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1.
J Enzyme Inhib Med Chem ; 34(1): 438-450, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30734609

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Ribonucleotídeo Redutases/metabolismo , Relação Estrutura-Atividade
2.
Oncogene ; 38(13): 2364-2379, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30518875

RESUMO

DNA replication machinery is responsible for accurate and efficient duplication of the chromosome. Since inhibition of DNA replication can lead to replication fork stalling, resulting in DNA damage and apoptotic death, inhibitors of DNA replication are commonly used in cancer chemotherapy. Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the biosynthesis of deoxyribonucleoside triphosphates (dNTPs) that are essential for DNA replication and DNA damage repair. Gemcitabine, a nucleotide analog that inhibits RNR, has been used to treat various cancers. However, patients often develop resistance to this drug during treatment. Thus, new drugs that inhibit RNR are needed to be developed. In this study, we identified a synthetic analog of resveratrol (3,5,4'-trihydroxy-trans-stilbene), termed DHS (trans-4,4'-dihydroxystilbene), that acts as a potent inhibitor of DNA replication. Molecular docking analysis identified the RRM2 (ribonucleotide reductase regulatory subunit M2) of RNR as a direct target of DHS. At the molecular level, DHS induced cyclin F-mediated down-regulation of RRM2 by the proteasome. Thus, treatment of cells with DHS reduced RNR activity and consequently decreased synthesis of dNTPs with concomitant inhibition of DNA replication, arrest of cells at S-phase, DNA damage, and finally apoptosis. In mouse models of tumor xenografts, DHS was efficacious against pancreatic, ovarian, and colorectal cancer cells. Moreover, DHS overcame both gemcitabine resistance in pancreatic cancer and cisplatin resistance in ovarian cancer. Thus, DHS is a novel anti-cancer agent that targets RRM2 with therapeutic potential either alone or in combination with other agents to arrest cancer development.


Assuntos
Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Neoplasias/patologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Estilbenos/farmacologia , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Simulação de Acoplamento Molecular , Subunidades Proteicas/efeitos dos fármacos , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Estilbenos/química , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Curr Genet ; 65(2): 477-482, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30519713

RESUMO

The molecular chaperones Hsp70 and Hsp90 bind and fold a significant proportion of the proteome. They are responsible for the activity and stability of many disease-related proteins including those in cancer. Substantial effort has been devoted to developing a range of chaperone inhibitors for clinical use. Recent studies have identified the oncogenic ribonucleotide reductase (RNR) complex as an interactor of chaperones. While several generations of RNR inhibitor have been developed for use in cancer patients, many of these produce severe side effects such as nausea, vomiting and hair loss. Development of more potent, less patient-toxic anti-RNR strategies would be highly desirable. Inhibition of chaperones and associated co-chaperone molecules in both cancer and model organisms such as budding yeast result in the destabilization of RNR subunits and a corresponding sensitization to RNR inhibitors. Going forward, this may form part of a novel strategy to target cancer cells that are resistant to standard RNR inhibitors.


Assuntos
Chaperonas Moleculares/metabolismo , Ribonucleotídeo Redutases/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Dano ao DNA , Replicação do DNA , Ativação Enzimática , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/metabolismo , Ligação Proteica , Proteômica/métodos , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/metabolismo
4.
ACS Chem Biol ; 13(12): 3251-3258, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30133247

RESUMO

Understanding the mechanism of action (MOA) of new antimicrobial agents is a critical step in drug discovery but is notoriously difficult for compounds that appear to inhibit multiple cellular pathways. We recently described image-based approaches [bacterial cytological profiling and rapid inducible profiling (RIP)] for identifying the cellular pathways targeted by antibiotics. Here we have applied these methods to examine the effects of proteolytically degrading enzymes involved in pyrimidine nucleotide biosynthesis, a pathway that produces intermediates for transcription, DNA replication, and cell envelope synthesis. We show that rapid removal of enzymes directly involved in deoxyribonucleotide synthesis blocks DNA replication. However, degradation of cytidylate kinase (CMK), which catalyzes reactions involved in the synthesis of both ribonucleotides and deoxyribonucleotides, blocks both DNA replication and wall teichoic acid biosynthesis, producing cytological effects identical to those created by simultaneously inhibiting both processes with the antibiotics ciprofloxacin and tunicamycin. Our results suggest that RIP can be used to identify and characterize potential keystone enzymes like CMK whose inhibition dramatically affects multiple pathways, thereby revealing important metabolic connections. Identifying and understanding the role of keystone targets might also help to determine the MOAs of drugs that appear to inhibit multiple targets.


Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA/fisiologia , Núcleosídeo-Fosfato Quinase/metabolismo , Ribonucleotídeo Redutases/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/citologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Proteínas de Transporte/metabolismo , Análise Discriminante , Endopeptidase Clp/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Núcleosídeo-Fosfato Quinase/antagonistas & inibidores , Núcleosídeo-Fosfato Quinase/genética , Proteínas Recombinantes de Fusão , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/genética , Ácidos Teicoicos/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
5.
J Biol Chem ; 293(26): 10413-10414, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29959279

RESUMO

Ribonucleotide reductases (RNRs) are essential enzymes producing de novo deoxynucleotide (dNTP) building blocks for DNA replication and repair and regulating dNTP pools important for fidelity of these processes. A new study reveals that the class Ia Escherichia coli RNR is regulated by dATP via stabilization of an inactive α4ß4 quaternary structure, slowing formation of the active α2ß2 structure. The results support the importance of the regulatory α4ß4 complex providing insight in design of experiments to understand RNR regulation in vivo.


Assuntos
Nucleotídeos de Desoxiadenina/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Domínio Catalítico , Escherichia coli/enzimologia , Modelos Moleculares , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo
6.
J Biol Chem ; 293(26): 10404-10412, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29700111

RESUMO

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α2 subunit that houses the active site and allosteric regulatory sites and a ß2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact α2ß2 state allowing for radical transfer from ß2 to α2 RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied Escherichia coli class Ia RNR, dATP binding to an allosteric site on α promotes formation of an α4ß4 ring-like state. Here, we investigate whether the α4ß4 formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-ß interface (S37D/S39A-α2, S39R-α2, S39F-α2, E42K-α2, or L43Q-α2) that disrupt the α4ß4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that α4ß4 formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the α-ß interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the α-ß interface whose substitution has previously been shown to cause a mutator phenotype in Escherichia coli (i.e. S39F-α2 or E42K-α2) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.


Assuntos
Substituição de Aminoácidos , Escherichia coli/enzimologia , Multimerização Proteica/genética , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Nucleotídeos de Desoxiadenina/farmacologia , Modelos Moleculares , Estrutura Quaternária de Proteína/genética , Ribonucleotídeo Redutases/química
7.
Mol Cancer Ther ; 17(6): 1240-1250, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29592883

RESUMO

Gallium, a metal with antineoplastic activity, binds transferrin (Tf) and enters tumor cells via Tf receptor1 (TfR1); it disrupts iron homeostasis leading to cell death. We hypothesized that TfR1 on brain microvascular endothelial cells (BMEC) would facilitate Tf-Ga transport into the brain enabling it to target TfR-bearing glioblastoma. We show that U-87 MG and D54 glioblastoma cell lines and multiple glioblastoma stem cell (GSC) lines express TfRs, and that their growth is inhibited by gallium maltolate (GaM) in vitro After 24 hours of incubation with GaM, cells displayed a loss of mitochondrial reserve capacity followed by a dose-dependent decrease in oxygen consumption and a decrease in the activity of the iron-dependent M2 subunit of ribonucleotide reductase (RRM2). IHC staining of rat and human tumor-bearing brains showed that glioblastoma, but not normal glial cells, expressed TfR1 and RRM2, and that glioblastoma expressed greater levels of H- and L-ferritin than normal brain. In an orthotopic U-87 MG glioblastoma xenograft rat model, GaM retarded the growth of brain tumors relative to untreated control (P = 0.0159) and reduced tumor mitotic figures (P = 0.045). Tumors in GaM-treated animals displayed an upregulation of TfR1 expression relative to control animals, thus indicating that gallium produced tumor iron deprivation. GaM also inhibited iron uptake and upregulated TfR1 expression in U-87 MG and D54 cells in vitro We conclude that GaM enters the brain via TfR1 on BMECs and targets iron metabolism in glioblastoma in vivo, thus inhibiting tumor growth. Further development of novel gallium compounds for brain tumor treatment is warranted. Mol Cancer Ther; 17(6); 1240-50. ©2018 AACR.


Assuntos
Antineoplásicos/farmacologia , Glioblastoma/metabolismo , Ferro/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Compostos Organometálicos/farmacologia , Pironas/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Animais , Antineoplásicos/química , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glioblastoma/patologia , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Compostos Organometálicos/química , Pironas/química , Ratos , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores
8.
Infect Genet Evol ; 60: 58-65, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29454978

RESUMO

Leprosy is a chronic infection of skin and nerve caused by Mycobacterium leprae. The treatment is based on standard multi drug therapy consisting of dapsone, rifampicin and clofazamine. The use of rifampicin alone or with dapsone led to the emergence of rifampicin-resistant Mycobacterium leprae strains. The emergence of drug-resistant leprosy put a hurdle in the leprosy eradication programme. The present study aimed to predict the molecular model of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of nucleotides, to screen new drugs for treatment of drug-resistant leprosy. The study was conducted by retrieving RNR of M. leprae from GenBank. A molecular 3D model of M. leprae was predicted using homology modelling and validated. A total of 325 characters were included in the analysis. The predicted 3D model of RNR showed that the ϕ and φ angles of 251 (96.9%) residues were positioned in the most favoured regions. It was also conferred that 18 α-helices, 6 ß turns, 2 γ turns and 48 helix-helix interactions contributed to the predicted 3D structure. Virtual screening of Food and Drug Administration approved drug molecules recovered 1829 drugs of which three molecules, viz., lincomycin, novobiocin and telithromycin, were taken for the docking study. It was observed that the selected drug molecules had a strong affinity towards the modelled protein RNR. This was evident from the binding energy of the drug molecules towards the modelled protein RNR (-6.10, -6.25 and -7.10). Three FDA-approved drugs, viz., lincomycin, novobiocin and telithromycin, could be taken for further clinical studies to find their efficacy against drug resistant leprosy.


Assuntos
Proteínas de Bactérias/química , Farmacorresistência Bacteriana , Hansenostáticos/metabolismo , Mycobacterium leprae/enzimologia , Ribonucleotídeo Redutases/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sistemas de Liberação de Medicamentos , Hansenostáticos/química , Simulação de Acoplamento Molecular , Ligação Proteica , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/metabolismo
9.
PLoS One ; 13(1): e0191866, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29370277

RESUMO

Chemotherapy-induced cognitive impairment (CICI) occurs in a substantial proportion of treated cancer patients, with no drug currently available for its therapy. This study investigated whether PAN-811, a ribonucleotide reductase inhibitor, can reduce cognitive impairment and related suppression of neurogenesis following chemotherapy in an animal model. Young adult rats in Chemo and Chemo+PAN-811 groups received 3 intraperitoneal (i.p.) injections of methotrexate (MTX) and 5-fluorouracil (5-FU), and those in Saline and Saline+PAN-811 groups received equal volumes of physiological saline at 10-day intervals. PAN-811 in saline was delivered through i.p. injection, 10 min following each saline (Saline+PAN-811 group) or MTX/5-FU (Chemo+PAN-811 group) treatment, while equal volumes of saline were delivered to Saline and Chemo groups. Over Days 31-66, rats were administered tests of spatial memory, nonmatching-to-sample rule learning, and discrimination learning, which are sensitive to dysfunction in hippocampus, frontal lobe and striatum, respectively. On Day 97, neurogenesis was immnunohistochemically evaluated by counting doublecortin-positive (DCX+) cells in the dentate gyrus (DG). The results demonstrated that the Chemo group was impaired on the three cognitive tasks, but co-administration of PAN-811 significantly reduced all MTX/5-FU-induced cognitive impairments. In addition, MTX/5-FU reduced DCX+ cells to 67% of that in Saline control rats, an effect that was completely blocked by PAN-811 co-administration. Overall, we present the first evidence that PAN-811 protects cognitive functions and preserves neurogenesis from deleterious effects of MTX/5-FU. The current findings provide a basis for rapid clinical translation to determine the effect of PAN-811 on CICI in human.


Assuntos
Disfunção Cognitiva/prevenção & controle , Neurogênese/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Piridinas/farmacologia , Tiossemicarbazonas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Disfunção Cognitiva/induzido quimicamente , Giro Denteado/efeitos dos fármacos , Aprendizagem por Discriminação/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Aprendizagem/efeitos dos fármacos , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Ratos , Ratos Long-Evans , Ribonucleotídeo Redutases/antagonistas & inibidores , Memória Espacial/efeitos dos fármacos
10.
J Med Chem ; 61(3): 666-680, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29253340

RESUMO

Ribonucleotide reductase (RR), an established cancer target, is usually inhibited by antimetabolites, which display multiple cross-reactive effects. Recently, we discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH or E-3a) of human RR (hRR) binding at the catalytic site (C-site) and inhibiting hRR reversibly. We herein report the synthesis and biochemical characterization of 25 distinct analogs. We designed each analog through docking to the C-site of hRR based on our 2.7 Å X-ray crystal structure (PDB ID: 5TUS). Broad tolerance to minor structural variations preserving inhibitory potency is observed. E-3f (82% yield) displayed an in vitro IC50 of 5.3 ± 1.8 µM against hRR, making it the most potent in this series. Kinetic assays reveal that E-3a, E-3c, E-3t, and E-3w bind and inhibit hRR through a reversible and competitive mode. Target selectivity toward the R1 subunit of hRR is established, providing a novel way of inhibition of this crucial enzyme.


Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Hidrazonas/síntese química , Hidrazonas/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Técnicas de Química Sintética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Hidrazonas/química , Simulação de Acoplamento Molecular , Conformação Proteica , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Relação Estrutura-Atividade
11.
Gene ; 642: 249-255, 2018 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-29126924

RESUMO

INTRODUCTION: p53R2 is a p53-inducible protein that contributes to DNA repair by providing dNTPs in response to DNA damage. The roles of p53R2 in cancer cells and malignancies still remain controversial. Herein, we examined the effects of p53R2 silencing on HepG2 human hepatocellular carcinoma (HHC) cell line (wild-type p53) viability, apoptosis and cell cycle arrest in the presence and absence of doxorubicin. METHODS: Cell transfection was performed using a liposomal approach. Gene silencing was determined by quantitative real-time PCR and western blot analysis. To evaluate the cell growth rate after transfection, trypan blue dye exclusion assay was employed. The cytotoxicity of the doxorubicin and p53R2 siRNA as single agents or in combination against HepG2 cell was analyzed by MTT assay and the drug combination effects was evaluated by calculating the combination index. The effects of treatments on different stages of cell cycle were analyzed by flow cytometry using propidium iodide (PI) and induction of apoptosis was assessed using DNA-histone ELISA. RESULTS: We found that silencing of p53R2 alone had a strong effect on growth inhibition and spontaneous apoptosis in HepG2 cells. p53R2 siRNA synergistically enhanced the cytotoxic effect of doxorubicin. Furthermore, when used in combination with doxorubicin (0.4µM), a significant increase in the rate of apoptosis was observed (P<0.05). Moreover, cell cycle at S and G2/M phases progressed at a lower rate after p53R2 combination treatment compared with doxorubicin mono-therapy. CONCLUSION: These findings suggest that siRNA-mediated silencing of p53R2 has great potential as a therapeutic tool and adjuvant in chemotherapy.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Doxorrubicina/farmacologia , Neoplasias Hepáticas/genética , RNA Interferente Pequeno/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Carcinoma Hepatocelular/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo
12.
J Cell Physiol ; 233(6): 4677-4687, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29111571

RESUMO

Breast cancer is the first cause of cancer death in women. Many patients are resistant to current therapies, and even those were sensitive at first may eventually become resistant later. Thiosemicarbazones (TSCs) are synthetic compounds that exhibit several pharmacological activities. In this study, we investigated the potential anti-tumor activity of a set of N4 -arylsubstituted TSCs (N4 -TSCs) on human breast cancer cell lines. Studies on the effect of N4 -TSCs (T1, T2, and T3) were carried on MCF-7, MDA-MB 231, and BT 474 cell lines which differ in their expression of ER, PR, and Her2/neu. Non-transformed MCF-10A breast cell line were used as normal cells. Action of N4 -TSCs were evaluated by proliferation assay, quantification of apoptosis and cell cycle analysis. Modulation of clonogenic efficiency and migratory capacity by N4 -TSCs were also evaluated. We further investigated the effects of N4 -TSCs on ROS level and Ribonucleotide Reductase (RR) activity. We analyzed the action of these compounds on cellular mammosphere-forming capacity. We found that T1 and T2 had specific anti-tumor effect on all breast cancer cell lines based on their pro-apoptotic action and inhibitory effect on clonogenic efficiency and cell migration capacity. We also showed that both compounds increased ROS level and inhibited RR activity. Finally, we found that all N4 -TSCs diminished mammospehere-forming capacity of MCF-7 and BT 474 cells. N4 -TSCs showed specific anti-tumor action on human breast cancer cells independently their biomarkers expression pattern. Our results place these compounds as promising novel anti-tumor drugs with potential therapeutic application against different types of breast cancer.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Indanos/farmacologia , Tiossemicarbazonas/farmacologia , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Indanos/síntese química , Células MCF-7 , Necrose , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiossemicarbazonas/síntese química
13.
Artigo em Inglês | MEDLINE | ID: mdl-28893786

RESUMO

We have previously reported that an erg11 mutation affecting ergosterol synthesis and a hem13 mutation in the heme synthesis pathway significantly sensitize the fission yeast Schizosaccharomyces pombe to hydroxyurea (HU) (1, 2). Here we show that treatment with inhibitors of Erg11 and heme biosynthesis phenocopies the two mutations in sensitizing wild-type cells to HU. Importantly, HU synergistically interacts with the heme biosynthesis inhibitor sampangine and several Erg11 inhibitors, the antifungal azoles, in causing cell lethality. Since the synergistic drug interactions are also observed in the phylogenetically divergent Saccharomyces cerevisiae and the opportunistic fungal pathogen Candida albicans, the synergism is likely conserved in eukaryotes. Interestingly, our genetic data for S. pombe has also led to the discovery of a robust synergism between sampangine and the azoles in C. albicans Thus, combinations of HU, sampangine, and the azoles can be further studied as a new method for the treatment of fungal infections.


Assuntos
Alcaloides/farmacologia , Antifúngicos/farmacologia , Azóis/farmacologia , Inibidores Enzimáticos/farmacologia , Hidroxiureia/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Coproporfirinogênio Oxidase/genética , Sistema Enzimático do Citocromo P-450/genética , Citocinese/efeitos dos fármacos , Sinergismo Farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Testes de Sensibilidade Microbiana , Naftiridinas , Ribonucleotídeo Redutases/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética
14.
J Pharm Biomed Anal ; 146: 154-160, 2017 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-28881312

RESUMO

The ribonucleotide reductase inhibitor and radiosensitizer triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), NSC 663249) is clinically being evaluated via the intravenous (IV) route for the treatment of cervical and vulvar cancer in combination with primary cisplatin chemoradiation. The need for a 2-h infusion and frequent administration of triapine is logistically challenging, prompting us to pursue oral (PO) administration. In support of the clinical trial investigating oral triapine in combination with chemoradiation, we developed and validated a novel LC-MS/MS assay for the quantification of triapine in 50µL human plasma. After protein precipitation, chromatographic separation of the supernatant was achieved with a Shodex ODP2 column and an isocratic acetonitrile-water mobile phase with 10% ammonium acetate. Detection with an ABI 4000 mass spectrometer utilized electrospray positive mode ionization. The assay was linear from 3 to 3,000ng/mL and proved to be accurate (97.1-103.1%) and precise (<7.4% CV), and met the U.S. FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be an essential tool to further define the pharmacokinetics and oral bioavailability of triapine.


Assuntos
Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/química , Plasma/química , Piridinas/sangue , Piridinas/química , Ribonucleotídeo Redutases/antagonistas & inibidores , Tiossemicarbazonas/sangue , Tiossemicarbazonas/química , Bioensaio/métodos , Cromatografia Líquida/métodos , Humanos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos
15.
mBio ; 8(5)2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28928213

RESUMO

African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens.IMPORTANCE The development of drugs to treat infections with eukaryotic pathogens is challenging because many key virulence factors have closely related homologues in humans. Drug toxicity greatly limits these development efforts. For pathogens that replicate at a high rate, especially in the blood, an alternative approach is to target the cell cycle directly, much as is done to treat some hematologic malignancies. The results presented here indicate that targeting the cell cycle via inhibition of ribonucleotide reductase is effective at killing trypanosomes and prolonging the survival of infected animals.


Assuntos
Ciclo Celular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Hidroxiureia/uso terapêutico , Ribonucleotídeo Redutases/antagonistas & inibidores , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Animais , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Humanos , Hidroxiureia/administração & dosagem , Hidroxiureia/farmacologia , Camundongos , Ribonucleotídeo Redutases/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiologia , Tripanossomíase Africana/parasitologia
16.
Proc Natl Acad Sci U S A ; 114(31): 8241-8246, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28716944

RESUMO

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.


Assuntos
Hidrazonas/farmacologia , Naftalenos/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Salicilatos/farmacologia , Domínio Catalítico , Ciclo Celular/efeitos dos fármacos , Cristalografia por Raios X , Nucleotídeos de Desoxiadenina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Hidrazonas/química , Naftalenos/química , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Salicilatos/química , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo
17.
Food Chem Toxicol ; 108(Pt A): 53-62, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28716444

RESUMO

Cellular growth inhibition exerted by thiosemicarbazones is mainly attributed to down-regulation of ribonucleotide reductase (RNR) activity, with RNR being responsible for the rate-limiting step of de novo DNA synthesis. In this study, we investigated the antineoplastic effects of three newly synthesized thiosemicarbazone derivatives, thiazolyl hydrazones, in human HL-60 promyelocytic leukemia cells. The cytotoxicity of compounds alone and in combination with arabinofuranosylcytosine (AraC) was determined by growth inhibition assays. Effects on deoxyribonucleoside triphosphate (dNTP) concentrations were quantified by HPLC, and the incorporation of radio-labeled 14C-cytidine into nascent DNA was measured using a beta counter. Cell cycle distribution was analyzed by FACS, and protein levels of RNR subunits and checkpoint kinases were evaluated by Western blotting. VG12, VG19, and VG22 dose-dependently decreased intracellular dNTP concentrations, impaired cell cycle progression and, consequently, inhibited the growth of HL-60 cells. VG19 also lowered the protein levels of RNR subunits R1 and R2 and significantly diminished the incorporation of radio-labeled 14C-cytidine, being equivalent to an inhibition of DNA synthesis. Combination of thiazolyl hydrazones with AraC synergistically potentiated the antiproliferative effects seen with each drug alone and might therefore improve conventional chemotherapeutic regimens for the treatment of human malignancies such as acute promyelocytic or chronic myelogenous leukemia.


Assuntos
Citarabina/farmacocinética , Regulação para Baixo/efeitos dos fármacos , Hidrazonas/farmacocinética , Ribonucleotídeo Redutases/antagonistas & inibidores , Tiossemicarbazonas/farmacocinética , Ciclo Celular/efeitos dos fármacos , Citarabina/administração & dosagem , Citarabina/farmacologia , Sinergismo Farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Hidrazonas/administração & dosagem , Hidrazonas/química , Hidrazonas/farmacologia , Estrutura Molecular , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Tiossemicarbazonas/administração & dosagem , Tiossemicarbazonas/química , Tiossemicarbazonas/farmacologia
18.
J Cancer Res Clin Oncol ; 143(8): 1499-1529, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28624910

RESUMO

PURPOSE: Ribonucleotide reductase (RR) enzymes (RR1 and RR2) play an important role in the reduction of ribonucleotides to deoxyribonucleotides which is involved in DNA replication and repair. Augmented RR activity has been ascribed to uncontrolled cell growth and tumorigenic transformation. METHODS: This review mainly focuses on several biological and chemical RR inhibitors (e.g., siRNA, GTI-2040, GTI-2501, triapine, gemcitabine, and clofarabine) that have been evaluated in clinical trials with promising anticancer activity from 1960's till 2016. A summary on whether their monotherapy or combination is still effective for further use is discussed. RESULTS: Among the RR2 inhibitors evaluated, GTI-2040, siRNA, gallium nitrate and didox were more efficacious as a monotherapy, whereas triapine was found to be more efficacious as combination agent. Hydroxyurea is currently used more in combination therapy, even though it is efficacious as a monotherapy. Gallium nitrate showed mixed results in combination therapy, while the combination activity of didox is yet to be evaluated. RR1 inhibitors that have long been used in chemotherapy such as gemcitabine, cladribine, fludarabine and clofarabine are currently used mostly as a combination therapy, but are equally efficacious as a monotherapy, except tezacitabine which did not progress beyond phase I trials. CONCLUSIONS: Based on the results of clinical trials, we conclude that RR inhibitors are viable treatment options, either as a monotherapy or as a combination in cancer chemotherapy. With the recent advances made in cancer biology, further development of RR inhibitors with improved efficacy and reduced toxicity is possible for treatment of variety of cancers.


Assuntos
Carcinogênese/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Ribonucleotídeo Redutases/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos como Assunto , Humanos , Neoplasias/genética , Neoplasias/patologia , Oligodesoxirribonucleotídeos/uso terapêutico , Farmacologia Clínica , Ribonucleotídeo Redutases/genética
19.
Oncogene ; 36(35): 5068-5074, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28459467

RESUMO

Primary effusion lymphoma (PEL) is a highly aggressive B-cell malignancy that is closely associated with one of oncogenic viruses infection, Kaposi's sarcoma-associated herpesvirus. PEL prognosis is poor and patients barely survive >6 months even following active chemotherapy interventions. There is therefore an urgent need to discover more effective targets for PEL management. We recently found that the ribonucleotide reductase (RR) subunit M2 is potentially regulated by the key oncogenic hepatocyte growth factor/c-MET pathway in PEL. In this study, we set to investigate the role of RR in PEL pathogenesis and to evaluate its potential as a therapeutic target. We report that the RR inhibitor 3-AP actively induces PEL cell cycle arrest through inhibiting the activity of the nuclear factor-κB pathway. Using a xenograft model, we found that 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) effectively suppresses PEL progression in immunodeficient mice. Transcriptome analysis of 3-AP-treated PEL cell lines reveals altered cellular genes, most of whose roles in PEL have not yet been reported. Taken together, we propose that RR and its signaling pathway may serve as novel actionable targets for PEL management.


Assuntos
Linfoma de Efusão Primária/tratamento farmacológico , Linfoma de Efusão Primária/enzimologia , Piridinas/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/metabolismo , Tiossemicarbazonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Transdução de Sinais , Transcriptoma/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Inorg Chem ; 56(6): 3532-3549, 2017 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-28252952

RESUMO

As ribonucleotide reductase (RNR) plays a crucial role in nucleic acid metabolism, it is an important target for anticancer therapy. The thiosemicarbazone Triapine is an efficient R2 inhibitor, which has entered ∼20 clinical trials. Thiosemicarbazones are supposed to exert their biological effects through effectively binding transition-metal ions. In this study, six iminodiacetate-thiosemicarbazones able to form transition-metal complexes, as well as six dicopper(II) complexes, were synthesized and fully characterized by analytical, spectroscopic techniques (IR, UV-vis; 1H and 13C NMR), electrospray ionization mass spectrometry, and X-ray diffraction. The antiproliferative effects were examined in several human cancer and one noncancerous cell lines. Several of the compounds showed high cytotoxicity and marked selectivity for cancer cells. On the basis of this, and on molecular docking calculations one lead dicopper(II) complex and one thiosemicarbazone were chosen for in vitro analysis as potential R2 inhibitors. Their interaction with R2 and effect on the Fe(III)2-Y· cofactor were characterized by microscale thermophoresis, and two spectroscopic techniques, namely, electron paramagnetic resonance and UV-vis spectroscopy. Our findings suggest that several of the synthesized proligands and copper(II) complexes are effective antiproliferative agents in several cancer cell lines, targeting RNR, which deserve further investigation as potential anticancer drugs.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos Organometálicos/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cobre/química , Cobre/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Iminoácidos/química , Iminoácidos/farmacologia , Camundongos , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Ribonucleotídeo Redutases/isolamento & purificação , Ribonucleotídeo Redutases/metabolismo , Relação Estrutura-Atividade , Tiossemicarbazonas/química , Tiossemicarbazonas/farmacologia , Células Tumorais Cultivadas
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