Synergistic therapeutics: Co-targeting histone deacetylases and ribonucleotide reductase for enhanced cancer treatment.

The development of cancer is influenced by several variables, including altered protein expression, and signaling pathways. Cancers are inherently heterogeneous and exhibit genetic and epigenetic aberrations; therefore, developing therapies that act on numerous biological targets is encouraged. To achieve this, two approaches are employed: combination therapy and dual/multiple targeting chemotherapeutics. Two enzymes, histone deacetylases (HDACs) and ribonucleotide reductase (RR), are crucial for several biological functions, including replication and repair of DNA, division of cells, transcription of genes, etc. However, it has been noted that different cancers exhibit abnormal functions of these enzymes. Potent inhibitors for each of these proteins have been extensively researched. Many medications based on these inhibitors have been successfully food and drug administration (FDA) approved, and the majority are undergoing various stages of clinical testing. This review discusses various studies of HDAC and RR inhibitors in combination therapy and dual-targeting chemotherapeutics.

Probing Nonadiabaticity of Proton-Coupled Electron Transfer in Ribonucleotide Reductase.

The enzyme ribonucleotide reductase, which is essential for DNA synthesis, initiates the conversion of ribonucleotides to deoxyribonucleotides via radical transfer over a 32 Å pathway composed of proton-coupled electron transfer (PCET) reactions. Previously, the first three PCET reactions in the α subunit were investigated with hybrid quantum mechanical/molecular mechanical (QM/MM) free energy simulations. Herein, the fourth PCET reaction in this subunit between C439 and guanosine diphosphate (GDP) is simulated and found to be slightly exoergic with a relatively high free energy barrier. To further elucidate the mechanisms of all four PCET reactions, we analyzed the vibronic and electron-proton nonadiabaticities. This analysis suggests that interfacial PCET between Y356 and Y731 is vibronically and electronically nonadiabatic, whereas PCET between Y731 and Y730 and between C439 and GDP is fully adiabatic and PCET between Y730 and C439 is in the intermediate regime. These insights provide guidance for selecting suitable rate constant expressions for these PCET reactions.

A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench.

We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn-Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.

Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs.

DNA regulation, replication and repair are processes fundamental to all known organisms and the sliding clamp proliferating cell nuclear antigen (PCNA) is central to all these processes. S-phase delaying protein 1 (Spd1) from S. pombe, an intrinsically disordered protein that causes checkpoint activation by inhibiting the enzyme ribonucleotide reductase, has one of the most divergent PCNA binding motifs known. Using NMR spectroscopy, in vivo assays, X-ray crystallography, calorimetry, and Monte Carlo simulations, an additional PCNA binding motif in Spd1, a PIP-box, is revealed. The two tandemly positioned, low affinity sites exchange rapidly on PCNA exploiting the same binding sites. Increasing or decreasing the binding affinity between Spd1 and PCNA through mutations of either motif compromised the ability of Spd1 to cause checkpoint activation in yeast. These results pinpoint a role for PCNA in Spd1-mediated checkpoint activation and suggest that its tandemly positioned short linear motifs create a neatly balanced competition-based system, involving PCNA, Spd1 and the small ribonucleotide reductase subunit, Suc22R2. Similar mechanisms may be relevant in other PCNA binding ligands where divergent binding motifs so far have gone under the PIP-box radar.

Class Ib Ribonucleotide Reductases: Activation of a Peroxido-MnIIMnIII to Generate a Reactive Oxo-MnIIIMnIV Oxidant.

In the postulated catalytic cycle of class Ib Mn2 ribonucleotide reductases (RNRs), a MnII2 core is suggested to react with superoxide (O2·-) to generate peroxido-MnIIMnIII and oxo-MnIIIMnIV entities prior to proton-coupled electron transfer (PCET) oxidation of tyrosine. There is limited experimental support for this mechanism. We demonstrate that [MnII2(BPMP)(OAc)2](ClO4) (1, HBPMP = 2,6-bis[(bis(2 pyridylmethyl)amino)methyl]-4-methylphenol) was converted to peroxido-MnIIMnIII (2) in the presence of superoxide anion that converted to (µ-O)(µ-OH)MnIIIMnIV (3) via the addition of an H+-donor (p-TsOH) or (µ-O)2MnIIIMnIV (4) upon warming to room temperature. The physical properties of 3 and 4 were probed using UV-vis, EPR, X-ray absorption, and IR spectroscopies and mass spectrometry. Compounds 3 and 4 were capable of phenol oxidation to yield a phenoxyl radical via a concerted PCET oxidation, supporting the proposed mechanism of tyrosyl radical cofactor generation in RNRs. The synthetic models demonstrate that the postulated O2/Mn2/tyrosine activation mechanism in class Ib Mn2 RNRs is plausible and provides spectral insights into intermediates currently elusive in the native enzyme.

3-AP inhibits the growth of human osteosarcoma by decreasing the activity of the iron-dependent pathway.

3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) has broad-spectrum antitumor activity. However, its role in osteosarcoma (OS) remains unclear. Therefore, this study explored the effects of 3-AP on OS in vitro and in vivo using three human OS cell lines (MG-63, U2-OS, and 143B) and a nude mice model generated by transplanting 143B cells. The cells and mice were treated with DMSO (control) or gradient concentrations of 3-AP. Then, various assays (e.g., cell counting kit-8, flow cytometry, immunohistochemistry, and western blotting) were performed to assess cell viability and apoptosis levels, as well as γH2A.X (DNA damage correlation), ribonucleotide reductase catalytic subunit M1 and M2 (RRM1 and RRM2, respectively) protein levels (iron-dependent correlation). 3-AP time- and dose-dependably suppressed growth and induced apoptosis in all three OS cell lines, and ferric ammonium citrate (FAC) blocked these effects. Moreover, 3-AP decreased RRM2 and total ribonucleotide reductase (RRM1 plus RRM2) protein expression but significantly increased γH2A.X expression; treatment did not affect RRM1 expression. Again, FAC treatment attenuated these effects. In vivo, the number of apoptotic cells in the tumor slices increased in the 3-AP-treated mice compared to the control mice. 3-AP treatment also decreased Ki-67 and p21 expression, suggesting inhibited OS growth. Furthermore, the expression of RRM1, RRM2, and transferrin receptor protein 1 (i.e., Tfr1) indicated that 3-AP inhibited OS growth via an iron-dependent pathway. In conclusion, 3-AP exhibits anticancer activity in OS by decreasing the activity of iron-dependent pathways, which could be a promising therapeutic strategy for OS.

Theoretical Insights into the Generation Mechanism of the Tyr122 Radical Catalyzed by Intermediate X in Class Ia Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides in all organisms. There is an ∼35 Å long-range electron-hole transfer pathway during the catalytic process of class Ia RNR, which can be described as Tyr122ß â†” [Trp48ß]? ↔ Tyr356ß â†” Tyr731α ↔ Tyr730α ↔ Cys439α. The formation of the Y122• radical initiates this long-range radical transfer process. However, the generation mechanism of Y122• is not yet clear due to confusion over the intermediate X structures. Based on the two reported X structures, we examined the possible mechanisms of Y122• generation by density functional theory (DFT) calculations. Our examinations revealed that the generation of the Y122• radical from the two different core structures of X was via a similar two-step reaction, with the first step of proton transfer for the formation of the proton receptor of Y122 and the second step of a proton-coupled long-range electron transfer reaction with the proton transfer from the Y122 hydroxyl group to the terminal hydroxide ligand of Fe1III and simultaneously electron transfer from the side chain of Y122 to Fe2IV. These findings provide an insight into the formation mechanism of Y122• catalyzed by the double-iron center of the ß subunit of class Ia RNR.

Ribonucleotide reductase as a therapeutic target for drug repurposing as anthelmintics.

Visceral cestodiases, like echinococcoses and cysticercoses, are zoonoses of worldwide distribution and are responsible for public health problems in many countries, especially in underdeveloped regions. Current treatments have low efficiency and there are few drugs currently in use for chemotherapy, making the development of new anthelmintics an urgent matter. The nucleotide salvage pathways are the only ones available for nucleotide synthesis in cestodes and other parasitic helminths, and, here, we used in silico approaches to assess the potential of the enzymes in these pathways as targets for drug repurposing as anthelminthics. First, a genomic survey allowed to identify a repertoire of 28 enzymes of the purine and pyrimidine salvage pathways from the cestode Echinococcus granulosus sensu stricto. Regarding purines, the parasite relies on salvaging free bases rather than salvaging nucleosides. Pyrimidines, on the other hand, can be salvaged from both bases and nucleosides. Druggability of the parasite enzymes was assessed, as well as the availability of commercial inhibitors for them. Druggable enzymes were then ranked according to their potential for drug repurposing and the 17 most promising enzymes were selected for evolutionary analyses. The constructed phylogenetic trees allowed to assess the degree of conservation among ortholog enzymes from parasitic helminths and their mammalian hosts. Positive selection is absent in all assessed flatworm enzymes. A potential target enzyme for drug repurposing, ribonucleotide reductase (RNR), was selected for further assessment. RNR 3D-modelling showed structural similarities between the E. granulosus and the human orthologs suggesting that inhibitors of the human RNR should be effective against the E. granulosus enzyme. In line with that, E. granulosus protoscolices treated in vitro with the inhibitor hydroxyurea had their viability and DNA synthesis reduced. These results are consistent with nucleotide synthesis inhibition and confirm the potential of a nucleotide salvage inhibitors for repurposing as an anthelmintic.

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 ß. This pathway connects the C-terminal loop of ß2, transfers the activation signal from the large catalytic subunit α to ß 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.

Structure of a ribonucleotide reductase R2 protein radical.

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.

Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide to activate AMPK for driving chemoresistance of glioblastomas.

Temozolomide (TMZ) is a standard treatment for glioblastoma (GBM) patients. However, TMZ has moderate therapeutic effects due to chemoresistance of GBM cells through less clarified mechanisms. Here, we demonstrate that TMZ-derived 5-aminoimidazole-4-carboxamide (AICA) is converted to AICA ribosyl-5-phosphate (AICAR) in GBM cells. This conversion is catalyzed by hypoxanthine phosphoribosyl transferase 1 (HPRT1), which is highly expressed in human GBMs. As the bona fide activator of AMP-activated protein kinase (AMPK), TMZ-derived AICAR activates AMPK to phosphorylate threonine 52 (T52) of RRM1, the catalytic subunit of ribonucleotide reductase (RNR), leading to RNR activation and increased production of dNTPs to fuel the repairment of TMZ-induced-DNA damage. RRM1 T52A expression, genetic interruption of HPRT1-mediated AICAR production, or administration of 6-mercaptopurine (6-MP), a clinically approved inhibitor of HPRT1, blocks TMZ-induced AMPK activation and sensitizes brain tumor cells to TMZ treatment in mice. In addition, HPRT1 expression levels are positively correlated with poor prognosis in GBM patients who received TMZ treatment. These results uncover a critical bifunctional role of TMZ in GBM treatment that leads to chemoresistance. Our findings underscore the potential of combined administration of clinically available 6-MP to overcome TMZ chemoresistance and improve GBM treatment.

Ribonucleotide reductase regulatory subunit M2 (RRM2) as a potential sero-diagnostic biomarker in non-small cell lung cancer.

OBJECTIVES: Non-small cell lung cancer (NSCLC) is a major cause of cancer-related death worldwide. Most cases are diagnosed at an advanced stage using current tumor markers. Here, we aimed to identify potential novel potential biomarkers for NSCLC. MATERIAL/METHODS: Four independent datasets from the Gene Expression Omnibus database were analyzed. The relative expression of ribonucleotide reductase regulatory subunit M2 (RRM2) mRNA in 30 paired of NSCLC paired tissues was measured by reverse transcription quantitative PCR. Serum levels of cytokeratin fragment 21-1 (CYFRA21-1), pro-gastrin-releasing peptide (ProGRP), carcinoembryonic antigen (CEA), and neuron-specific enolase (NSE) were measured using electrochemiluminescence immunoassays, and serum RRM2 levels were evaluated by an enzyme-linked immunosorbent assay. RESULTS: The mRNA expression level of RRM2 was significantly increased in most NSCLC lesions compared to para-adjacent tissues. Serum RRM2 levels in NSCLC patients were significantly elevated compared to healthy controls and were also associated with distant metastasis and histological type, but not with tumor size or lymph node metastasis. Receiver operating characteristic curve analysis showed a higher diagnostic ratio for NSCLC using RRM2 alone compared to other traditional tumor markers. CONCLUSIONS: RRM2 is a potential sero-diagnostic biomarker for NSCLC.

Activator Protein-1 (AP-1) Signaling Inhibits the Growth of Ewing Sarcoma Cells in Response to DNA Replication Stress.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleosides and is required for DNA replication. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors have complicated the identification of the mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of the RRM1 protein results in the upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos, and downregulation of c-Myc. Notably, overexpression of c-Jun and c-Fos in Ewing sarcoma cells is sufficient to inhibit cell growth and downregulate the expression of the c-Myc oncogene. We also identified that the upregulation of AP-1 is mediated, in part, by SLFN11, which is a replication stress response protein that is expressed at high levels in Ewing sarcoma. In addition, small-molecule inhibitors of RNR, including gemcitabine, and histone deacetylase inhibitors, which reduce the level of the RRM1 protein, also activate AP-1 signaling and downregulate the level of c-Myc in Ewing sarcoma. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR. Significance: RNR is the rate-limiting enzyme in the synthesis of deoxyribonucleotides. Although RNR is the target of multiple chemotherapy drugs, polypharmacology and off-target effects have complicated the identification of the precise mechanism of action of these drugs. In this work, using a knockout-rescue approach, we identified that inhibition of RNR upregulates AP-1 signaling and downregulates the level of c-Myc in Ewing sarcoma tumors.

Inhibition of Ribonucleotide Reductase Induces Endoplasmic Reticulum Stress and Apoptosis, Leading to the Death of Docetaxel-resistant Prostate Cancer Cells.

BACKGROUND: The development of chemotherapy resistance in prostate cancer (PCa) patients poses a significant obstacle to disease progression. Ribonucleotide reductase is a crucial enzyme for cell division and tumor growth. Triapine, an inhibitor of ribonucleotide reductase, has shown strong anti-tumor activity in various types of cancers. However, the effect of triapine on docetaxel-resistant (DR) human PCa cells has not been explored previously. AIM: This study aimed to examine the potential anti-proliferative effects of triapine in PC3-DR (docetaxel-resistant) cells. METHODS: Cell viability was determined by the MTT test, and apoptosis and cell cycle progression were analyzed by image-based cytometer. mRNA and protein expression were assessed by RT-qPCR and western blot, respectively. RESULTS: Triapine administration significantly reduced PC3 and PC3-DR cells' survival, while the cytotoxic effect was higher in PC3-DR cells. Cell death resulting from inhibition of ribonucleotide reductase was mediated by endoplasmic reticulum stress, induction of apoptosis, and cell cycle arrest. The findings were supported by the upregulation of caspases, Bax, Bak, P21, P27, P53, TNF-α, FAS, and FASL, and downregulation of Bcl2, Bcl-XL, cyclin-dependent kinase 2 (CDK2), CDK4, cyclins, and heat shock proteins expression. According to the data, the reduction of ABC transporter proteins and NF-ĸB expression may play a role in triapine-mediated cytotoxicity in docetaxel-resistant cells. CONCLUSION: Based on our findings, triapine emerges as a promising chemotherapeutic approach for combating docetaxel- resistant prostate cancer.

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism.

The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCE Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.

Analysis of the Arabidopsis venosa4-0 mutant supports the role of VENOSA4 in dNTP metabolism.

Human Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) functions as a dNTPase to maintain dNTP pool balance. In eukaryotes, the limiting step in de novo dNTP biosynthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR). In Arabidopsis, the RNR1 subunit of RNR is encoded by CRINKLED LEAVES 8 (CLS8), and RNR2 by three paralogous genes, including TSO MEANING 'UGLY' IN CHINESE 2 (TSO2). In plants, DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1 (DOV1) catalyzes the first step of the de novo biosynthesis of purines. Here, to explore the role of VENOSA4 (VEN4), the most likely Arabidopsis ortholog of human SAMHD1, we studied the ven4-0 point mutation, whose leaf phenotype was stronger than those of its insertional alleles. Structural predictions suggested that the E249L substitution in the mutated VEN4-0 protein rigidifies its 3D structure. The morphological phenotypes of the ven4, cls8, and dov1 single mutants were similar, and those of the ven4 tso2 and ven4 dov1 double mutants were synergistic. The ven4-0 mutant had reduced levels of four amino acids related to dNTP biosynthesis, including glutamine and glycine, which are precursors in the de novo purine biosynthesis. Our results reveal high functional conservation between VEN4 and SAMHD1 in dNTP metabolism.

Effect of siRNA-mediated silencing of p53R2 gene on sensitivity of T-ALL cellsto Daunorubicin.

INTRODUCTION: p53R2 is a p53-inducible protein that, as one of the subunits of ribonucleotide reductase, plays an important role in providing dNTPs for DNA repair. Although p53R2 is associated with cancer progression, its role in T-cell acute lymphoblastic leukemia (T-ALL) cells is unknown. Therefore, in this study, we evaluated the effect of p53R2 silencing on double-stranded DNA breaks, apoptosis and cell cycle of T-ALL cells treated with Daunorubicin. METHODS: Transfection was performed using Polyethyleneimine (PEI). Gene expression was measured using real-time PCR and protein expression was evaluated using Western blotting. Cell metabolic activity and IC50 were calculated using MTT assay, formation of double-stranded DNA breaks was checked using immunohistochemistry for γH2AX, and cell cycle and apoptosis were evaluated using flow cytometry. RESULTS: We found that p53 silencing synergistically inhibited the growth of T-ALL cells by Daunorubicin. p53R2 siRNA in combination with Daunorubicin but not alone increases the rate of DNA double-strand breaks in T-ALL cells. In addition, p53R2 siRNA significantly increased Daunorubicin-induced apoptosis. p53R2 siRNA also caused a non-significant increase in cells in G2 phase. CONCLUSION: The results of the present study showed that silencing of p53R2 using siRNA can significantly increase the antitumor effects of Daunorubicin on T-ALL cells. Therefore, p53R2 siRNA has the potential to be used as an adjuvant therapy in combination with Daunorubicin in T-ALL.

In vitro anti-proliferative, and in silico ribonucleotide reductase and pharmacokinetics studies of heteroleptic silver(I), nickel(II) and copper(II) complexes of 4-methyl-3-thiosemicarbazones and ibuprofen.

OBJECTIVES: The present research focuses on the in vitro anti-proliferative, and in silico ribonucleotide reductase and pharmacokinetics studies of twelve heteroleptic metal complexes of the general formulae [Ag(L1-4)(ibu)] (1-4) and [M(L1-4)(ibu)2] (5-12), where L1-4 = 2-(1-(4-substitutedphenyl)ethylidene)-N-methylhydrazinecarbothioamide, ibu = non-steroidal anti-inflammatory drug (ibuprofen), and M = Cu(II) and Ni(II). METHODS: Various spectroscopic techniques were used to authenticate the structure of the synthesized complexes. UV-Vis and cyclic voltammetry techniques were used to analyse the stability and the reducing ability of the complexes. In vitro anti-proliferative studies by MTT assay, apoptotic behaviour and cellular uptake studies were investigated followed by the in silico interaction with ribonucleotide reductase (RNR) enzyme. RESULTS: The spectral studies predicted distorted tetrahedral geometry around silver(I) ion and distorted octahedral geometry around nickel(II) and copper(II) ions. The reducing ability of the copper(II) complexes was analysed using ascorbic acid by UV-Vis and cyclic voltammetry techniques, which authenticate the reducing ability of the complexes and the possible interactions within the cells. The in vitro anti-proliferative activity of the synthesized complexes against three cancerous (estrogen positive (MCF-7), estrogen negative (MDA-MB-231) and pancreatic (PANC-1)) and one normal (MCF-10a) cell lines by MTT assay showed enhanced activity for copper(II) complexes 11 and 12 containing the hydrophobic substituents. The apoptotic and cellular uptake studies showed that the complex 12 is readily taken up by PANC-1 cell lines and induces ROS-mediated mitochondrial and caspase-dependent apoptosis. The in silico studies indicated hydrogen bonding, hydrophobic and π-pair (π-π, π-σ and π-cation) interactions between the complexes and the ribonucleotide reductase (RNR) enzyme. The in silico pharmacokinetics studies of the complexes predicted the drug-likeness characteristics of the complexes. CONCLUSION: The synthesized complexes are found to be less toxic to normal cells and inhibit the growth of cancerous cells by inducing mitochondrial-mediated and caspase dependent apoptotic pathway in PANC-1 cells.

Iron necessity for chlamydospore germination in Fusarium oxysporum f. sp. cubense TR4.

Fusarium wilt disease of banana, caused by the notorious soil-borne pathogen Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), is extremely difficult to manage. Manipulation of soil pH or application of synthetic iron chelators can suppress the disease through iron starvation, which inhibits the germination of pathogen propagules called chlamydospores. However, the effect of iron starvation on chlamydospore germination is largely unknown. In this study, scanning electron microscopy was used to assemble the developmental sequence of chlamydospore germination and to assess the effect of iron starvation and pH in vitro. Germination occurs in three distinct phenotypic transitions (swelling, polarized growth, outgrowth). Outgrowth, characterized by formation of a single protrusion (germ tube), occurred at 2 to 3 h, and a maximum value of 69.3% to 76.7% outgrowth was observed at 8 to 10 h after germination induction. Germination exhibited plasticity with pH as over 60% of the chlamydospores formed a germ tube between pH 3 and pH 11. Iron-starved chlamydospores exhibited polarized-growth arrest, characterized by the inability to form a germ tube. Gene expression analysis of rnr1 and rnr2, which encode the iron-dependent enzyme ribonucleotide reductase, showed that rnr2 was upregulated (p < 0.0001) in iron-starved chlamydospores compared to the control. Collectively, these findings suggest that iron and extracellular pH are crucial for chlamydospore germination in Foc TR4. Moreover, inhibition of germination by iron starvation may be linked to a different mechanism, rather than repression of the function of ribonucleotide reductase, the enzyme that controls growth by regulation of DNA synthesis.

Protein kinase ATR inhibits E3 ubiquitin ligase CRL4PRL1 to stabilize ribonucleotide reductase in response to replication stress.

The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide (dNTP), the universal building block for DNA replication and repair. However, the relationship between ATR and RNR is not well understood. Here, we show that ATR promotes the protein stability of RNR in Arabidopsis. Through an activation tagging-based genetic screen, we found that overexpression of TSO2, a small subunit of RNR, partially suppresses the hypersensitivity of the atr mutant to replication stress. Biochemically, TSO2 interacts with PRL1, a central subunit of the Cullin4-based E3 ubiquitin ligase CRL4PRL1, which polyubiquitinates TSO2 and promotes its degradation. ATR inhibits CRL4PRL1 to attenuate TSO2 degradation. Our work provides an important insight into the replication stress responses and a post-translational regulatory mechanism for RNR. Given the evolutionary conservation of the proteins involved, the ATR-PRL1-RNR module may act across eukaryotes.