Ribonucleotide Reductase Activity Research Articles

Altered dNTP pools accelerate tumor formation in mice.

Alterations in deoxyribonucleoside triphosphate (dNTP) pools have been linked to increased mutation rates and genome instability in unicellular organisms and cell cultures. However, the role of dNTP poolchanges in tumor development in mammals remains unclear. In this study, we present a mouse model with a point mutation at the allosteric specificity site of ribonucleotide reductase, RRM1-Y285A. This mutation reduced ribonucleotide reductase activity, impairing the synthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosinetriphosphate (dGTP). Heterozygous Rrm1+/Y285A mice exhibited distinct alterations in dNTP pools across various organs, shorter lifespans and earlier tumor onset compared with wild-type controls. Mutational spectrum analysis of tumors revealed two distinct signatures, one resembling a signature extracted from a human cancer harboring a mutation of the same amino acid residue in ribonucleotide reductase, RRM1Y285C. Our findings suggest that mutations in enzymes involved in dNTP metabolism can serve as drivers of cancer development.

How ATP and dATP Act as Molecular Switches to Regulate Enzymatic Activity in the Prototypical Bacterial Class Ia Ribonucleotide Reductase.

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β2 and α2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β2 to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.

The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i’s) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

Protein engineering a PhotoRNR chimera based on a unifying evolutionary apparatus among the natural classes of ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5'-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating β subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y356[β], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any β metallocofactor highlights the adaptability of the 10-stranded αβ barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs.

Abstract B024: The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity

Abstract The replication stress (RS) response protein ataxia telangiectasia and Rad3-related protein (ATR) and its main downstream factor, checkpoint kinase 1 (CHK1), play important roles in cell survival under RS. The inhibitors targeting CHK1 (CHK1i’s) have been shown to be a powerful strategy for treating solid tumors and hematological malignancies in preclinical studies. However, in most clinical trials, including those for treating non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer cases, CHKi’s have failed to achieve their primary endpoints and have shown cumulative tissue toxicities in normal tissues. These findings significantly limit the clinical benefit of these agents. Thus, identifying novel combinational strategies that can enhance the sensitivity of tumor cells to CHKi’s, while limiting their toxicities, might be the key to improve the safety and efficacy of these compounds. To explore novel combinational strategies that can overcome these limitations, we performed an unbiased high-throughput screen in a NSCLC cell line and identified thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a novel determinant of CHK1i sensitivity. We established a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated RS. In addition, we also demonstrate that CHK1-mediated E2F1-RRM2 induction was triggered during Trx1 or TrxR1 depletion-induced RS. CHK1 inhibition abrogates the Trx1 or TrxR1 depletion induced RRM2 expression and leads to the more profound dNTP pool scarcity. Further, the TrxR1 inhibitor auronafin, an anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. In summary, our study reveals an underappreciated mechanism responsible for the Trx or TrxR1 inhibition-induced RS. We propose that increased RRM1 oxidation following Trx system inhibition and the abrogation of RRM2 pathway by CHKi contributes to the synergistic interaction by the severe loss of RNR function. Our findings identify a new pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity. Citation Format: Chandra Bhushan Prasad, Adrian Oo, Zhaojun Qiu, Na Li, Deepika Singh, Xiwen Xin, Young-Jae Cho, Zaibo Li, Xiaoli Zhang, Chunhong Yan, Qingfei Zheng, Jimin Shao, Qi-En Wang, Baek Kim, Junran Zhang. The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B024.

Impact of N221S missense mutation in human ribonucleotide reductase small subunit b on mitochondrial DNA depletion syndrome

The impact of N221S mutation in hRRM2B gene, which encodes the small subunit of human ribonucleotide reductase (RNR), on RNR activity and the pathogenesis of mitochondrial DNA depletion syndrome (MDDS) was investigated. Our results demonstrate that N221 mutations significantly reduce RNR activity, suggesting its role in the development of MDDS. We proposed an allosteric regulation pathway involving a chain of three phenylalanine residues on the αE helix of RNR small subunit β. This pathway connects the C-terminal loop of β2, transfers the activation signal from the large catalytic subunit α to β active site, and controls access of oxygen for radical generation. N221 is near this pathway and likely plays a role in regulating RNR activity. Mutagenesis studies on residues involved in the phenylalanine chain and the regulation pathway were conducted to confirm our proposed mechanism. We also performed molecular dynamic simulation and protein contact network analysis to support our findings. This study sheds new light on RNR small subunit regulation and provides insight on the pathogenesis of MDDS.

Sevoflurane inhibited reproductive function in male mice by reducing oxidative phosphorylation through inducing iron deficiency.

Sevoflurane (Sev) is one of the commonly used inhalation anesthetic chemicals in clinics. It has great impact on spermatogenesis and fertilization in male animals. The underlying mechanism remains largely unexplored. Based on our previous research, we hypothesized that Sev induced iron metabolism disturbance in the testis and epididymis and inhibited the spermatogenesis. In this study, two-month-old C57BL/6 male mice were treated with 3% Sev for 6h, and their fertility (including sperm concentration, sperm mobility, and the number of offspring) was evaluated. Mice testis, epididymis, and sperm were harvested and subjected to Western blot analysis and immunofluorescence analysis. Iron levels were reflected by the gene expression of iron metabolism-related proteins (including ferritin, TfR1, and FpN1) and ICP-MS and Perl's iron staining. Electron transport and oxidative phosphorylation levels were measured by Oxygraph-2k and ATP contents. The activity of ribonucleotide reductase was evaluated by assay kit. DNA synthesis status in testis and/or epididymis was marked with BrdU. Cell proliferation was evaluated by double immunofluorescence staining of specific protein marker expression. Our results revealed that the mice exposed to Sev showed damaged testicular and epididymis structure and significantly reduced the sperm concentration, sperm motility, and fertility. Sev decreases the iron levels through down-regulating the expression of H-ferritin, L-ferritin, and FpN1, and up-regulating the expression of TfR1 in the testis and epididymis. Iron levels also significantly reduced in germ cells which decrease the number of germ cells, including sperm, Sertoli cells, and primary spermatocyte. Iron deficiency not only decreases electron transport, oxidative phosphorylation level, and ATP production but also suppresses the activity of ribonucleotide reductase and the expression of Ki67, DDX4, GATA1, and SCP3, indicating that Sev affects the spermatogenesis and development. Meanwhile, Sev impaired the blood-testis barrier by decreasing the ZO1 expression in the testis and epididymis. The damage effect induced by Sev can be significantly ameliorated by iron supplementation. In conclusion, our study illustrates a new mechanism by which Sev inhibits spermatogenesis and fertility through an oxidative phosphorylation pathway due to iron deficiency of epididymis and testis or sperm. Furthermore, the damaging effects could be ameliorated by iron supplementation.

Yeast Ribonucleotide Reductase Is a Direct Target of the Proteasome and Provides Hyper Resistance to the Carcinogen 4-NQO.

Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.

Biochemistry-Not Oncogenes-May Demystify and Defeat Cancer.

The presence of mutated genes strongly correlates with the incidence of cancer. Decades of research, however, has not yielded any specific causative gene or set of genes for the vast majority of cancers. The Cancer Genome Atlas program was supposed to provide clarity, but it only gave much more data without any accompanying insight into how the disease begins and progresses. It may be time to notice that epidemiological studies consistently show that the environment, not genes, has the principal role in causing cancer. Since carcinogenic chemicals in our food, drink, air, and water are the primary culprits, we need to look at the biochemistry of cancer, with a focus on enzymes that invariably facilitate transformations in a cell. In particular, attention should be paid to the rate-limiting enzyme in DNA synthesis, ribonucleotide reductase (RnR), whose activity is tightly linked to tumor growth. Besides circumstantial evidence that cancer is induced at this enzyme's vulnerable free-radical-containing active site by various carcinogens, its role in initiating retinoblastoma and human papillomavirus (HPV)-related cervical cancers has been well documented in recent years. Blocking the activity of malignant RnR is a certain way to arrest cancer.

A missense mutation in the suc22 gene encoding the small subunit of ribonucleotide reductase significantly sensitizes fission yeast to chronic treatment with hydroxyurea.

Ribonucleotide reductase (RNR) is essential for the biosynthesis of dNTPs and a therapeutic target. We have identified a missense mutation in suc22 , which encodes the small subunit of RNR in fission yeast. The suc22-S239F mutation significantly sensitizes the cells to chronic but not acute treatment with the RNR inhibitor hydroxyurea. Preliminary data indicate that the drug sensitivity is likely due to decreased RNR activity. Since S239F is the first missense mutation reported for suc22 and the mutated residue is highly conserved, the results will be useful for future yeast genetic studies and potentially, the development of new therapeutics targeting RNR.

PSUN347 A Novel Mechanism-based Inhibitor of Ribonucleotide Reductase is Highly Effective in Preclinical Models of Endocrine Therapy-Resistant Breast Cancer

Abstract Approximately 70% of breast cancers express Estrogen Receptor (ER), and patients with ER+ disease benefit from first-line targeted anti-hormone therapies (e. g. tamoxifen). However, at least 30% of patients develop therapeutic resistance. Second-line therapy includes CDK4/6 inhibitors, yet patients nearly uniformly develop resistance to these drugs as well, highlighting the need to identify additional targets in resistant tumors. In this regard, tamoxifen-resistant breast cancer cells and tumors display increased expression of several key cell cycle and chromosomal maintenance-related genes. This includes elevated expression of genes in the Chromosomal Instability (CIN70) signature, comprising of 70 genes that promote CIN and facilitate tumor progression. RRM2 is one such gene that encodes the small subunit of Ribonucleotide Reductase (RNR). In public RNA-seq data, RRM2 was one of the most differentially expressed CIN70 genes in tamoxifen-resistant cells and tumors, and increased expression is associated with poor patient outcomes. We hypothesized that elevated RRM2 drives CIN and subsequent tamoxifen resistance, and that targeting RRM2 or RNR activity will exacerbate CIN to intolerable levels, leading to drug-resensitization and cell death. We confirmed that RRM2 expression is elevated in multiple tamoxifen-resistant ER+ breast cancer cell lines compared to their parental counterparts. Moreover, we found that tamoxifen-resistant cells also exhibit increased CIN phenotypes as indicated by nuclear defects (micro-, multi-, and dysmorphic nuclei). As a model of intrinsic resistance to endocrine therapy, we examined RRM2 expression in triple-negative breast cancer (TNBC) cells that lack ER and found that these cells also have elevated RRM2 compared to ER+ cells. These data affirm the utility of targeting RNR for treating aggressive disease. While there are five currently approved RNR inhibitors, they cause significant patient toxicities due to their chelator mechanism of action. To address this limitation, we developed a novel mechanism-based inhibitor of the catalytic activity of RNR (RRM127) that is less toxic in mouse models. We demonstrate that RRM127 induces excessive CIN in breast cancer cell lines with high RRM2 expression. Most importantly, we have found that RRM127 suppresses growth of both cell line and patient-derived TNBC xenografts in mice. Together, these results indicate that elevated RRM2 and its associated RNR activity promote CIN and that catalytic inhibition with RRM127 has strong efficacy in preclinical models of breast cancer. Current studies are focused on identifying the mechanisms by which RRM127 blocks breast cancer cell growth. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Comprehensive bioinformatics analysis of ribonucleoside diphosphate reductase subunit M2(RRM2) gene correlates with prognosis and tumor immunotherapy in pan-cancer.

Ribonucleotide reductase (RNR) small subunit M2 (RRM2) levels are known to regulate the activity of RNR, a rate-limiting enzyme in the synthesis of deoxyribonucleotide triphosphates (dNTPs) and essential for both DNA replication and repair. The high expression of RRM2 enhances the proliferation of cancer cells, thereby implicating its role as an anti-cancer agent. However, little research has been performed on its role in the prognosis of different types of cancers. This pan-cancer study aimed to evaluate the effect of high expression of RRM2 the tumor prognosis based on clinical information collected from The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) databases. We found RRM2 gene was highly expressed in 30 types of cancers. And we performed a pan-cancer analysis of the genetic alteration status and methylation of RRM2. Results indicated that RRM2 existed hypermethylation, associated with m6A, m1A, and m5C related genes. Subsequently, we explored the microRNAs (miRNA), long non-coding RNAs (lncRNA), and the transcription factors responsible for the high expression of RRM2 in cancer cells. Results indicated that has-miR-125b-5p and has-miR-30a-5p regulated the expression of RRM2 along with transcription factors, such as CBFB, E2F1, and FOXM. Besides, we established the competing endogenous RNA (ceRNA) diagram of lncRNAs-miRNAs-circular RNAs (circRNA) involved in the regulation of RRM2 expression. Meanwhile, our study demonstrated that high-RRM2 levels correlated with patients’ worse prognosis survival and immunotherapy effects through the consensus clustering and risk scores analysis. Finally, we found RRM2 regulated the resistance of immune checkpoint inhibitors through the PI3K-AKT single pathways. Collectively, our findings elucidated that high expression of RRM2 correlates with prognosis and tumor immunotherapy in pan-cancer. Moreover, these findings may provide insights for further investigation of the RRM2 gene as a biomarker in predicting immunotherapy’s response and therapeutic target.

Identification of Monobenzone as a Novel Potential Anti-Acute Myeloid Leukaemia Agent That Inhibits RNR and Suppresses Tumour Growth in Mouse Xenograft Model.

Simple SummaryThe clinical treatment of acute myeloid leukaemia is still dominated by chemotherapy. Clinically used anti-leukaemia drugs have shortcomings such as myelosuppression, toxicity and drug resistance. Therefore, the need to develop other chemotherapeutic drugs to meet more clinical needs is urgent. Ribonucleotide reductase (RNR) consists of a catalytic large subunit M1 (RRM1) and a regulatory small subunit M2 (RRM2), which provides dNTPs for DNA synthesis. The rapid proliferation of cancer cells requires large amounts of dNTPs. Therefore, the use of RNR inhibitors is a promising strategy for the clinical treatment of various malignancies. Monobenzone is an FDA-approved depigmenting agent for vitiligo patients. In this study, we demonstrate that monobenzone is a potent inhibitor of RNR enzyme activity by targeting RRM2 protein, and thus has significant anti-leukaemia efficacy in vitro and in vivo. This finding suggests that monobenzone has the potential to be optimized as a novel anti-AML therapeutic drug in the future. Acute myeloid leukaemia (AML) is one of the most common types of haematopoietic malignancy. Ribonucleotide reductase (RNR) is a key enzyme required for DNA synthesis and cell proliferation, and its small subunit RRM2 plays a key role for the enzymatic activity. We predicted monobenzone (MB) as a potential RRM2 target compound based on the crystal structure of RRM2. In vitro, MB inhibited recombinant RNR activity (IC50 = 0.25 μM). Microscale thermophoresis indicated that MB inhibited RNR activity by binding to RRM2. MB inhibited cell proliferation (MTT IC50 = 6–18 μM) and caused dose-dependent DNA synthesis inhibition, cell cycle arrest, and apoptosis in AML cells. The cell cycle arrest was reversed by the addition of deoxyribonucleoside triphosphates precursors, suggesting that RNR was the intracellular target of the compound. Moreover, MB overcame drug resistance to the common AML drugs cytarabine and doxorubicin, and treatment with the combination of MB and the Bcl-2 inhibitor ABT-737 exerted a synergistic inhibitory effect. Finally, the nude mice xenografts study indicated that MB administration produced a significant inhibitory effect on AML growth with relatively weak toxicity. Thus, we propose that MB has the potential as a novel anti-AML therapeutic agent in the future.

A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates.

Ribonucleotide reductases (RNRs) use radical-based chemistry to catalyze the conversion of all four ribonucleotides to deoxyribonucleotides. The ubiquitous nature of RNRs necessitates multiple RNR classes that differ from each other in terms of the phosphorylation state of the ribonucleotide substrates, oxygen tolerance, and the nature of both the metallocofactor employed and the reducing systems. Although these differences allow RNRs to produce deoxyribonucleotides needed for DNA biosynthesis under a wide range of environmental conditions, they also present a challenge for establishment of a universal activity assay. Additionally, many current RNR assays are limited in that they only follow the conversion of one ribonucleotide substrate at a time, but in the cell, all four ribonucleotides are actively being converted into deoxyribonucleotide products as dictated by the cellular concentrations of allosteric specificity effectors. Here, we present a liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based assay that can determine the activity of both aerobic and anaerobic RNRs on any combination of substrates using any combination of allosteric effectors. We demonstrate that this assay generates activity data similar to past published results with the canonical Escherichia coli aerobic class Ia RNR. We also show that this assay can be used for an anaerobic class III RNR that employs formate as the reductant, i.e. Streptococcus thermophilus RNR. We further show that this class III RNR is allosterically regulated by dATP and ATP. Lastly, we present activity data for the simultaneous reduction of all four ribonucleotide substrates by the E. coli class Ia RNR under various combinations of allosteric specificity effectors. This validated LC-MS/MS assay is higher throughput and more versatile than the historically established radioactive activity and coupled RNR activity assays as well as a number of the published HPLC-based assays. The presented assay will allow for the study of a wide range of RNR enzymes under a wide range of conditions, facilitating the study of previously uncharacterized RNRs.

Glutathione-dependent thioredoxin reduction and lipoamide system support in-vitro mammalian ribonucleotide reductase catalysis: a possible antioxidant redundancy.

The thioredoxin system (Trx), comprising of Trx, Thioredoxin reductase (TrxR) and NADPH aids in donating hydrogen group to support Ribonucleotide reductase (RNR) catalysis during de-novo DNA biosynthesis. However, it has been observed that inhibiting TrxR does not affect the viability of cancer cells that are susceptible to pharmacological glutathione (GSH) depletion. This prompted us to study the potential antioxidant redundancies that might prolong RNR activity. To study the RNR activity assay, the RNR complex was reconstituted by mixing purified mouse recombinant RNR subunits and the conversion of [3H] CDP into [3H] dCDP was monitored. In the assay system, either purified Trx and GSH or Lipoamide system was supplemented as reducing agents to support RNR catalysis. Herein, we have found that GSH-dependent Trx reduction supports mammalian class I RNR catalysis in absence of TrxR in the system. Our data also presents the first report that the LAM system is capable of supporting in-vitro RNR activity in the complete absence of either Trx or Grx systems. We conclude that GSH-mediated Trx reduction and LAM systems support basal level RNR activity in vitro; in absence of TrxR and complete redoxin systems respectively and hypothesize that potential redundancy between the various antioxidant systems might synergize in sustaining RNR activity.

A nucleotide-sensing oligomerization mechanism that controls NrdR-dependent transcription of ribonucleotide reductases

Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of RNR genes and, often, its own, in most bacteria and some archaea. NrdR senses the concentration of nucleotides through its ATP-cone, an evolutionarily mobile domain that also regulates the enzymatic activity of many RNRs, while a Zn-ribbon domain mediates binding to NrdR boxes upstream of and overlapping the transcription start site of RNR genes. Here, we combine biochemical and cryo-EM studies of NrdR from Streptomyces coelicolor to show, at atomic resolution, how NrdR binds to DNA. The suggested mechanism involves an initial dodecamer loaded with two ATP molecules that cannot bind to DNA. When dATP concentrations increase, an octamer forms that is loaded with one molecule each of dATP and ATP per monomer. A tetramer derived from this octamer then binds to DNA and represses transcription of RNR. In many bacteria — including well-known pathogens such as Mycobacterium tuberculosis — NrdR simultaneously controls multiple RNRs and hence DNA synthesis, making it an excellent target for novel antibiotics development.

Preclinical validation and phase I trial of 4-hydroxysalicylanilide, targeting ribonucleotide reductase mediated dNTP synthesis in multiple myeloma

BackgroundAberrant DNA repair pathways contribute to malignant transformation or disease progression and the acquisition of drug resistance in multiple myeloma (MM); therefore, these pathways could be therapeutically exploited. Ribonucleotide reductase (RNR) is the rate-limiting enzyme for the biosynthesis of deoxyribonucleotides (dNTPs), which are essential for DNA replication and DNA damage repair. In this study, we explored the efficacy of the novel RNR inhibitor, 4-hydroxysalicylanilide (HDS), in myeloma cells and xenograft model. In addition, we assessed the clinical activity and safety of HDS in patients with MM.MethodsWe applied bioinformatic, genetic, and pharmacological approaches to demonstrate that HDS was an RNR inhibitor that directly bound to RNR subunit M2 (RRM2). The activity of HDS alone or in synergy with standard treatments was evaluated in vitro and in vivo. We also initiated a phase I clinical trial of single-agent HDS in MM patients (ClinicalTrials.gov: NCT03670173) to assess safety and efficacy.ResultsHDS inhibited the activity of RNR by directly targeting RRM2. HDS decreased the RNR-mediated dNTP synthesis and concomitantly inhibited DNA damage repair, resulting in the accumulation of endogenous unrepaired DNA double-strand breaks (DSBs), thus inhibiting MM cell proliferation and inducing apoptosis. Moreover, HDS overcame the protective effects of IL-6, IGF-1 and bone marrow stromal cells (BMSCs) on MM cells. HDS prolonged survival in a MM xenograft model and induced synergistic anti-myeloma activity in combination with melphalan and bortezomib. HDS also showed a favorable safety profile and demonstrated clinical activity against MM.ConclusionsOur study provides a rationale for the clinical evaluation of HDS as an anti-myeloma agent, either alone or in combination with standard treatments for MM.Trial registration: ClinicalTrials.gov, NCT03670173, Registered 12 September 2018.

Biochemical Characterization of a Human Ribonucleotide Reductase (HsRNR) Variant Lacking Activity Regulation

Ribonucleotide reductase (RNR) is responsible for the synthesis of deoxyribonucleotides used in DNA synthesis and repair. Class Ia RNRs are composed of two dimeric subunits: a catalytic subunit (α2) which contains the active site and two allosteric regulation sites, and radical generating (β2) subunit housing the diferric‐tyrosyl radical cofactor. The two subunits form an α2β2 heterotetrametric complex that allows the radical to travel from β to α subunit for catalysis. RNR activity is regulated by the binding of ATP/dATP to an N‐terminal cone domain. Human RNRs (HsRNRs) are class Ia enzymes that form a homohexameric α6in the presence of either dATP or ATP. Although, both α6 assemblies are morphologically identical, only the α6‐ATP can be disturbed by the addition of β to form the α2β2 complex necessary for catalysis. Recent studies have shown that α subunits contact each other via their cone domains in the α6 rings, but it is unknown if the cone domains contact β2 in the α2β2 active complex. Here, we aim to probe the role of the cone domains in catalysis through biochemical and biophysical studies of an α subunit variant lacking the cone domain. We used site‐directed mutagenesis to delete residues 2‐92 of α, and expressed and purified the variant a protein. The variant protein was used for HPLC‐MS based activity assays of HsRNR. Preliminary results show the cone domain is important for catalysis. Understanding more about RNR’s structure and activity regulation can facilitate development of new cancer therapies.

Front Cover: Gallium (III) Complexes in Cancer Chemotherapy (Eur. J. Inorg. Chem. 6/2022)

The Front Cover shows the major antineoplastic mechanisms of gallium compounds. Gallium, whose name originates from the Latin word “Gallus (the Rooster)”, has been considered as one of the main-group elements potentially used in chemotherapy for decades. Gallium compounds, such as Ga(NO3)3, inhibit tumors by relying on the similarity between Ga3+ and Fe3+, the latter of which plays a crucial role in enzyme catalysis and redox homeostasis. Ga3+ ions enter the cells through transferrin-mediated endocytosis and are released after acidification of endosomes. Later on, they interrupt Fe-dependent processes; for example, they inhibit the activity of ribonucleotide reductase (leading to cell cycle arrest) and Fe-containing proteins in the respiratory chain (leading to the generation of ROS). To solve the problem of complex Ga(III) speciation in the circulatory system, a large number of ligands were applied to form Ga(III) complexes, supplying multiple theranostic functions and novel modes of action. More information can be found in the Review by J.-L. Zhang and co-workers.

Ribonucleotide reductase mediated regulation of mitochondrial activity in the adult heart

Ribonucleotide Reductase (RNR) is the rate-limiting enzyme for the de novo dNTP synthesis, the building blocks of nuclear and mitochondrial DNA. RNR consists of two subunits, encoded by genes Rrm1 and Rrm2/Rrm2b. RNR activity is controlled by relative dNTP levels to maintain balanced nuclear and mitochondrial dNTP pools. The Rrm2 acts as a molecular switch that affects RNR activity, dNTP synthesis, and DNA replication. Previous work showed that reduced perinatal RNR activity caused cardiac dilation and early death.