Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): a combined experimental and theoretical study

Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper(II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron(II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical.     

Ribonucleotide reductase inhibition by p-alkoxyphenols studied by molecular docking and molecular dynamics simulations

Ribonucleotide reductase (RNR) is necessary for production of the precursor deoxyribonucleotides for DNA synthesis. Class Ia RNR functions via a stable free radical in one of the two components protein R2. The enzyme mechanism involves long range (proton coupled) electron transfer between protein R1 and the tyrosyl radical in protein R2. Earlier experimental studies showed that p-alkoxyphenols inhibit RNR. Here, molecular docking and molecular dynamics simulations involving protein R2 suggest an inhibition mechanism for p-alkoxyphenols . A low energy binding pocket is identified in protein R2. The preferred configuration provides a structural basis explaining their specific binding to the Escherichia coli and mouse R2 proteins. Trp48 (E. coli numbering), on the electron transfer pathway, is involved in the interactions with the inhibitors. The relative order of the binding energies calculated for the phenol derivatives to protein R2 is correlated with earlier experimental data on inhibition efficiency, in turn related to increasing size of the hydrophobic alkyl substituents. Using the configuration identified by molecular docking as a starting point for molecular dynamics simulations, we find that the p-allyloxyphenol interrupts the catalytic electron transfer pathway of the R2 protein by forming hydrogen bonds with Trp48 and Asp237, thus explaining the inhibitory activity of p-alkoxyphenols.     

The first holocomplex structure of ribonucleotide reductase gives new insight into its mechanism of action

Ribonucleotide reductase is an indispensable enzyme for all cells, since it catalyses the biosynthesis of the precursors necessary for both building and repairing DNA. The ribonucleotide reductase class I enzymes, present in all mammals as well as in many prokaryotes and DNA viruses, are composed mostly of two homodimeric proteins, R1 and R2. The reaction involves long-range radical transfer between the two proteins. Here, we present the first crystal structure of a ribonucleotide reductase R1/R2 holocomplex. The biological relevance of this complex is based on the binding of the R2 C terminus in the hydrophobic cleft of R1, an interaction proven to be crucial for enzyme activity, and by the fact that all conserved amino acid residues in R2 are facing the R1 active sites. We suggest that the asymmetric R1/R2 complex observed in the 4A crystal structure of Salmonella typhimurium ribonucleotide reductase represents an intermediate stage in the reaction cycle, and at the moment of reaction the homodimers transiently form a tight symmetric complex.     

Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd)

Motexafin gadolinium (MGd) is an expanded porphyrin anticancer agent which selectively targets tumor cells and works as a radiation enhancer, with promising results in clinical trials. Its mechanism of action is oxidation of intracellular reducing molecules and acting as a direct inhibitor of mammalian ribonucleotide reductase (RNR). This paper focuses on the mechanism of inhibition of RNR by MGd. Our experimental data present at least two pathways for inhibition of RNR; one precluding subunits oligomerization and the other direct inhibition of the large catalytic subunit of the enzyme. Co-localization of MGd and RNR in the cytoplasm particularly in the S-phase may account for its inhibitory properties. These data can elucidate an important effect of MGd on the cancer cells with overproduction of RNR and its efficacy as an anticancer agent and not only as a general radiosensitizer.     

Identification of A Free Radical and Oxygen Dependence of Ribonucleotide Reductase in Yeast

Ribonucleotide reductase is a key enzyme for DNA biosynthesis. The enzymes isolated from animal and plant cells possess a stable tyrosyl free radical which is essential for catalysis. Fungal ribonucleotide reductases are little known; the partially characterized enzyme from yeast cells proved exceptionally shortlived, and a free radical could not as yet be demonstrated. We here show that a doublet ESR signal centered at g = 2.0046 can be measured below 60°K in rapidly purified protein samples which is very similar to the ESR spectra of the tyrosine radicals present in other eukaryotic ribonucleotide reductases in structure, microwave saturation, and quenching by hydroxyurea. Because generation of these radicals requires oxygen, anaerobic yeast cultures were also studied. No change in ribonucleotide reductase was observed at 50ppm residual oxygen in the gas phase, but cell proliferation ceased entirely under complete anaerobiosis.     

Identification of A Free Radical and Oxygen Dependence of Ribonucleotide Reductase in Yeast

Ribonucleotide reductase is a key enzyme for DNA biosynthesis. The enzymes isolated from animal and plant cells possess a stable tyrosyl free radical which is essential for catalysis. Fungal ribonucleotide reductases are little known; the partially characterized enzyme from yeast cells proved exceptionally shortlived, and a free radical could not as yet be demonstrated. We here show that a doublet ESR signal centered at g = 2.0046 can be measured below 60°K in rapidly purified protein samples which is very similar to the ESR spectra of the tyrosine radicals present in other eukaryotic ribonucleotide reductases in structure, microwave saturation, and quenching by hydroxyurea. Because generation of these radicals requires oxygen, anaerobic yeast cultures were also studied. No change in ribonucleotide reductase was observed at 50ppm residual oxygen in the gas phase, but cell proliferation ceased entirely under complete anaerobiosis.     

Assay of UDP, GDP, CDP, and ADP reductase activities by column chromatography on polyethyleneimine cellulose

Deoxyribonucleosides were separated from ribonucleosides by chromatography on polyethyleneimine cellulose columns (Pasteur pipettes. The deoxyribonucleosides were quantitatively eluted with 25 mM boric acid in less than 10 ml while the ribonucleosides were retained. The ribonucleosides were eluted with 1 M NaCl. This method was utilized to assay for GDP, UDP, ADP, and CDP reductase activities after hydrolysis of the substrate and product nucleotides to the corresponding nucleosides. All four reductase activities were assayed using identical conditions of column size, eluting solution (25 mM boric acid), and elution volume. The use of polyethyleneimine cellulose columns with boric acid can be adapted to other enzyme assays such as purine nucleoside phosphorylase and for the isolation of deoxyribonucleotides from cellular extracts.     

Ribonucleotide Reductases: Radical Chemistry and Inhibition at the Active Site

Abstract

Ribonucleoside 5′-diphosphate reductases (RDPRs) have been studied for several decades. Increasingly sophisticated mechanisms have been proposed for the reduction of natural substrate ribonucleotides to their 2′-deoxy counterparts and for mechanism-based inactivation of RDPRs with 2′-substituted-ribonucleotides. We now discuss biomimetic reactions of model substrate and inhibitor analogues, which clarify three aspects of previously proposed mechanisms postulated to occur at the active site of RDPRs.     

Inhibition of aldose reductase by dietary antioxidant curcumin: Mechanism of inhibition, specificity and significance

Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. In this study we show that curcumin inhibits ALR2 with an IC₅₀ of 10 μM in a non-competitive manner, but is a poor inhibitor of closely-related members of the aldo-keto reductase superfamily, particularly aldehyde reductase. Results from molecular docking studies are consistent with the pattern of inhibition of ALR2 by curcumin and its specificity. Moreover, curcumin is able to suppress sorbitol accumulation in human erythrocytes under high glucose conditions, demonstrating an in vivo potential of curcumin to prevent sorbitol accumulation. These results suggest that curcumin holds promise as an agent to prevent or treat diabetic complications.     

Impact of tumor-specific targeting and dosing schedule on tumor growth inhibition after intravenous administration of siRNA-containing nanoparticles

This study addresses issues of relevance for siRNA nanoparticle delivery by investigating the functional impact of tumor-specific targeting and dosing schedule. The investigations are performed using an experimental system involving a syngeneic mouse cancer model and a theoretical system based on our previously described mathematical model of siRNA delivery and function. A/J mice bearing subcutaneous Neuro2A tumors approximately 100 mm(3) in size were treated by intravenous injection with siRNA-containing nanoparticles formed with cyclodextrin-containing polycations (CDP). Three consecutive daily doses of transferrin (Tf)-targeted nanoparticles carrying 2.5 mg/kg of two different siRNA sequences targeting ribonucleotide reductase subunit M2 (RRM2) slowed tumor growth, whereas non-targeted nanoparticles were significantly less effective when given at the same dose. Furthermore, administration of the three doses on consecutive days or every 3 days did not lead to statistically significant differences in tumor growth delay. Mathematical model calculations of siRNA-mediated target protein knockdown and tumor growth inhibition are used to elucidate possible mechanisms to explain the observed effects and to provide guidelines for designing more effective siRNA-based treatment regimens regardless of delivery methodology and tumor type.     

Manganese-dependent ribonucleotide reductase ofPropionibacterium freudenreichii subsp.shermanii: Partial purification, characterization, and role in DNA biosynthesis

LikeLactobacillus leichmanii, Rhizobium meliloti, andEuglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids,P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with partially purified ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein components, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficientP. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.     

Inhibition of chlamydial class Ic ribonucleotide reductase by C‐terminal peptides from protein R2

Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese–iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C‐terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical‐containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese–iron cofactor is similar to that of the tyrosyl‐radical‐containing RNRs, involving reversible long‐range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2‐derived oligopeptide blocks the transfer pathway. We have constructed three‐dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20‐mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C‐terminus of protein R2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase,an enzyme essential for the alleviation of oxidative stress

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H₂O₂. We show that the E. coli AhpC (EcAhpC, 187 residues) forms a decameric ring structure under reduced and close to physiological conditions, composed of five catalytic dimers. Single particle analysis of cryo-electron micrographs of C-terminal truncated (EcAhpC_1 -172 and EcAhpC_1 -182) and mutated forms of EcAhpC reveals the loss of decamer formation, indicating the importance of the very C-terminus of AhpC in dimer to decamer transition. The crystallographic structures of the truncated EcAhpC_1 -172 and EcAhpC_1 -182 demonstrate for the first time that, in contrast to the reduced form, the very C-terminus of the oxidized EcAhpC is oriented away from the AhpC dimer interface and away from the catalytic redox-center, reflecting structural rearrangements during redox-modulation and -oligomerization. Furthermore, using an ensemble of different truncated and mutated EcAhpC protein constructs the importance of the very C-terminus in AhpC activity and in AhpC–AhpF assembly has been demonstrated.     

Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase.

Mycobacterium tuberculosis ribonucleotide reductase (RNR) is a potential target for new antitubercular drugs. Herein we describe the synthesis and evaluation of peptide inhibitors of RNR derived from the C-terminus of the small subunit of M. tuberculosis RNR. An N-terminal truncation, an alanine scan and a novel statistical molecular design (SMD) approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied in this study. The alanine scan showed that Trp5 and Phe7 were important for inhibitory potency. A quantitative structure relationship (QSAR) model was developed based on the synthesized peptides which showed that a negative charge in positions 2, 3, and 6 is beneficial for inhibitory potency. Finally, in position 5 the model coefficients indicate that there is room for a larger side chain, as compared to Trp5.     

Benzoxazinone-thiosemicarbazones as antidiabetic leads via aldose reductase inhibition: Synthesis,biological screening and molecular docking study

Aldose reductase is an important enzyme in the polyol pathway, where glucose is converted to fructose, and sorbitol is released. Aldose reductase activity increases in diabetes as the glucose levels increase, resulting in increased sorbitol production. Sorbitol, being less cell permeable tends to accumulate in tissues such as eye lenses, peripheral nerves and glomerulus that are not insulin sensitive. This excessive build-up of sorbitol is responsible for diabetes associated complications such as retinopathy and neuropathy. In continuation of our interest to design and discover potent inhibitors of aldo-keto reductases (AKRs; aldehyde reductase ALR1 or AKR1A, and aldose reductase ALR2 or AKR1B), herein we designed and investigated a series of new benzoxazinone-thiosemicarbazones (3a-r) as ALR2 and ALR1 inhibitors. Most compounds exhibited excellent inhibitory activities with IC₅₀ values in lower micro-molar range. Compounds 3b and 3l were found to be most active ALR2 inhibitors with IC₅₀ values of 0.52 ± 0.04 and 0.19 ± 0.03 μM, respectively, both compounds were more effective inhibitors as compared to the standard ALR2 inhibitor (sorbinil, with IC₅₀ value of 3.14 ± 0.02 μM).     

Cellular knock-down of quinone reductase 2: a laborious road to successful inhibition by RNA interference

NRH:quinone oxidoreductase 2 (QR2) is a long forgotten oxidoreductive enzyme that metabolizes quinones and binds melatonin. We used the potency of the RNA interference (RNAi)-mediated gene silencing to build a cellular model in which the role of QR2 could be studied. Because standard approaches were poorly successful, we successively used: (1) two chemically synthesized fluorescent small interfering RNA (siRNA) duplexes designed and tested for their gene silencing capacity leading to a maximal 40% QR2 gene silencing 48h post-transfection; (2) double transfection and cell-sorting of high fluorescent siRNA-transfected HT22 cells further enhancing QR2 RNAi silencing to 88%; (3) stable QR2 knock-down HT22 cell lines established with H1and U6 promoter driven QR2 short hairpin RNA (shRNA) encoding vectors, resulting in a 71-80% reduction of QR2 enzymatic activity in both QR2 shRNA HT22 cells. Finally, as a first step in the study of this cellular model, we observed a 42-48% reduction of menadione/BNAH-mediated toxicity in QR2 shRNA cells compared to the wild-type HT22 cells. Although becoming widespread and in some cases effective, siRNA-mediated cellular knock-down proves in the present work to be of marginal efficiency. Much development is required for this technique to be of general application.     

Norepinephrine specifically stimulates ribonucleotide reductase subunit R2 gene expression in proliferating brown adipocytes: mediation via a cAMP/PKA pathway involving Src and Erk1/2 kinases

We have examined whether a qualitative switch occurs in the response of the ribonucleotide reductase (RNR) genes to the effect of the physiological cAMP-elevating agent norepinephrine (NE) during the development of brown adipocytes. Basal expression of the genes for both RNR subunits, R1 and R2, was high in proliferating cells, but was markedly down-regulated in parallel with adipocyte differentiation. NE stimulation, which promotes DNA synthesis and proliferation of brown preadipocytes, resulted in an increased expression of the R2 gene in proliferating cells (1.6-fold), but was without effect on R1 expression. In contrast, NE stimulation of confluent differentiating brown adipocytes reduced both R1 and R2 expression. The NE stimulation of R2 expression in preadipocytes was mimicked by forskolin and abolished by H89, demonstrating mediation via cAMP and protein kinase A (PKA). Also, inhibitors of Src and of Erk1/2 kinases markedly reduced NE-stimulated R2 expression. We conclude that adrenergic stimulation of brown adipocytes by NE specifically elevates expression of the RNR subunit R2 gene in the proliferative stage of brown adipocyte development, the mediating pathway being a cAMP/PKA cascade further involving Src and the MAP kinase Erk1/2. These results suggest that adrenergic stimulation of brown adipocyte proliferation may act at the level of gene expression of the limiting subunit for RNR activity, R2, and demonstrate a qualitative switch in the response of the R2 gene to cAMP-elevating agents as a consequence of the switch from proliferating to differentiating cell status.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

Interaction of adenanthin with glutathione and thiol enzymes: Selectivity for thioredoxin reductase and inhibition of peroxiredoxin recycling

The diterpenoid, adenanthin, represses tumor growth and prolongs survival in mouse promyelocytic leukemia models (Liu et al., Nat. Chem. Biol. 8, 486, 2012). It was proposed that this was done by inactivating peroxiredoxins (Prxs) 1 and 2 through the formation of an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidation to a disulfide-linked dimer. However, contrary to the original report, both the peroxidatic and the resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reacting with a second-order rate constant of 25±5 M^–1 s^–1. With 50 µM adenanthin, the peroxidatic and resolving Cys of Prx2 reacted with half-times of 7 and 40 min, respectively, compared with 10 min for GSH. When erythrocytes or Jurkat T cells were treated with adenanthin, we saw no evidence for a reaction with Prxs 1 or 2. Instead, adenanthin caused time- and concentration-dependent loss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normally recycled by thioredoxin reductase and thioredoxin. Our results indicate that Prx reduction was inhibited. We observed rapid inhibition of purified thioredoxin reductase (half-time 5 min with 2 µM adenanthin) and in cells, thioredoxin reductase was much more sensitive than GSH and loss of both preceded accumulation of oxidized Prxs. Thus, adenanthin is not a specific Prx inhibitor, and its reported antitumor and anti-inflammatory effects are more likely to involve more general inhibition of thioredoxin and/or glutathione redox pathways.