Methyl-Hydroxylamine as an Efficacious Antibacterial Agent That Targets the Ribonucleotide Reductase Enzyme

The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound''s activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA) in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RNR enzyme.     

Biofilm Modifies Expression of Ribonucleotide Reductase Genes in Escherichia coli

Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed.     

Bacillus anthracis Thioredoxin Systems,Characterization and Role as Electron Donors for Ribonucleotide Reductase

Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.     

Micro-Array Analysis of Resistance for Gemcitabine Results in Increased Expression of Ribonucleotide Reductase Subunits

To study in detail the relation between gene expression and resistance against gemcitabine, a cell line was isolated from a tumor for which gemcitabine resistance was induced in vivo. Similar to the in vivo tumor, resistance in this cell line, C 26-G, was not related to deficiency of deoxycytidine kinase (dCK). Micro-array analysis showed increased expression of ribonucleotide reductase (RR) subunits M1 and M2 as confirmed by real time PCR analysis (28- and 2.7-fold, respectively). In cell culture, moderate cross-resistance (about 2-fold) was observed to 1-ß-D-arabinofuranosylcytosine (ara-C), 2-chloro-2’deoxyadenosine (CdA), LY231514 (ALIMTA), and cisplatin (CDDP), and pronounced cross-resistance (>23-fold) to 2′,2′-difluorodeoxyuridine (dFdU) and 2′,2′-difluorodeoxyguanosine (dFdG). Culture in the absence of gemcitabine reduced resistance as well as RRM1 RNA expression, demonstrating a direct relationship of RRM1 RNA expression with acquired resistance to gemcitabine.     

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 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.     

Mechanistic Insights into Ferredoxin-NADP(H) Reductase Catalysis Involving the Conserved Glutamate in the Active Site

Plant-type ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes harboring one molecule of noncovalently bound flavin adenine dinucleotide that catalyze reversible reactions between obligatory one-electron carriers and obligatory two-electron carriers. A glutamate next to the C-terminus is strictly conserved in FNR and has been proposed to function as proton donor/acceptor during catalysis. However, experimental studies of this proposed function led to contradicting conclusions about the role of this glutamate in the catalytic mechanism. In the present work, we study the titration behavior of the glutamate in the active site of FNR using theoretical methods. Protonation probabilities for maize FNR were computed for the reaction intermediates of the catalytic cycle by Poisson-Boltzmann electrostatic calculations and Metropolis Monte Carlo titration. The titration behavior of the highly conserved glutamate was found to vary depending on the bound substrates NADP(H) and ferredoxin and also on the redox states of these substrates and the flavin adenine dinucleotide. Our results support the involvement of the glutamate in the FNR catalytic mechanism not only as a proton donor but also as a key residue for stabilizing and destabilizing reaction intermediates. On the basis of our findings, we propose a model rationalizing the function of the glutamate in the reaction cycle, which allows reinterpretation of previous experimental results.     

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.     

Cephaloglycin and Its Biologically Active Metabolite Desacetylcephaloglycin

Chromatographic studies and microbiological assays show that, after oral administration, cephaloglycin is partially converted in man to a biologically active metabolite desacetylcephaloglycin. The antibacterial activity of this metabolite compared to that of cephaloglycin is equivalent against gram-positive organisms but is lower against gram-negative bacilli. Successful therapy of urinary tract infections with cephaloglycin must be mainly attributed to the antibacterial activity of this metabolite. At the present time, it is not possible to assess what influence low amounts of unaltered cephaloglycin have on the outcome of therapy.     

Mechanism-Based Inhibition of Ribonucleotide Reductases: New Mechanistic Considerations and Promising Biological Applications

Abstract

Ribonucleotide reductases (RNRs) perform the de novo biosynthesis of 2′-deoxynucleoside 5′-(di or tri)phosphates. Inhibition of RNRs removes a crucial source of genetic components and enhances the probability of salvage incorporation of analogues into DNA. Several laboratories have clarified aspects of the reaction cascades initiated by generation of substrate nucleotide C3′ free radicals by RNRs. New considerations for radical-mediated mechanism-based inhibition and biological applications are discussed.     

Identification, Development, and Regional Distribution of Ribonucleotide Reductase in Adult Rat Brain

The development and regional distribution of ribonucleotide reductase (EC 1.17.4.1) were determined in rat brain. Ribonucleotide reductase was partially purified by ammonium sulfate fractionation (20-40% saturation). Enzyme activity was measured by a specific radiochemical assay. This method involved the reduction of [¹⁴C]cytidine diphosphate (CDP) to [¹⁴C]deoxy-cytidine diphosphate with subsequent hydrolysis and separation of the product ([¹⁴C]deoxycytidine) from substrate ([¹⁴C]cytidine) by Dowex-1-borate ion-exchange chro-matography. The specific activity of ribonucleotide reductase in whole brain of newborn rats was 3.78 ± 0.55 units (pmol/h)/mg protein (SEM; n = 6) and declined to 0.17 ± 0.01 units/mg protein (n = 7) at 10-12 weeks of age, with a further decline to 0.11 ± 0.01 units/mg protein (n = 3) at 1 year. Ribonucleotide reductase activity in rat liver decreased from 4.58 ± 0.62 units/mg protein (n = 3) in newborn animals to 0.06 ± 0.01 units/mg protein (n = 7) at 10-12 weeks and was present at trace levels at 6 months of age. The decline in specific activity with age was not due to a change in the K_m for CDP. The K_m for CDP in brain of newborn and adult rats was 80-90 μM. In 10- to 12-week-old rats, the specific activity of ribonucleotide reductase was similar in the various regions of the brain tested except for the brainstem, which had 50% lower specific activity than the whole brain. These results indicate that ribonucleotide reductase activity is present and widely distributed in adult rat brain.     

Enzymes Involved in Pyrophosphate and Calcium Metabolism as Targets for Anti‐scuticociliate Chemotherapy

Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca²⁺ homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the in vitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+‐PPases (H+‐PPases). CQ and ART inhibited the in vitro growth of ciliates with IC50 values of respectively 74 ± 9 μM and 80 ± 8 μM. CQ inhibits the H+ translocation (with an IC50 of 13.4 ± 0.2 μM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+‐PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca²⁺ induced by ATP, indicating an effect on the Ca²⁺‐ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca²⁺ metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis.     

Molecular Cloning and Expression of Biologically Active Human Glia Maturation Factor-β

Glia maturation factor-beta, a protein found in the brains of all vertebrates thus far examined, appears to play a role in the differentiation, maintenance, and regeneration of the nervous system. Using oligonucleotide probes based on the sequences of three tryptic peptides derived from bovine glia maturation factor-beta, we screened a human brainstem cDNA library in lambda gt11. A 0.7-kb clone was isolated, sequenced in its entirety, and found to encode a polypeptide of 142 amino acids which contained regions identical to the three bovine peptides. This polypeptide, human recombinant glia maturation factor-beta, has been expressed in Escherichia coli and found to possess structural characteristics and biological activity indistinguishable from those of the native bovine protein.     

Synthesis of a C-8 Modified Adenosine Analogue as a Potential Mechanistic Probe for Ribonucleotide Reductases

Abstract

O-2′ phenoxythionocarbonate of 8-vinyladenosine gives under Barton-McCombie conditions a C-2′ radical that reacts intramolecularly with the vinyl group to afford a 6-endocycloproduct.     

Designing Lipid Nanostructures for Local Delivery of Biologically Active Macromolecules

This study focuses on the possible therapeutic utility of liposomes in the local treatment of inflammatory disorders, specifically rheumatoid arthritis (RA). Our purpose was to design a depot delivery system of an anti-inflammatory glycoprotein, lactoferrin (Lf), using positive multivesicular liposomes and to investigate its in vivo efficiency. Lactoferrin (Lf) has previously been shown to have therapeutic potential in mice with collagen-induced arthritis (CIA) after intra-articular (i.a.) injection. In order to protect Lf from enzymatic degradation and to maintain an adequate concentration in the joint, liposomes have been used as carriers for controlled drug delivery. Based on our previous findings we compared the ability of free Lf and Lf encapsulated in liposomes to suppress established joint inflammation and to modulate the cytokine response of lymph node (LN) T lymphocytes in DBA/1 mice with CIA. The anti-inflammatory effect of Lf formulated in positive liposomes was more pronounced compared with the free protein. After a single i.a. injection of liposomal Lf the arthritic score significantly decreased continuously for 2 weeks while in the case of free Lf for only 3–4 days. The cytokine levels produced by LN T cells showed decreased pro-inflammatory cytokines (TNF-α and IFN-γ) accompanied by increased anti-inflammatory cytokines (IL-5 and especcialy IL-10) in encapsulated compared with free Lf. When compared with free Lf, liposomal Lf decreased the expression of costimulatory molecules on DCs, reduced pro-inflammatory (TNF) and increased anti-inflammatory (IL-10) cytokine production. Using CIA model we have studied the liposome trafficking following i.a. administration and we have identified DCs as a target for liposomes in the draining LN. Our results suggest that the entrapment of Lf in liposomes may modify its pharmacodynamic profile and could have great potential as controlled delivery system in the treatment of RA and other local inflammatory conditions.     

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.