ABSTRACT
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.
Subject(s)
Auranofin , Carcinoma, Non-Small-Cell Lung , Checkpoint Kinase 1 , Lung Neoplasms , Oxidation-Reduction , Thioredoxins , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 1/antagonists & inhibitors , Humans , Oxidation-Reduction/drug effects , Thioredoxins/metabolism , Cell Line, Tumor , Auranofin/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Protein Kinase Inhibitors/pharmacology , Ribonucleoside Diphosphate Reductase/metabolism , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleotide Reductases/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , Drug Synergism , AnimalsABSTRACT
The development of copper(II) thiosemicarbazone complexes as potential anticancer agents, possessing dual functionality as inhibitors of R2 ribonucleotide reductase (RNR) and tubulin polymerization by binding at the colchicine site, presents a promising avenue for enhancing therapeutic effectiveness. Herein, we describe the syntheses and physicochemical characterization of four isomeric proligands H2L3-H2L6, with the methylmorpholine substituent at pertinent positions of the pyridine ring, along with their corresponding Cu(II) complexes 3-6. Evidently, the position of the morpholine moiety and the copper(II) complex formation have marked effects on the in vitro antiproliferative activity in human uterine sarcoma MES-SA cells and the multidrug-resistant derivative MES-SA/Dx5 cells. Activity correlated strongly with quenching of the tyrosyl radical (Yâ¢) of mouse R2 RNR protein, inhibition of RNR activity in the cancer cells, and inhibition of tubulin polymerization. Insights into the mechanism of antiproliferative activity, supported by experimental results and molecular modeling calculations, are presented.
Subject(s)
Antineoplastic Agents , Copper , Morpholines , Ribonucleotide Reductases , Thiosemicarbazones , Tubulin , Thiosemicarbazones/chemistry , Thiosemicarbazones/pharmacology , Thiosemicarbazones/chemical synthesis , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/metabolism , Tubulin/metabolism , Animals , Morpholines/pharmacology , Morpholines/chemistry , Morpholines/chemical synthesis , Copper/chemistry , Mice , Cell Line, Tumor , Cell Proliferation/drug effects , Structure-Activity Relationship , Polymerization/drug effects , Coordination Complexes/pharmacology , Coordination Complexes/chemistry , Coordination Complexes/chemical synthesis , Pyridines/pharmacology , Pyridines/chemistry , Pyridines/chemical synthesis , Tubulin Modulators/pharmacology , Tubulin Modulators/chemical synthesis , Tubulin Modulators/chemistry , Drug Screening Assays, Antitumor , Models, MolecularABSTRACT
BACKGROUND: Aberrant expression of histone deacetylases (HDACs) and ribonucleotide reductase (RR) enzymes are commonly observed in various cancers. Researchers are focusing on these enzymes in cancer studies with the aim of developing effective chemotherapeutic drugs for cancer treatment. Targeting both HDAC and RR simultaneously with a dual HDAC/RR inhibitor has exhibited enhanced effectiveness compared to monotherapy in cancer treatment, making it a promising strategy. OBJECTIVES: The objective of the study is to synthesize and assess the anti-cancer properties of a 1,10-phenanthroline-based hydroxamate derivative, characterizing it as a novel dual HDAC/RR inhibitor. METHODS: The N1-hydroxy-N8-(1,10-phenanthrolin-5-yl)octanediamide (PA), a 1,10-phenanthroline-based hydroxamate derivative, was synthesized and structurally characterized. The compound was subjected to in vitro assessments of its anti-cancer, HDAC, and RR inhibitory activities. In silico docking and molecular dynamics simulations were further studied to explore its interactions with HDACs and RRM2. RESULTS: The structurally confirmed PA exhibited antiproliferative activity in SiHa cells with an IC50 of 16.43 µM. It displayed potent inhibitory activity against HDAC and RR with IC50 values of 10.80 µM and 9.34 µM, respectively. Co-inhibition of HDAC and RR resulted in apoptosis-induced cell death in SiHa cells, mediated by the accumulation of reactive oxygen species (ROS). In silico docking studies demonstrated that PA can effectively bind to the active sites of HDAC isoforms and RRM2. Furthermore, PA demonstrated a more favorable interaction with HDAC7, displaying a docking score of -9.633 kcal/mol, as compared to the standard HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), which exhibited a docking score of -8.244 kcal/mol against HDAC7. CONCLUSION: The present study emphasizes the prospect of designing a potential 1,10-phenanthroline hydroxamic acid derivative as a novel dual HDAC and RR-inhibiting anti-cancer molecule.
Subject(s)
Antineoplastic Agents , Cell Proliferation , Histone Deacetylase Inhibitors , Hydroxamic Acids , Molecular Docking Simulation , Phenanthrolines , Humans , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Phenanthrolines/chemistry , Phenanthrolines/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Molecular Dynamics Simulation , Histone Deacetylases/metabolism , Histone Deacetylases/chemistry , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/chemistry , Apoptosis/drug effectsABSTRACT
Alzheimer's disease is the main cause of aging-associated dementia, for which there is no effective treatment. In this work, we reanalyze the information of a previous genome wide association study, using a new pipeline design to identify novel potential drugs. With this approach, ribonucleoside-diphosphate reductase gene (RRM2B) emerged as a candidate target and its inhibitor, 2', 2'-difluoro 2'deoxycytidine (gemcitabine), as a potential pharmaceutical drug against Alzheimer's disease. We functionally verified the effect of inhibiting the RRM2B homolog, rnr-2, in an Alzheimer's model of Caenorhabditis elegans, which accumulates human Aß1-42 peptide to an irreversible paralysis. RNA interference against rnr-2 and also treatment with 200â ng/ml of gemcitabine, showed an improvement of the phenotype. Gemcitabine treatment increased the intracellular ATP level 3.03 times, which may point to its mechanism of action. Gemcitabine has been extensively used in humans for cancer treatment but at higher concentrations. The 200â ng/ml concentration did not exert a significant effect over cell cycle, or affected cell viability when assayed in the microglia N13 cell line. Thus, the inhibitory drug of the RRM2B activity could be of potential use to treat Alzheimer's disease and particularly gemcitabine might be considered as a promising candidate to be repurposed for its treatment.
Subject(s)
Alzheimer Disease , Caenorhabditis elegans , Deoxycytidine , Disease Models, Animal , Caenorhabditis elegans/drug effects , Alzheimer Disease/drug therapy , Animals , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Amyloid beta-Peptides/metabolism , Humans , Gemcitabine , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/metabolism , Adenosine Triphosphate/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , RNA InterferenceABSTRACT
Ribonucleotide reductase (RNR) is composed of two non-identical subunits, R1 and R2, and plays a crucial role in balancing the cellular dNTP pool, establishing it as an attractive cancer target. Herein, we report the discovery of a highly potent and selective small-molecule inhibitor, TAS1553, targeting protein-protein interaction between R1 and R2. TAS1553 is also expected to demonstrate superior selectivity because it does not directly target free radical or a substrate binding site. TAS1553 has shown antiproliferative activity in human cancer cell lines, dramatically reducing the intracellular dATP pool and causing DNA replication stress. Furthermore, we identified SLFN11 as a biomarker that predicts the cytotoxic effect of TAS1553. Oral administration of TAS1553 demonstrated robust antitumor efficacy against both hematological and solid cancer xenograft tumors and also provided a significant survival benefit in an acute myelogenous leukemia model. Our findings strongly support the evaluation of TAS1553 in clinical trials.
Subject(s)
Antineoplastic Agents , Enzyme Inhibitors , Ribonucleotide Reductases , Animals , Antineoplastic Agents/pharmacology , DNA Replication , Enzyme Inhibitors/pharmacology , Humans , Nuclear Proteins/metabolism , Ribonucleotide Reductases/antagonists & inhibitorsABSTRACT
USP2 contributes to the quality control of multiple oncogenic proteins including cyclin D1, Mdm2, Aurora-A, etc., and it is a potential target for anti-cancer drug development. However, currently only a few inhibitors with moderate inhibition activities against USP2 have been discovered. USP2-targeted active compounds with either new scaffolds or enhanced activities are in need. Here in this study, Ub-AMC hydrolysis assay-based screening against ~4000 commercially available drugs and drug candidates was performed to identify USP2-targeted inhibitors. COH29, which was originally developed as an anti-cancer agent by blocking the function of human ribonucleotide reductase (RNR, IC50 = 16 µM), was found to exhibit an inhibition activity against USP2 with the IC50 value at 2.02 ± 0.16 µM. The following conducted biophysical and biochemical experiments demonstrated that COH29 could specifically interact with USP2 and inhibit its enzymatic activity in a noncompetitive inhibition mode (Ki = 1.73 ± 0.14 µM). Since COH29 shows similar inhibitory potencies against RNR (RRM2) and USP2, USP2 inhibition-dependent cellular consequences of COH29 are expected. The results of cellular assays confirmed that the application of COH29 could downregulate the level of cyclin D1 by enhancing its degradation via ubiquitin-proteasome system (UPS), and the modulation effect of COH29 on cyclin D1 is independent of RRM2. Since cyclin D1 acts as an oncogenic driver in human cancer, our findings suggest that USP2 might be a promising therapeutic target for cyclin D1-addicted cancers, and COH29 could serve as a starting compound for high selectivity inhibitor development against USP2.
Subject(s)
Benzamides , Cyclin D1 , Neoplasms , Ribonucleotide Reductases , Thiazoles , Ubiquitin Thiolesterase , Benzamides/pharmacology , Cyclin D1/genetics , Cyclin D1/metabolism , Down-Regulation , Enzyme Inhibitors/pharmacology , Holoenzymes , Humans , Neoplasms/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/metabolism , Thiazoles/pharmacology , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific ProteasesABSTRACT
Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. SIGNIFICANCE: A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.
Subject(s)
Cystadenocarcinoma, Serous/genetics , Protein Phosphatase 2/genetics , Ribonucleotide Reductases/genetics , Synthetic Lethal Mutations/genetics , Uterine Neoplasms/genetics , Animals , Antimetabolites, Antineoplastic/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Clofarabine/pharmacology , Cystadenocarcinoma, Serous/drug therapy , Cystadenocarcinoma, Serous/metabolism , Female , Humans , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Protein Phosphatase 2/metabolism , Rats, Sprague-Dawley , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/metabolism , Synthetic Lethal Mutations/drug effects , Tumor Burden/drug effects , Tumor Burden/genetics , Uterine Neoplasms/drug therapy , Uterine Neoplasms/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.
Subject(s)
Adenosine Deaminase/physiology , Agammaglobulinemia/immunology , Induced Pluripotent Stem Cells/cytology , Neutrophils/cytology , Severe Combined Immunodeficiency/immunology , Adenosine Deaminase/genetics , Cells, Cultured , Embryoid Bodies/cytology , Fibroblasts/enzymology , Granulocytes/cytology , Humans , Hydroxamic Acids/pharmacology , Hydroxyurea/pharmacology , Infant , Male , Mutation, Missense , Myelopoiesis , Niacinamide/pharmacology , Point Mutation , Ribonucleotide Reductases/antagonists & inhibitorsABSTRACT
Ribonucleotide reductase (RNR), which supplies the building blocks for DNA biosynthesis and its repair, has been linked to human diseases and is emerging as a therapeutic target. Here, we present a mechanistic investigation of triapine (3AP), a clinically relevant small molecule that inhibits the tyrosyl radical within the RNR ß2 subunit. Solvent kinetic isotope effects reveal that proton transfer is not rate-limiting for inhibition of Y122· of E. coli RNR ß2 by the pertinent 3AP-Fe(II) adduct. Vibrational spectroscopy further demonstrates that unlike inhibition of the ß2 tyrosyl radical by hydroxyurea, a carboxylate containing proton wire is not at play. Binding measurements reveal a low nanomolar affinity (Kd â¼ 6 nM) of 3AP-Fe(II) for ß2. Taken together, these data should prompt further development of RNR inactivators based on the triapine scaffold for therapeutic applications.
Subject(s)
Enzyme Inhibitors/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Ferrous Compounds/chemistry , Pyridines/chemistry , Ribonucleotide Reductases/metabolism , Thiosemicarbazones/chemistry , Enzyme Inhibitors/metabolism , Escherichia coli Proteins/antagonists & inhibitors , Free Radicals/chemistry , Free Radicals/metabolism , Hydroxyurea/chemistry , Protein Binding , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform InfraredABSTRACT
Hydroxyurea (HU) is mostly referred to as an inhibitor of ribonucleotide reductase (RNR) and as the agent that is commonly used to arrest cells in the S-phase of the cycle by inducing replication stress. It is a well-known and widely used drug, one which has proved to be effective in treating chronic myeloproliferative disorders and which is considered a staple agent in sickle anemia therapy and-recently-a promising factor in preventing cognitive decline in Alzheimer's disease. The reversibility of HU-induced replication inhibition also makes it a common laboratory ingredient used to synchronize cell cycles. On the other hand, prolonged treatment or higher dosage of hydroxyurea causes cell death due to accumulation of DNA damage and oxidative stress. Hydroxyurea treatments are also still far from perfect and it has been suggested that it facilitates skin cancer progression. Also, recent studies have shown that hydroxyurea may affect a larger number of enzymes due to its less specific interaction mechanism, which may contribute to further as-yet unspecified factors affecting cell response. In this review, we examine the actual state of knowledge about hydroxyurea and the mechanisms behind its cytotoxic effects. The practical applications of the recent findings may prove to enhance the already existing use of the drug in new and promising ways.
Subject(s)
Hydroxyurea/metabolism , Hydroxyurea/pharmacology , Hydroxyurea/therapeutic use , Animals , DNA Replication/drug effects , Humans , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/metabolism , S Phase/drug effectsABSTRACT
Fluorouracil (5FU) is converted to its active metabolite fluorodeoxyuridine monophosphate (FdUMP) through the orotate phosphoribosyl transferase (OPRT)ribonucleotide reductase (RR) pathway and thymidine phosphatase (TP)thymidine kinase (TK) pathway and inhibits thymidylate synthase (TS), leading to inhibition of thymidine monophosphate (dTMP) synthesis through a de novo pathway. We investigated the mechanism of 5FU resistance and strategies to overcome it by focusing on 5FU metabolism. Colon cancer cell lines SW48 and LS174T and 5FUresistant cell lines SW48/5FUR and LS174T/5FUR were used. FdUMP amount was measured by western blotting. The FdUMP synthetic pathway was investigated by combining TP inhibitor (tipiracil hydrochloride; TPI) or RR inhibitor (hydroxyurea; HU) with 5FU. Drug cytotoxicity was observed by crystal violet staining assay. FdUMP was synthesized through the OPRTRR pathway in SW48 cells but was scarcely synthesized through either the OPRTRR or TPTK pathway in SW48/5FUR cells. FdUMP amount in SW48/5FUR cells was reduced by 87% vs. SW48 cells. Expression levels of OPRT and TP were lower in SW48/5FUR when compared with these levels in the SW48 cells, indicating decreased synthesis of FdUMPled 5FU resistance. These results indicated that fluorodeoxyuridine (FdU) rather than 5FU promotes FdUMP synthesis and overcomes 5FU resistance. Contrastingly, FdUMP was synthesized through the OPRTRR and TPTK pathways in LS174T cells but mainly through the TPTK pathway in LS174T/5FUR cells. FdUMP amount was similar in LS174T/5FUR vs. the LS174T cells. OPRT and RR expression was lower and TK expression was higher in LS174T/5FUR vs. the LS174T cells, indicating that dTMP synthesis increased through the salvage pathway, thus leading to 5FU resistance. LS174T/5FUR cells also showed crossresistance to FdU and TS inhibitor, suggesting that nucleoside analogs such as trifluorothymidine should be used to overcome 5FU resistance in these cells. 5FU metabolism and mechanisms of 5FU resistance are different in each cell line. Both synthesized FdUMP amount and FdUMP sensitivity should be considered in 5FUresistant cells.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Colonic Neoplasms/drug therapy , Drug Resistance, Neoplasm/drug effects , Fluorouracil/pharmacology , Antimetabolites, Antineoplastic/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Colonic Neoplasms/pathology , Drug Screening Assays, Antitumor , Floxuridine/pharmacology , Floxuridine/therapeutic use , Fluorouracil/therapeutic use , Humans , Hydroxyurea/pharmacology , Metabolic Networks and Pathways/drug effects , Pyrrolidines/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/metabolism , Thymidine Phosphorylase/antagonists & inhibitors , Thymidine Phosphorylase/metabolism , Thymine/pharmacology , Trifluridine/pharmacology , Trifluridine/therapeutic useABSTRACT
Resveratrol, the most widely studied natural phytochemical, has been shown to interact with different target proteins. Previous studies show that resveratrol binds and inhibits DNA polymerases and some other enzymes; however, the binding and functioning mechanisms remain unknown. The elucidated knowledge of inhibitory mechanisms of resveratrol will assist us in new drug discovery. We utilized molecular docking and molecular dynamics (MD) simulation to reveal how resveratrol and structurally similar compounds bind to various nucleotide-dependent enzymes, specifically, DNA polymerases, HIV-1 reverse transcriptase, and ribonucleotide reductase. The results show that resveratrol and its analogs exert their inhibitory effects by competing with the substrate dNTPs in these enzymes and blocking elongation of chain polymerization. In addition, the results imply that resveratrol binds to a variety of other ATP-/NTP-binding proteins.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Resveratrol/analogs & derivatives , Binding, Competitive , Catalytic Domain , DNA Polymerase I/antagonists & inhibitors , DNA Polymerase I/chemistry , DNA Polymerase III/antagonists & inhibitors , DNA Polymerase III/chemistry , Drug Discovery , HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/chemistry , Humans , Hydrogen Bonding , In Vitro Techniques , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Protein Conformation , Resveratrol/chemistry , Resveratrol/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/chemistry , Structure-Activity RelationshipABSTRACT
Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
Subject(s)
Anti-Bacterial Agents/chemistry , Antineoplastic Agents/chemistry , Escherichia coli Infections/drug therapy , Neoplasms/drug therapy , Nucleotides/metabolism , Ribonucleotide Reductases/chemistry , Anti-Bacterial Agents/therapeutic use , Antineoplastic Agents/therapeutic use , Biocatalysis , Drug Discovery/methods , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Infections/enzymology , Escherichia coli Infections/genetics , Escherichia coli Infections/microbiology , Humans , Molecular Docking Simulation , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/pathology , Nucleotides/chemistry , Oxidation-Reduction , Protein Structure, Secondary , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/genetics , Ribonucleotide Reductases/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/therapeutic use , Structure-Activity RelationshipABSTRACT
The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.
Subject(s)
Cytarabine/pharmacology , Leukemia, Myeloid, Acute , Pyrophosphatases/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , SAM Domain and HD Domain-Containing Protein 1/metabolism , Animals , Arabinofuranosylcytosine Triphosphate/metabolism , MiceABSTRACT
Ribonucleotide reductase (RNR) is an essential enzyme in DNA biogenesis and a target of several chemotherapeutics. Here, we investigate how anti-leukemic drugs (e.g., clofarabine [ClF]) that target one of the two subunits of RNR, RNR-α, affect non-canonical RNR-α functions. We discovered that these clinically approved RNR-inhibiting dATP-analogs inhibit growth by also targeting ZRANB3-a recently identified DNA synthesis promoter and nuclear-localized interactor of RNR-α. Remarkably, in early time points following drug treatment, ZRANB3 targeting accounted for most of the drug-induced DNA synthesis suppression and multiple cell types featuring ZRANB3 knockout/knockdown were resistant to these drugs. In addition, ZRANB3 plays a major role in regulating tumor invasion and H-rasG12V-promoted transformation in a manner dependent on the recently discovered interactome of RNR-α involving select cytosolic-/nuclear-localized protein players. The H-rasG12V-promoted transformation-which we show requires ZRANB3-supported DNA synthesis-was efficiently suppressed by ClF. Such overlooked mechanisms of action of approved drugs and a previously unappreciated example of non-oncogene addiction, which is suppressed by RNR-α, may advance cancer interventions.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cell Nucleus/drug effects , Clofarabine/pharmacology , DNA Helicases/antagonists & inhibitors , Ribonucleotide Reductases/antagonists & inhibitors , Signal Transduction/drug effects , Cell Line, Tumor , Cell Nucleus/metabolism , DNA Helicases/deficiency , DNA Helicases/metabolism , DNA, Neoplasm/antagonists & inhibitors , DNA, Neoplasm/biosynthesis , HeLa Cells , Humans , Ribonucleotide Reductases/metabolismABSTRACT
The quantification of cellular deoxyribonucleoside triphosphate (dNTP) levels is important for studying pathologies, genome integrity, DNA repair, and the efficacy of pharmacological drug treatments. Current standard methods, such as enzymatic assays or high-performance liquid chromatography, are complicated, costly, and labor-intensive, and alternative techniques that simplify dNTP quantification would present very useful complementary approaches. Here, we present a dNTP assay based on isothermal rolling circle amplification (RCA) and rapid time-gated Förster resonance energy transfer (TG-FRET), which used a commercial clinical plate reader system. Despite the relatively simple assay format, limits of detection down to a few picomoles of and excellent specificity for each dNTP against the other dNTPs, rNTPs, and dUTP evidenced the strong performance of the assay. Direct applicability of RCA-FRET to applied nucleic acid research was demonstrated by quantifying all dNTPs in CEM-SS leukemia cells with and without hydroxyurea or auranofin treatment. Both pharmacological agents could reduce the dNTP production in a time- and dose-dependent manner. RCA-FRET provides simple, rapid, sensitive, and specific quantification of intracellular dNTPs and has the potential to become an advanced tool for both fundamental and applied dNTP research.
Subject(s)
Deoxyribonucleotides/analysis , Fluorescence Resonance Energy Transfer/methods , Nucleic Acid Amplification Techniques/methods , Auranofin/pharmacology , Base Sequence , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Humans , Hydroxyurea/pharmacology , Limit of Detection , Proof of Concept Study , Ribonucleotide Reductases/antagonists & inhibitors , Sensitivity and Specificity , Thioredoxin-Disulfide Reductase/antagonists & inhibitorsABSTRACT
The antioxidant, antimalarial, antibacterial, and antitumor activities of thiosemicarbazones have made this class of compounds important for medicinal chemists. In addition, thiosemicarbazones are among the most potent and well-known ribonucleotide reductase inhibitors. In this study, 24 new thiosemicarbazone derivatives were synthesized, and the structures and purity of the compounds were determined by IR, 1 H NMR, 13 C NMR, mass spectroscopy, and elemental analysis. The IC50 values of these 24 compounds were determined with an assay for ribonucleotide reductase inhibition. Compounds 19, 20, and 24 inhibited ribonucleotide reductase enzyme activity at a higher level than metisazone as standard. The cytotoxic effects of these compounds were measured on the MCF7 (human breast adenocarcinoma) and HEK293 (human embryonic kidney) cell lines. Similarly, compounds 19, 20, and 24 had a selective effect on the MCF7 and HEK293 cell lines, killing more cancer cells than cisplatin as standard. The compounds (especially 19, 20, and 24 as the most active ones) were then subjected to docking experiments to identify the probable interactions between the ligands and the enzyme active site. The complex formation was shown qualitatively. The ADME (absorption, distribution, metabolism, and excretion) properties of the compounds were analyzed using in-silico techniques.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Thiazoles/pharmacology , Thiosemicarbazones/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , HEK293 Cells , Humans , Ligands , MCF-7 Cells , Molecular Structure , Ribonucleotide Reductases/metabolism , Structure-Activity Relationship , Thiazoles/chemistry , Thiosemicarbazones/chemical synthesis , Thiosemicarbazones/chemistryABSTRACT
Triapine (3-AP), is an iron-binding ligand and anticancer drug that is an inhibitor of human ribonucleotide reductase (RNR). Inhibition of RNR by 3-AP results in the depletion of dNTP precursors of DNA, thereby selectively starving fast-replicating cancer cells of nucleotides for survival. The redox-active form of 3-AP directly responsible for inhibition of RNR is the Fe(II)(3-AP)2 complex. In this work, we synthesize 12 analogs of 3-AP, test their inhibition of RNR in vitro, and study the electronic properties of their iron complexes. The reduction and oxidation events of 3-AP iron complexes that are crucial for the inhibition of RNR are modeled with solution studies. We monitor the pH necessary to induce reduction in iron complexes of 3-AP analogs in a reducing environment, as well as the kinetics of oxidation in an oxidizing environment. The oxidation state of the complex is monitored using UV-Vis spectroscopy. Isoquinoline analogs of 3-AP favor the maintenance of the biologically active reduced complex and possess oxidation kinetics that allow redox cycling, consistent with their effective inhibition of RNR seen in our in vitro experiments. In contrast, methylation on the thiosemicarbazone secondary amine moiety of 3-AP produces analogs that form iron complexes with much higher redox potentials, that do not redox cycle, and are inactive against RNR in vitro. The catalytic subunit of human Ribonucleotide Reductase (RNR), contains a tyrosyl radical in the enzyme active site. Fe(II) complexes of 3-AP and its analogs can quench the radical and, subsequently, inactivate RNR. The potency of RNR inhibitors is highly dependent on the redox properties of the iron complexes, which can be tuned by ligand modifications. Complexes are found to be active within a narrow redox window imposed by the cellular environment.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Coordination Complexes/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Iron/chemistry , Pyridines/chemistry , Thiosemicarbazones/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Electrochemistry/methods , Humans , Molecular Structure , Oxidation-Reduction/drug effects , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/metabolism , Tyrosine/chemistryABSTRACT
Ribonucleotide reductase (RR) catalyses the rate-limiting step of dNTP synthesis, establishing it as an important cancer target. While RR is traditionally inhibited by nucleoside-based antimetabolites, we recently discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH) that binds reversibly to the catalytic site (C-site). Here we report the synthesis and in vitro evaluation of 13 distinct compounds (TP1-13) with improved binding to hRR over NSAH (TP8), with lower KD's and more predicted residue interactions. Moreover, TP6 displayed the greatest growth inhibiting effect in the Panc1 pancreatic cancer cell line with an IC50 of 0.393 µM. This represents more than a 2-fold improvement over NSAH, making TP6 the most potent compound against pancreatic cancer emerging from the hydrazone inhibitors. NSAH was optimised by the addition of cyclic and polar groups replacing the naphthyl moiety, which occupies the phosphate-binding pocket in the C-site, establishing a new direction in inhibitor design.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Molecular Structure , Ribonucleotide Reductases/metabolism , Structure-Activity RelationshipABSTRACT
We synthesized five iron chelator derived from 2,6-diacetylpyridine bis(acylhydrazones) and proved their iron complexes structure by X-ray single crystal diffraction. These ligands have a significant anticancer proliferative activity and low cytotoxicity against normal cells. The Fe(III) complexes show reduced cytotoxic activity compared to the metal-free ligands. Anticancer mechanism studies indicate that ligands with a potential anticancer proliferation activity by inhibiting the activity of ribonucleotide reductase. Ligand rather than iron complexes regulate the expression of cell cycle associated proteins and inhibit cell cycle arrest in S phase. Apoptosis mechanism results showed that both ligand and iron complexes did not significantly promote apoptosis.