inhibition of β₂ by 3-AP does not result in significant modification of β₂ primary structure.

Mechanism of 3-AP Inhibition in Mammalian Cells

Our attention turned to determine if a similar mechanism occurs in COS-1-, K562-, and HU-resistant TA3 cells exposed to 3-AP.

Loss of β Activity in Cell Lysates from COS-1 and K562 and HU-resistant TA3 Cells Treated with 3-AP

Studies from independent laboratories have reported that cultured cells (HL60 and L1210) exposed to 1–10 μm 3-AP for 0.5–4 h result in complete inhibition of DNA synthesis long before loss of cell viability (2–4 days) (13, 22). Thus our experiments with COS-1 and K562 cells to study the RNR inhibition mechanism were carried out on cells treated with 3-AP (5- 500 μm) over 0.5–3 h where the cells maintained high viability (supplemental Figs. S3 and S4). As shown in supplemental Fig. S5, 30 min of exposure of cells to 3-AP at 5 μm, similar to the in vivo concentration for β₂ (∼2.5 μm monomer) (6, 31–33), resulted in no detectable β₂ activity in the resulting lysates. Under the same conditions, α₂-activity was unaffected. Similar experiments were also carried out with HU-resistant ΤΑ3 cells, previously shown to overexpress β₂ and to have elevated concentrations of Y· by whole-cell EPR analysis (37, 46). Consistent with the higher levels of Y·, our lysate assay data in these cells showed that β₂ activity is ∼4-fold higher than that in COS-1 cells (Fig. 3A versus supplemental Fig. S5A). Treatment of TA3 cells with 5 μm 3-AP for 30 min still completely abolished β₂ activity (Fig. 3A). Our lysate assay data in all three cell lines thus provided direct evidence that 3-AP promotes rapid loss of RNR activity in live cells and is consistent with previous reports on the early inhibition of DNA synthesis in other cell lines studied (13, 22).

FIGURE 3.

3-AP treatment of HU-resistant TA3 cells results in complete loss of β₂-specific activity (A) and Y· (B) but no change in β₂ protein levels (C). A, β₂ activity in resulting lysates from 30-min-3-AP-treated cells (black square, vehicle; purple inverted triangle, 500 μm; orange triangle, 50 μm; green diamond, 5 μm; blue circle, background). See supplemental Fig. S5 for the results with COS-1 and K562 cells. Error bars are S.D. over two independent experiments. B, Y· signal in treated (3 h) (green lines, 5 μm; red lines, 50 μm) and untreated (blue dots) intact TA3 cells analyzed by whole cell EPR. See supplemental Fig. S 6, C and D for the data at 30 min of treatment in TA3 and K562 cells. C, Western blot of β₂ protein levels in treated and untreated TA3 cells at 30 min. See supplemental Fig. S7 for the data from COS-1 and K562 cells. GAPDH was used as the loading control.

Whole Cell EPR Analysis Links β₂ Inhibition to Y· Loss

Whole cell EPR experiments were undertaken using analogous procedures to the previously published reports (36, 37, 46) to probe whether Y· loss from the [(Fe^III₂-Y·)(Fe^III₂)] cofactor of β₂ could account for RNR inactivation. Y· was first shown to be detectable in untreated K562 and TA3 cells at 30 K (supplemental Fig. S6). In the TA3 cells, the higher level of Y· was reflected in a strong signal that are easily monitored even at 77 K (supplemental Fig. S6A), and the signal was identical to that from recombinant [(Fe^III₂-Y·)(Fe^III₂)]-β₂ reconstituted in vitro (supplemental Fig. S6B). Treatment of either TA3 or K562 cells with 5 μm 3-AP abolished the Y· in as early as 30 min (Fig. 3B and supplemental Fig. S6, C and D). Note that the lower concentration of Y· in β₂ from K562 cells is accompanied by higher background signals (supplemental Fig. S6D) as previously reported (36). Thus whole cell EPR analyses and lysate activity assays together demonstrate that 3-AP targets β₂ in these cells.

Levels of β₂ Are Maintained in 3-AP-treated Cells 12 h Subsequent to Activity Loss

The absence of β₂ activity and loss of Y· could be accompanied by conversion of β₂ to an iron-loaded or apo form (Fig. 1). To address this issue, Western blot analysis was carried out on non-synchronized TA3, K562, and COS-1 cells at 30 min (supplemental Fig. S7). These studies showed that in all three cell lines, β₂ levels were similar to untreated cells (Fig. 3C and supplemental Fig. S7). In addition, a time course study showed that β₂ expression was unchanged up to a 12-h period of exposure to 5 μm 3-AP (Fig. 4B). Cells also maintained their high viability over this time (supplemental Fig. S3).

FIGURE 4.

Selective degradation of apoβ2 over holo-β₂ by the proteasome in COS-1 lysates (A) and the prolonged stability of inhibited β₂ in 3-AP-treated COS-1 cells (B). A, apoβ2 is selectively primed for degradation with t½ = 25 min, but holo-β₂ persists. Specific proteasome inhibiting drug, bortezomib (Bo), prevents apoβ2 degradation. B, β₂ protein levels were maintained over the 12-h treatment of COS-1 cells with 5 μm 3-AP. GAPDH was used as the loading control. High cell viability is maintained throughout this period (supplemental Fig. S3A). Activity depletion occurs within 30 min of exposure to 3-AP (supplemental Fig. S5A). Shown here in each diagram are representative Western blots from duplicate sets of experiments.

Analysis of Stability of Apoβ2 and Holo-β₂ in Mammalian Lysates to Proteasomal Degradation

The in vivo data thus far suggest that 3-AP-dependent inhibition is associated with formation of iron-loaded-β₂ but with Y· reduced or apoβ2. However, our in vitro analyses suggest that Y· loss alone is sufficient to account for β₂ inhibition (Fig. 2, B–D, and supplemental Fig. S1). Two approaches were undertaken to determine the cofactor status in inhibited β₂. The cell cycle-dependent periodicity of mammalian β₂ is regulated by ubiquitin-mediated proteasomal degradation during the passage into mitotic (M) phase (6, 47). This knowledge suggests that β₂ can be primed for degradation by the 26 S proteasome, although the cofactor status of the β₂ being degraded during the M phase remains an outstanding interesting question. We thus started our study using a proteasomal degradation assay (48). COS-1 lysates were chosen to allow comparison with Western blot analysis above. Freshly prepared lysates (1 mg/ml) were supplemented with an ATP-regenerating system and incubated with either apoβ2 or [(Fe^III₂-Y·)(Fe^III₂)]-β₂. Aliquots of each reaction were removed at the indicated time points from 0 to 60 min and quenched in Laemmli buffer. Western blot analysis revealed that apoβ2 had a t½ of 25 min, whereas the holo-β₂ level remained unaltered at 1 h (Fig. 4A). To verify that the proteasome was involved in this degradation, bortezomib, a clinically used proteasome inhibitor (49), was added to separate lysates from the same batch as those shown to degrade apoβ2, and the experiment was repeated (Fig. 4A). No degradation was observed, confirming that apoβ2 is targeted by the proteasome.

Stability of Inhibited β₂ Over Prolonged Periods

At this point it is unclear whether β₂ levels (Fig. 4B) are unchanged or they appear unchanged due to up-regulation of β₂ expression resulting from 3-AP treatment compensated for by increased β₂ degradation (consistent with apoβ2 formation). Up-regulation in expression of target enzymes upon inhibitor treatment has been reported in mammalian cells (50 and 68) and in many cases and can occur on a 3-h time scale (51). Cycloheximide (CHX), an inhibitor of new protein synthesis (52), was used with 3-AP-treated and untreated cells to gain mechanistic insight. Because β₂ has a t½ ∼ 3 h in mammalian cells (6, 46, 53), we opted to compare the effect of 3-AP on the levels of β₂ expression in the presence of CHX. Non-synchronized COS-1 cells in logarithmic phase were treated with CHX (0.2 mg/ml) or DMSO for 0.5 h, at which time the media in half of the CHX-treated samples as well as half of DMSO-treated samples were replaced with media that contained 5 μm 3-AP and CHX (0.2 mg/ml) (Fig. 5A, lane c) or 5 μm 3-AP only (lane d). The data from this set were labeled (3-AP + CHX; Fig. 5B). The other half of the samples was retreated with CHX (Fig. 5A, lane a) or DMSO only (lane b). The data from this set were labeled CHX (Fig. 5B). All samples were then incubated for an additional 3 h. This time frame also limits the off-target effects from CHX-induced cytotoxicity (54). Western blot analyses showed that the addition of CHX, as expected, decreased the steady-state level of β₂ by ∼60% over 3 h (Fig. 5, A, compare lanes a and b, and B, CHX), consistent with the reported t½ of β₂ in TA3 and 3T6 cells (6, 46, 53). When this experiment was repeated with 5 μm 3-AP (Fig. 5, A, compare lanes c and d, and B, 3-AP + CHX), a similar ∼60% decrease in β₂ levels was also observed. Thus β₂ stability was unaffected by 3-AP treatment. Given that apoβ2 appears to be unstable in the lysate degradation assay, iron-loaded β₂ is the most probable species remaining in the 3-AP-treated cells.

FIGURE 5.

The observed stability of inhibited β₂ is not a coincidental result of reduced stability of β₂ from 3-AP treatment that is offset by the new protein synthesis. A, COS-1 cells were treated with either CHX (0.2 mg/ml) (lanes a and c) or DMSO (lanes b and d) for 0.5 h, at which time media were replaced. Half of the plates were re-treated with either 5 μm 3-AP + DMSO (lane d) or 5 μm 3-AP + 0.2 mg/ml CHX (lane c) for 3-h-labeled, 3-AP+CHX in B. The other half was retreated with either 0.2 mg/ml CHX (lane a) or DMSO (lane b)-labeled CHX in B. B, shown is a histogram of data from density analysis of four independent experiments, a representative of which is shown in A. Error bars are S.D. over quadruplicate sets of experiments.

⁵⁵Fe-Pulse Labeling and Immunoprecipitation of β₂ Demonstrates No Change in Iron Loss ± 3-AP-treated Cells

The second approach undertaken to examine the state of inhibited β₂ involves immunoprecipitation from 3-AP-treated and -untreated cells that have been prelabeled with ⁵⁵Fe. The labile nature of the diferric center revealed by our in vitro studies on ⁵⁵Fe-labeled β₂ (Fig. 2, B–D) mandates rapid isolation of β₂. An optimized protocol typically affords ∼50 ng of homogeneous β from ∼80 × 10⁶ K562 cells (Fig. 6A). Pulse-labeling was performed according to published protocols (55) using in vitro ⁵⁵Fe-labeled human transferrin (Tf) (2.4 × 10⁵ cpm/nmol of iron with 2 iron/Tf), which was added (at 25 μg/ml) to logarithmically growing K562 cells. Subsequent to 18 h of incubation, the ⁵⁵Fe-labeled Tf-containing media was replaced with fresh media, and the cells were treated with 5 μm 3-AP for 0.5 h or DMSO (control). The cells were then harvested, and the relative levels of ⁵⁵Fe-labeled β₂ between 3-AP-treated and control cells were determined using immunoprecipitation. The results are shown in Fig. 6. Importantly, the observed values were appreciably above the non-specifically bound background values obtained in the replica pulldown experiments in which the antibody was omitted, but the resin maintained (Fig. 6, A, lane d, and B, lane c). As an additional control, the lysate from the untreated, labeled cells was treated with 10 mm HU during the antibody incubation step, and the immunoprecipitation process was repeated. HU is known to labilize the diiron center from the mouse and hRNR β₂ in vitro (56)³ and in mammalian lysates (57). Consistent with these reports, only background levels of iron are associated with β₂ when HU is added (Fig. 6B, lane d). These data in addition to the degradation studies suggest that the iron of β₂ is largely retained during the 30 min of exposure of cells to 5 μm 3-AP, although Y· is reduced.

FIGURE 6.

Effect of 3-AP on the diferric cluster of β₂ immunopurified from treated and untreated ⁵⁵Fe-pulse-labeled K562 cells. A, rapid immunoprecipitation was performed with anti-β₂ antibody (Abcam) conjugated to Protein G-Dyna beads (Invitrogen). a, immunoprecipitated human β₂ band (*, monomer, 45 kDa) was confirmed with Western blotting using human β₂-specific antibody (Ab57653). b, M_r ladders (Bio-Rad). c, PVDF membrane in a was stained with Coomassie Brilliant Blue. * indicates immunoprecipitated β monomer. d, control as in a except buffer replaced Ab57653 but Protein G-Dyna beads were kept. B, shown is the amount of radioactivity associated with isolated β₂ subsequent to 30-min treatment of the prelabeled cells with 5 μm inhibitor. Shown are the results from 3-AP-untreated (a) and -treated cells (b). c, treatment was identical to a, except anti-β₂ antibody was omitted but the beads were retained. d, treatment was a except 10 mm HU was added to the lysate. Error bars are S.D. over duplicate sets of experiments.

An Oxyblot of Lysates of 3-AP-treated Cells Shows the Absence of Protein Carbonyls

As shown above in vitro (supplemental Fig. S2A), further validation of the absence of oxidative carbonylation was probed in whole cell lysates resulting from 3-AP-treated cells and a comparison was made with untreated controls. No change in the extent of carbonylation was observed in any of the cell lines studied (supplemental Fig. S2B), suggesting that 3-AP treatment of the mammalian cells over this time period does not induce oxidative damage.

3-AP Is Specific for hRNR over Other Iron/Sulfur-requiring Enzymes: Lack of Inhibition of Aconitase

Given the recent report on the interconnections between iron/sulfur centers, non-heme iron and heme cofactor biogenesis, and iron homeostasis (58, 59), we examined whether 3-AP was selective for β₂ or whether it might affect the activity of other iron-requiring enzymes. Aconitase that contains an essential [4Fe4S] cluster (60) was chosen as a representative enzyme, as its activity is readily assayed in lysates, the cluster is very sensitive to ROS, and one iron is readily lost from the cluster (61). The proposed chelation property of 3-AP could thus affect the availability of labile iron pools from which the iron/sulfur cluster is biosynthesized and repaired (58, 62). Thus, aconitase is a sensitive probe of several mechanisms postulated for 3-AP. However, subsequent to 3 h of treatment of COS-1 cells with 5 μm 3-AP, no changes in aconitase activity relative to the control were observed. These observations are consistent with other data in this study which indicate that 3-AP plays an active role in RNR inhibition. These results further show that the action of 3-AP is β₂-specific.

DISCUSSION

Phase II clinical trials on 3-AP have sparked a resurgence of interest in the mechanisms by which 3-AP and other semicarbazones result in cell cytotoxicity (16, 17). The studies are complex, as 3-AP and similar species are typically excellent iron chelators (19, 20, 25, 26), and the reduction potential of these complexes allows redox cycling under physiological conditions (19, 25, 26), which can result in production of ROS (21, 27–29). 3-AP, in contrast to other iron chelators, does not appear to sequester iron effectively in the cell (18); thus, it is unlikely to be involved in disruption of biosynthesis and repair of many metallo-cofactor-requiring proteins (30, 62, 63). Early studies in cultured cells established that their treatment with 3-AP resulted in rapid DNA inhibition within the initial hours (13, 22) and that this effect was related to loss of activity of RNR known to require a [(Fe^III₂-Y·)(Fe^III₂)] cofactor (13, 14). This cofactor is common to all class Ia RNRs (holo-β₂, Fig. 1) (3, 4, 30), although the in vitro the half-life of the Y· varies from days (E. coli) to minutes (Pseudomonas aeruginosa and human) (11, 64). In addition, the mouse β₂ under conditions in which deoxynucleoside diphosphates are produced has been reported to spontaneously lose iron (∼60% loss in 30 min at 37 °C) (37). Because the [(Fe^III₂-Y·)(Fe^III₂)] cofactor is essential for RNR activity (3, 4), loss of either Y· or iron cluster results in inhibition.

Studies in cultured cells and xenograft models treated with 3-AP on the other hand have in general focused on assessment of 3-AP-promoted late-stage cytotoxicity (13–15, 18–24, 28, 29). These data suggest that multiple pathways are operative. Postulated mechanisms include deficiency in DNA lesion repair (15), depletion of intracellular iron storage pools (18, 20), ROS-mediated oxidative damage (21, 24, 27–29), and initiation of apoptotic signals (21, 23, 24). Our data now provide a window into the initial period of 3-AP treatment of cells before loss of regulation at multiple levels impact cell viability. A unified model results from comparison of in vitro and cell culture studies despite many remaining questions that need to be resolved.

In vitro

The currently favored model for the mechanism of RNR inhibition by 3-AP in vitro is that Fe(III)-(3-AP) can be reduced to Fe(II)-(3-AP) by DTT in assay buffers (27–29) or β₂ itself (29) and generate ROS that reduce the Y·. Yen and co-workers (27) studying hRNR β₂ alone by EPR analysis showed complete loss of Y· subsequent to a 30-min incubation with a catalytic amount of Fe(II)-(3-AP) (preassembled in the presence of excess DTT). Under the same conditions, Fe(III)-(3-AP) without DTT resulted in ∼60% Y· loss, whereas 3-AP alone resulted in ∼40% Y· loss (see Fig. 4 in Yen and co-workers (27)). These results are similar to our EPR data shown in Fig. 2E. Yen and co-workers (27) further showed using a 5,5-dimethyl-1-pyrroline-N-oxide as a radical trap that incubation of Fe(II)-(3-AP) with O₂ generates ROS and that superoxide dismutase and catalase partially prevented Y· loss. From all their data they concluded that O₂ is important in Y· destruction. Their complex experimental protocol, however, involved preincubation of Fe(II)-(3-AP) for 30 min with catalase alone or in combination with superoxide dismutase before addition to β₂ followed by a further 30-min incubation. The experimental design (27) and the observation that DTT, Fe(II), and O₂ can rapidly regenerate Y· (65) suggest that an alternative interpretation of their results is possible; that is, that Y· is regenerated.

Gräslund and co-workers (29) has also reported time-dependent Y· loss on mouse β₂ alone. Their EPR data in the presence of DTT (see Fig. 6 in Gräslund and co-workers (29)) support Fe(II)-(3-AP) as the active species in Y· reduction, consistent with Yen and co-workers (27, 28) and our own data. However, as in Yen and co-workers (27, 28), Gräslund and co-workers (29) favor the importance of ROS in Y· reduction. This proposal was based on the observation that they observed 100% loss in Y· in 5 min at 37 °C with substoichiometric amounts of Fe(III)-(3-AP) relative to β₂ but only 20% loss under anaerobic conditions (see Fig. 6a in Gräslund and co-workers (29)). The source of reducing equivalents essential for reduction of Fe(III)-(3-AP) to Fe(II)-(3-AP) was suggested to be mouse β₂ itself. We note that with Fe(III)-(3-AP) and O₂, neither our laboratory (Fig. 2E) nor Yen and co-workers (27) see 100% Y· loss, although the possibility remains that hRNR-β₂ is distinct from the mouse enzyme. The basis for the differences in the results remains unresolved. Finally, although both the Yen (27, 28) and Gräslund laboratories (29) have suggested that 3-AP can bind to β₂, in the former case based on binding experiments with 3-[³H]AP and the latter case based on molecular docking and computational analysis, in neither case is the proposal compelling.

We propose an alternative model, consistent with our biochemical studies, that accommodates most of the previously reported results. Our kinetic data on Y· and ⁵⁵Fe loss from β₂ suggest that rapid inhibition of hRNR-β₂ results from reduction of Y· by Fe(II)-(3-AP) by outer sphere electron transfer (Fig. 2, B–E). This mechanism also can account for catalytic cycling of this reductant but does not involve O₂ (Fig. 2E). Our studies further suggest that met-β₂ is likely generated (Fig. 2B) and that this state persists for 20 min, the duration of the assay. Given recent studies on the P. aeruginosa β (64), however, we cannot rule out the possibility that Fe^II₂ is formed and remains tightly bound. Our additional experiments reveal that presence of α₂ appears to allow 3-AP to remove the iron from the holo-complex, although the rate of iron loss is slower than Y· loss (Fig. 2, C and D, supplemental Fig. S1B). Finally, efforts to detect evidence for damage to the inhibited β₂ using oxyblot methods (supplemental Fig. S2A) and mass spectroscopy analysis of peptides of in-gel-trypsinized β₂ (supplemental Table S1) failed to reveal protein damage that could account for its inhibition. Thus our data in vitro show that the inhibition of β₂ occurs without the need to invoke ROS formation. We also observe a distinction between the effects of 3-AP on Y· reduction and chelation of active-site iron (Fig. 2, B--E). A detailed mechanism of Fe(II)-(3-AP) reduction of the Y· remains to be established in vitro.

In Vivo

Our studies in cell culture further support our in vitro mechanism for 3-AP-mediated β₂ inhibition that occurs in the initial hours of drug treatment (0.5–4 h) (13, 22). Evidence is provided from whole cell EPR methods to monitor Y· (Fig. 3B, supplemental Fig. S6, C and D), ⁵⁵Fe-labeled Tf labeling, and immunoprecipitation of the inhibited β₂ to assess iron content (Fig. 6) and oxyblot experiments of the 3-AP treated cells (supplemental Fig. S2B). Our results demonstrate that Y· loss occurs in multiple cell lines after only a 30-min exposure to 3-AP (Fig. 3B, supplemental Fig. S6, C and D). Assays of accompanying cell lysates revealed no detectable RNR activity (Fig. 3A, supplemental Fig. S5) or iron loss in the same time frame (Fig. 6B). Finally, no enhanced oxidation due to 3-AP treatment of the live cells was detected (supplemental Fig. S6B).

In the cell the actual state(s) of β₂ (holo-complex versus β₂ alone) has not been reported. Our studies indicate that the final state of β₂ is iron-loaded with its Y· reduced (Figs. 4A and 6). The inhibited state of β₂ is surprisingly stable even under conditions where new protein synthesis is inhibited (Fig. 5). Our findings suggest that apoβ₂ is not the end product in 3-AP-treated cells. Finally, α₂ is also present in cells, conditions where 3-AP can remove iron from β₂ in a holo-complex based on our in vitro findings (Fig. 2, C and D). Thus, although a portion of β₂ may undergo iron loss, it may be reloaded, giving rise to the observed protein stability in 3-AP-treated cells (Figs. 4B and 5). Preservation of the diiron center in β₂ is consistent with previous studies with SK-N-MC cells treated with 50 μm 3-AP for 3 h which revealed that iron uptake from ⁵⁹Fe-labeled Tf (∼20%) and iron release from ⁵⁹Fe pulse-labeled cells (25%) were both minimal relative to the other iron chelators studied (18). Consistent with these results, whole cell EPR analyses of 3-AP-treated K562 cells (5 or 500 μm) after 0.5, 3, and 12 h showed no change in the size of the intracellular g = 4.3 signals between the treated and untreated cells (data not shown). These observations contrast to the phenotype typically observed when chelation and depletion of intracellular iron pools are in operation (36).

Our studies thus raise interesting questions about the mechanisms by which the diiron cluster of β₂ persists and whether the [(Fe^III₂-Y·)(Fe^III₂)]-β₂ can be regenerated by a maintenance pathway in vivo, with the Y· continually being re-reduced by cycling Fe(II)-(3-AP). The identification of a small molecule that can negatively intercept the regeneration of active cofactor in living mammalian cells (Fig. 1) may ultimately shed light on the [(Fe^III₂-Y·)(Fe^III₂)] cluster biosynthetic and maintenance pathways (30).

Our model is that Fe(II)-(3-AP) is the active species involved in β₂ inhibition (Fig. 1). This proposal is in accord with the reported detection of EPR signals (g = 2) in peripheral blood mononuclear cells collected from patients treated with 3-AP (66) within 2 h of treatment, proposed to be iron- and copper-bound 3-AP. Given the intracellular prevalence of iron relative to Cu (63) and the stability constants for 3-AP (19, 24, 25, 29), Fe(II)-(3-AP) is the most likely active species. Our in vitro studies with ⁵⁵Fe-labeled β₂ suggest the protein itself is capable of providing at least catalytic amounts of iron in all states of β₂ (Fig. 2, B–D); in vivo, however, multiple sources of iron are present. In the continued presence of free ligand in cultured cells, RNR inhibition can persist by the redox cycling capacity of Fe(II)/(III) complex (19, 25, 26), facilitated by intracellular reductants such as GSH (38). Finally, although our model (Fig. 1) primarily focuses on targeting β₂ alone, our data in vitro and in non-synchronized cells suggest that the holo-complex is also susceptible to 3-AP-promoted inhibition by direct Y· reduction (Fig. 2, C and D and supplemental Fig. S1B).

Our findings demonstrate that Y· within hRNR β₂ in any state (alone or non-cycling or cycling holo-complex) is highly susceptible to reduction while being stable to a range of reactive small-molecule oxidants. This observation is significant because a pool of β₂ unbound to (α₂)_m almost certainly exists in cells, and our in vitro data show that this form of β₂ is relatively inert to iron chelation, at least by 3-AP. Thus to design more effective semicarbazone anticancer agents, the efficiency of the reduction of hRNR Y· by the semicarbazone Fe(III)/(II) complexes should be considered alongside other pharmacokinetic properties. Furthermore, because Y· is highly susceptible to reduction, alternative reductants/radical quenchers should be investigated as potential β₂ inhibitors (19). Finally, our studies provide a simple series of experiments that can be used to distinguish between ROS effects and Y· reduction both in vitro and in cells, allowing a more thorough evaluation/optimization of other semicarbazones as potential therapeutics.