RESUMO
PARP15, or ARTD7, is an enzyme carrying out mono-ADP-ribosylation and regulating activities of a range of cellular proteins. This enzyme belongs to the family of the poly(ADP-ribose) polymerases (PARPs), which comprises of proteins with various potential disease indications. Due to their involvement in a number of cellular processes and important role in DNA repair and regulation, PARPs have been considered attractive therapeutic targets over the past few years. The pursuit of small molecule PARP inhibitors has resulted in several FDA approved drugs for multiple cancers so far. As the use of PARP inhibitors as drug scaffolds is actively explored recently, there is increasing interest in the design of selective inhibitors based on the structural features of the PARP proteins. Here, we solved high-resolution crystal structures of the human PARP15 catalytic domain in complex with three marketed drugs of PARP inhibitors, which includes compounds 3-AB, iniparib and niraparib. The structures reported here contribute to our understanding of the ligand binding modes and structural features in the PARP15 catalytic domain, which can be employed to guide the rational design of selective inhibitors of PARPs.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases , ADP Ribose Transferases/antagonistas & inibidores , Domínio Catalítico , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
The scaffold of TIQ-A, a previously known inhibitor of human poly-ADP-ribosyltransferase PARP1, was utilized to develop inhibitors against human mono-ADP-ribosyltransferases through structure-guided design and activity profiling. By supplementing the TIQ-A scaffold with small structural changes, based on a PARP10 inhibitor OUL35, selectivity changed from poly-ADP-ribosyltransferases towards mono-ADP-ribosyltransferases. Binding modes of analogs were experimentally verified by determining complex crystal structures with mono-ADP-ribosyltransferase PARP15 and with poly-ADP-ribosyltransferase TNKS2. The best analogs of the study achieved 10-20-fold selectivity towards mono-ADP-ribosyltransferases PARP10 and PARP15 while maintaining micromolar potencies. The work demonstrates a route to differentiate compound selectivity between mono- and poly-ribosyltransferases of the human ARTD family.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Isoquinolinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Tiofenos/farmacologia , ADP Ribose Transferases/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Isoquinolinas/síntese química , Isoquinolinas/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.
Assuntos
ADP Ribose Transferases , Produtos Biológicos/imunologia , Dependovirus , Vetores Genéticos , Depleção Linfocítica , Receptores Purinérgicos P2X7/imunologia , Anticorpos de Domínio Único , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/imunologia , Animais , Camundongos , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologiaRESUMO
Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Inibidores Enzimáticos/isolamento & purificação , Ensaios de Triagem em Larga Escala , Inibidores de Poli(ADP-Ribose) Polimerases/isolamento & purificação , Processamento de Proteína Pós-Traducional/genética , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP-Ribosilação/genética , Adenosina Difosfato Ribose/genética , Inibidores Enzimáticos/farmacologia , Humanos , NAD/química , Poli ADP Ribosilação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Especificidade por SubstratoRESUMO
: PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. SIGNIFICANCE: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/química , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mono-ADP-ribosyltransferases of the PARP/ARTD enzyme family are enzymes catalyzing the transfer of a single ADP-ribose unit to target proteins. The enzymes have various roles in vital cellular processes such as DNA repair and transcription, and many of the enzymes are linked to cancer-relevant functions. Thus inhibition of the enzymes is a potential way to discover and develop new drugs against cancer. Here we describe an activity-based screening assay for mono-ADP-ribosyltransferases. The assay utilizes the natural substrate of the enzymes, NAD+, and it is based on chemically converting the leftover substrate to a fluorophore and measuring its relative concentration after the enzymatic reaction. The assay is homogenous, robust, and cost-effective and, most importantly, applicable to mono-ADP-ribosyltransferases as well as poly-ADP-ribosyltransferases for screening of small-molecule inhibitors against the enzymes.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , ADP Ribose Transferases/química , Reparo do DNA/efeitos dos fármacos , Humanos , NAD/química , Bibliotecas de Moléculas Pequenas/química , Especificidade por SubstratoRESUMO
Poly-ADP-ribose polymerases (also known as ADP-ribosyltransferases or ARTDs) are a family of 17 enzymes in humans that catalyze the reversible posttranslational modification known as ADP-ribosylation. PARPs are implicated in diverse cellular processes, from DNA repair to the unfolded protein response. Small-molecule inhibitors of PARPs have improved our understanding of PARP-mediated biology and, in some cases, have emerged as promising treatments for cancers and other human diseases. However these advancements are hindered, in part, by a poor understanding of inhibitor selectivity across the PARP family. Here, we describe a simple, sensitive, and generalizable plate assay to test the potency and selectivity of small molecules against several PARP enzymes in vitro. In principle, this assay can be extended to all active PARPs, providing a convenient and direct comparison of inhibitors across the entire PARP enzyme family.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/química , ADP Ribose Transferases/química , Humanos , NAD/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
Human Diphtheria toxin-like ADP-ribosyltranferases (ARTD) 10 is an enzyme carrying out mono-ADP-ribosylation of a range of cellular proteins and affecting their activities. It shuttles between cytoplasm and nucleus and influences signaling events in both compartments, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and S phase DNA repair. Furthermore, overexpression of ARTD10 induces cell death. We recently reported on the discovery of a hit compound, OUL35 (compound 1), with 330â¯nM potency and remarkable selectivity towards ARTD10 over other enzymes in the human protein family. Here we aimed at establishing a structure-activity relationship of the OUL35 scaffold, by evaluating an array of 4-phenoxybenzamide derivatives. By exploring modifications on the linker between the aromatic rings, we identified also a 4-(benzyloxy)benzamide derivative, compound 32, which is potent (IC50â¯=â¯230â¯nM) and selective, and like OUL35 was able to rescue HeLa cells from ARTD10-induced cell death. Evaluation of an enlarged series of derivatives produced detailed knowledge on the structural requirements for ARTD10 inhibition and allowed the discovery of further tool compounds with submicromolar cellular potency that will help in understanding the roles of ARTD10 in biological systems.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Benzamidas/química , Benzamidas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Morte Celular/efeitos dos fármacos , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-AtividadeRESUMO
Iota toxin is produced by Clostridium perfringens type E strains and associated with diarrhea in cattle and lambs. This binary protein toxin comprises the enzyme component iota a (Ia), which ADP-ribosylates G-actin, and the separate transport component iota b (Ib), which delivers Ia into the cytosol of target cells. Ib binds to cell receptors and forms biologically active toxin complexes with Ia, which cause rounding of adherent cells due to the destruction of the actin cytoskeleton. Here, we report that the human peptide α-defensin-1 protects cultured cells including human colon cells from intoxication with iota toxin. In contrast, the related ß-defensin-1 had no effect, indicating a specific mode of action. The α-defensin-1 did not inhibit ADP-ribosylation of actin by Ia in vitro. Pretreatment of Ib with α-defensin-1 prior to addition of Ia prevented intoxication. Additionally, α-defensin-1 protected cells from cytotoxic effects mediated by Ib in the absence of Ia, implicating that α-defensin-1 interacts with Ib to prevent the formation of biologically active iota toxin on cells. In conclusion, the findings contribute to a better understanding of the functions of α-defensin-1 and suggest that this human peptide might be an attractive starting point to develop novel pharmacological options to treat/prevent diseases associated with iota toxin-producing Clostridium perfringens strains.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/toxicidade , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/toxicidade , Clostridium perfringens/patogenicidade , Células Epiteliais/fisiologia , alfa-Defensinas/metabolismo , Animais , Células CACO-2 , Chlorocebus aethiops , Células Epiteliais/efeitos dos fármacos , Humanos , Células VeroRESUMO
ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.
Assuntos
ADP Ribose Transferases/análise , ADP Ribose Transferases/metabolismo , Ensaios Enzimáticos/métodos , Ensaio de Imunoadsorção Enzimática , Toxina Pertussis/metabolismo , Vacinas Conjugadas/metabolismo , ADP Ribose Transferases/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão , Clostridium/enzimologia , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Humanos , Peptídeos/química , Peptídeos/metabolismo , Toxina Pertussis/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Vacinas Conjugadas/análiseRESUMO
Colorectal adenocarcinoma is the third most common cancer worldwide. PARP6, a novel member of the poly(ADP-ribose) polymerases (PARPs) and survivin, a member of the family of inhibitor of apoptosis (IAP) proteins are associated with a poor prognosis in various types of cancers. However, limited evidence exists regarding the interaction between PARP6 and survivin in colorectal adenocarcinoma. In the present study, we used the paired samples of 20 patients with colorectal adenocarcinoma to detect the expression of PARP6 and survivin in both tumor and adjacent normal colorectal mucosa. Their interaction and roles in cell viability, cell cycle, cell apoptosis and cell invasion were further investigated. Our results showed that both PARP6 and survivin exhibited higher expression in colorectal adenocarcinoma tissues and SW620 cells when compared with levels in adjacent non-tumor tissues and a normal colon cell line FHC. Co-immunoprecipitation assay showed that a significant correlation existed between PARP6 and survivin. We also showed that sole treatment of PARP6 siRNA or survivin siRNA partially inhibited the cell survival and invasion, induced cell G0/G1 arrest, and cell apoptosis at the early and late stages. The combined treatment of PARP6 siRNA and survivin siRNA suppressed the cell survival and cell invasion, further induced cell cycle phase G0/G1 arrest, and cell apoptosis at the early and late stages. Taken together, knockdown of PARP6 or survivin promotes cell apoptosis and inhibits the cell invasion of colorectal adenocarcinoma cells. A significant correlation exists between PARP6 and survivin, and both are promising targets for the development of new strategies for the diagnosis and treatment of advanced or metastatic colorectal adenocarcinoma.
Assuntos
ADP Ribose Transferases/genética , Adenocarcinoma/patologia , Neoplasias Colorretais/patologia , Técnicas de Silenciamento de Genes/métodos , Proteínas Inibidoras de Apoptose/genética , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/metabolismo , Invasividade Neoplásica , RNA Interferente Pequeno/farmacologia , Survivina , Regulação para CimaRESUMO
The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.
Assuntos
ADP Ribose Transferases/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , DNA de Cadeia Simples/metabolismo , Mycobacterium tuberculosis/genética , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Difosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Antitoxinas/química , Antitoxinas/genética , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Timidina/metabolismoRESUMO
Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.
Assuntos
Biflavonoides/farmacologia , Catequina/análogos & derivados , Toxina da Cólera/antagonistas & inibidores , Fenóis/farmacologia , Proantocianidinas/farmacologia , ADP Ribose Transferases/antagonistas & inibidores , Animais , Toxinas Bacterianas/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Células CHO , Catequina/farmacologia , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Toxina da Cólera/metabolismo , Cricetulus , Toxina Diftérica/antagonistas & inibidores , Exotoxinas/antagonistas & inibidores , Frutas/química , Extrato de Sementes de Uva/farmacologia , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Ricina/antagonistas & inibidores , Células Vero , Fatores de Virulência/antagonistas & inibidores , Vitis/química , Exotoxina A de Pseudomonas aeruginosaRESUMO
Members of the human diphtheria toxin-like ADP-ribosyltransferase (ARTD or PARP) family play important roles in regulating biological activities by mediating either a mono-ADP-ribosylation (MARylation) of a substrate or a poly-ADP-ribosylation (PARylation). ARTD10/PARP10 belongs to the MARylating ARTDs (mARTDs) subfamily, and plays important roles in biological processes that range from cellular signaling, DNA repair, and cell proliferation to immune response. Despite their biological and disease relevance, no selective inhibitors for mARTDs are available. Here we describe a small-molecule ARTD10 inhibitor, OUL35, a selective and potent inhibitor for this enzyme. We characterize its selectivity profile, model its binding, and demonstrate activity in HeLa cells where OUL35 rescued cells from ARTD10 induced cell death. Using OUL35 as a cell biology tool we show that ARTD10 inhibition sensitizes the cells to the hydroxyurea-induced genotoxic stress. Our study supports the proposed role of ARTD10 in DNA-damage repair and provides a tool compound for selective inhibition of ARTD10-mediated MARylation.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/metabolismo , Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Inibidores Enzimáticos/química , Células HeLa , Humanos , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Arginine-specific ADP-ribosytransferases 1 (ART1) is able to modify the arginine of specific proteins by mono-ADP-ribosylation. We previously reported that the expression of ART1 in human colon adenocarcinoma tissues was higher than in adjacent tissues. Herein, we primarily revealed that ART1 could regulate the epithelial-mesenchymal transition (EMT) and, therefore, the development of colon carcinoma. In CT26 cells, which overexpressed ART1 by lentiviral transfection, the following were promoted: alterations of spindle-like non-polarization, expression of EMT inducers and mesenchymal markers, migration, invasion and adhesion. However, epithelial marker expression was decreased. Correspondingly, knockdown of ART1 in CT26 cells had the opposite effects. The effect of ART1 on EMT and carcinoma metastasis was also verified in a liver metastasis model of BALB/c mice. To further explore the molecular mechanism of ART1 in EMT, CT26 cells were treated with several specific inhibitors and gene silencing. Our data suggest that ART1 could regulate EMT by regulating the RhoA/ROCK1/AKT/ß-catenin pathway and its downstream factors (snail1, vimentin, N-cadherin and E-cadherin) and that it therefore plays an important role in the progression of colon carcinoma.
Assuntos
ADP Ribose Transferases/genética , Carcinoma/genética , Neoplasias do Colo/genética , Transição Epitelial-Mesenquimal/genética , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/biossíntese , Animais , Arginina/genética , Carcinoma/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/patologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Técnicas de Silenciamento de Genes , Humanos , Lentivirus , Camundongos , Proteína Oncogênica v-akt/genética , Fuso Acromático/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Poly(ADP-ribos)ylation (PARylation) is a major posttranslational modification and signaling event in most eukaryotes. Fundamental processes like DNA repair and transcription are coordinated by this transient polymer and its binding to proteins. ADP-ribosyltransferases (ARTs) build complex ADP-ribose chains from NAD(+) onto various acceptor proteins. Molecular studies of PARylation thus remain challenging. Herein, we present the development of bioorthogonally functionalized NAD(+) analogues for the imaging of PARylation inâ vitro and in cells. Our results show that 2-modified NAD(+) analogues perform remarkably well and can be applied to the in-cell visualization of PARylation simultaneously in two colors. This tool gives insight into the substrate scope of ARTs and will help to further elucidate the biological role of PARylation by offering fast optical, multichannel read-outs.
Assuntos
NAD/análogos & derivados , Poli Adenosina Difosfato Ribose/química , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Microscopia Confocal , NAD/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Diphtheria toxin-like ADP-ribosyltransferases 1 and 5 (ARTD-1, ARTD-5) are poly ADP-ribose enzymes (PARP) involved in non-homologous end-joining (NHEJ), which is the major pathway of double-strand break (DSB) repair. In addition, ARTD-5, or Tankyrase (TNKS), is a positive regulator of the WNT signaling implicated in the development and biological behavior of many neoplasms, such as Medulloblastoma (MB), in which radiotherapy is an essential part of the treatment. The use of radiosensitizing agents may improve the therapeutic index in MB patients by increasing the efficacy of radiotherapy, while reducing toxicity to the neuroaxis. ARTD-5 seems to be a good molecular target for improving the current treatment of MB. In this study, we used the small molecule XAV939, a potent ARTD-5 inhibitor with a slight affinity for ARTD-1, in different human MB cell lines. XAV939 inhibited the WNT pathway and DNA-PKcs in our MB cells, with many biological consequences. The co-administration of XAV939 and ionizing radiations (IR) inhibited MB cells proliferation and clonogenic capacity, decreased their efficacy in repairing DNA damage, and increased IR-induced cell mortality. In conclusion, our in vitro data show that XAV939 could be a very promising small molecule in MB treatment, and these results lay the basis for further in vivo studies with the aim of improving the current therapy available for MB patients.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/farmacologia , Radiossensibilizantes/farmacologia , Tanquirases/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/genética , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/radioterapia , Proteínas Nucleares/antagonistas & inibidores , Via de Sinalização Wnt/genéticaRESUMO
Protein ADP-ribosylation is a post-translational modification involved in DNA repair, protein degradation, transcription regulation, and epigenetic events. Intracellular ADP-ribosylation is catalyzed predominantly by ADP-ribosyltransferases with diphtheria toxin homology (ARTDs). The most prominent member of the ARTD family, poly(ADP-ribose) polymerase-1 (ARTD1/PARP1) has been a target for cancer drug development for decades. Current PARP inhibitors are generally non-selective, and inhibit the mono-ADP-ribosyltransferases with low potency. Here we describe the synthesis of acylated amino benzamides and screening against the mono-ADP-ribosyltransferases ARTD7/PARP15, ARTD8/PARP14, ARTD10/PARP10, and the poly-ADP-ribosyltransferase ARTD1/PARP1. The most potent compound inhibits ARTD10 with sub-micromolar IC50.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50RESUMO
Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 µM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Botulínicas/antagonistas & inibidores , Ciclofilinas/antagonistas & inibidores , ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/toxicidade , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/toxicidade , Chlorocebus aethiops , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Células HeLa , Humanos , Transporte Proteico/efeitos dos fármacos , Células VeroRESUMO
BACKGROUND/AIMS: Colorectal carcinoma is one of the most common cancers world-wide, with high morbidity and mortality rates. Arginine ADP-ribosyltransferase 1(ART1) is an important ecto-ADP-ribose transferase and has been proven to be intimately involved in a number of biological processes. However, the influence of ART1 on survival and apoptosis of colorectal carcinoma cells and the potential mechanism of action of ART1 remain uncharacterized. METHODS: ART1 was silenced via lentiviral vector-mediated short hairpin RNA (shRNA) in CT26 colon carcinoma cells, and cisplatin (CDDP) was applied to induce apoptosis. Survival and apoptosis rate of CT26 cells was assessed by CCK8 assay, flow cytometry and Hoechst 33342 staining. Expression and activity of signaling proteins were detected by Western blot. RESULTS: ART1 knockdown enhanced the inhibition of cell survival and increased the apoptosis induced by CDDP. Furthermore, the reduced survival rate correlated with reduced levels of phos-Akt(Thr308) and phos-IκBα and reduced NF-κB p65 nuclear translocation. A decline in Bcl-2 and Bcl-xl expression and an increase in Bax expression may explain the enhanced apoptosis. CONCLUSION: This study provides a molecular mechanism for the function of ART1 in colorectal carcinoma and defines a potential therapeutic target for the enhanced treatment of this prominent world-wide disease.