RESUMEN
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.
Asunto(s)
Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas , ADP Ribosa Transferasas/antagonistas & inhibidores , Dominio Catalítico , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Isoquinolinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tiofenos/farmacología , ADP Ribosa Transferasas/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Isoquinolinas/síntesis química , Isoquinolinas/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Poli(ADP-Ribosa) Polimerasas/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/químicaRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas , Productos Biológicos/inmunología , Dependovirus , Vectores Genéticos , Depleción Linfocítica , Receptores Purinérgicos P2X7/inmunología , Anticuerpos de Dominio Único , ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/inmunología , Animales , Ratones , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunologíaRESUMEN
The targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify milligram-levels of active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 µM and 6.8 µM) were identified in the in vitro NAD+ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove to be useful hit compounds in targeted B. pertussis-selective drug development.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/metabolismo , Descubrimiento de Drogas , Toxina del Pertussis/antagonistas & inhibidores , Toxina del Pertussis/metabolismo , Bordetella pertussis/efectos de los fármacos , Bordetella pertussis/patogenicidad , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , NAD/metabolismoRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Inhibidores de Poli(ADP-Ribosa) Polimerasas/aislamiento & purificación , Procesamiento Proteico-Postraduccional/genética , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/genética , ADP-Ribosilación/genética , Adenosina Difosfato Ribosa/genética , Inhibidores Enzimáticos/farmacología , Humanos , NAD/química , Poli ADP Ribosilación/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/genética , Especificidad por SustratoRESUMEN
Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Exotoxinas/antagonistas & inhibidores , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Anticuerpos de Cadena Única/farmacología , Factores de Virulencia/antagonistas & inhibidores , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/inmunología , ADP Ribosa Transferasas/metabolismo , Antibacterianos/inmunología , Antibacterianos/uso terapéutico , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Dominio Catalítico/genética , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/farmacología , Exotoxinas/genética , Exotoxinas/inmunología , Exotoxinas/metabolismo , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Biblioteca de Péptidos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéutico , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMEN
DNA-encoded chemical libraries are increasingly used in pharmaceutical research because they enable the rapid discovery of synthetic protein ligands. Here we explored whether target-class focused DNA-encoded chemical libraries can be cost-effective tools to achieve robust screening productivity for a series of proteins. The study revealed that a DNA-encoded library designed for NAD+-binding pockets (NADEL) effectively sampled the chemical binder space of enzymes with ADP-ribosyltransferase activity. The extracted information directed the synthesis of inhibitors for several enzymes including PARP15 and SIRT6. The high dissimilarity of NADEL screening fingerprints for different proteins translated into inhibitors that showed selectivity for their target. The discovery of patterns of enriched structures for six out of eight tested proteins is remarkable for a library of 58â¯302 DNA-tagged structures and illustrates the prospect of focused DNA-encoded libraries as economic alternatives to large library platforms.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , ADN/química , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Sirtuinas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , ADP Ribosa Transferasas/metabolismo , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Sirtuinas/metabolismo , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
: 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.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Neoplasias de la Mama/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , ADP Ribosa Transferasas/química , Reparación del ADN/efectos de los fármacos , Humanos , NAD/química , Bibliotecas de Moléculas Pequeñas/química , Especificidad por SustratoRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/química , ADP Ribosa Transferasas/química , Humanos , NAD/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Benzamidas/química , Benzamidas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/metabolismo , Muerte Celular/efectos de los fármacos , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/toxicidad , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/toxicidad , Clostridium perfringens/patogenicidad , Células Epiteliales/fisiología , alfa-Defensinas/metabolismo , Animales , Células CACO-2 , Chlorocebus aethiops , Células Epiteliales/efectos de los fármacos , Humanos , Células VeroRESUMEN
During infection, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa employs its type III secretion system to translocate the toxin exoenzyme S (ExoS) into the eukaryotic host cell cytoplasm. ExoS is an essential in vivo virulence factor that enables P. aeruginosa to avoid phagocytosis and eventually kill the host cell. ExoS elicits its pathogenicity mainly via ADP-ribosyltransferase (ADPRT) activity. We recently identified a new class of ExoS ADPRT inhibitors with in vitro IC50 of around 20 µM in an enzymatic assay using a recombinant ExoS ADPRT domain. Herein, we report structure-activity relationships of this compound class by comparing a total of 51 compounds based on a thieno [2,3-d]pyrimidin-4(3H)-one and 4-oxo-3,4-dihydroquinazoline scaffolds. Improved inhibitors with in vitro IC50 values of 6 µM were identified. Importantly, we demonstrated that the most potent inhibitors block ADPRT activity of native full-length ExoS secreted by viable P. aeruginosa with an IC50 value of 1.3 µM in an enzymatic assay. This compound class holds promise as starting point for development of novel antibacterial agents.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Pseudomonas aeruginosa/enzimología , Pirimidinonas/farmacología , Quinazolinas/farmacología , ADP Ribosa Transferasas/metabolismo , Toxinas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Estructura Molecular , Inhibidores de Poli(ADP-Ribosa) Polimerasas/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Pirimidinonas/síntesis química , Pirimidinonas/química , Quinazolinas/síntesis química , Quinazolinas/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/análisis , ADP Ribosa Transferasas/metabolismo , Pruebas de Enzimas/métodos , Ensayo de Inmunoadsorción Enzimática , Toxina del Pertussis/metabolismo , Vacunas Conjugadas/metabolismo , ADP Ribosa Transferasas/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión , Clostridium/enzimología , Escherichia coli/enzimología , Escherichia coli/metabolismo , Humanos , Péptidos/química , Péptidos/metabolismo , Toxina del Pertussis/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Vacunas Conjugadas/análisisRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that can be very hard to treat because of high resistance to different antibiotics and alternative treatment regimens are greatly needed. An alternative or a complement to traditional antibiotic is to inhibit virulence of the bacteria. The salicylidene acylhydrazide, INP0341, belongs to a class of compounds that has previously been shown to inhibit virulence in a number of Gram-negative bacteria. In this study, the virulence blocking effect of INP0341 on P. aeruginosa was studied in vitro and in vivo. Two important and closely related virulence system were examined, the type III secretion system (T3SS) that translocates virulence effectors into the cytosol of the host cell to evade immune defense and facilitate colonization and the flagella system, needed for motility and biofilm formation. INP0341 was shown to inhibit expression and secretion of the T3SS toxin exoenzyme S (ExoS) and to prevent bacterial motility on agar plates and biofilm formation. In addition, INP0341 showed an increased survival of P. aeruginosa-infected mice. In conclusion, INP0341 attenuates P. aeruginosa virulence.
Asunto(s)
Antibacterianos/uso terapéutico , Biopelículas/efectos de los fármacos , Flagelos/efectos de los fármacos , Hidrazinas/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Infección de Heridas/tratamiento farmacológico , ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/metabolismo , Administración Cutánea , Animales , Antibacterianos/administración & dosificación , Antibacterianos/efectos adversos , Antibacterianos/farmacología , Fenómenos Fisiológicos Bacterianos/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Quemaduras/complicaciones , Quemaduras/microbiología , Farmacorresistencia Bacteriana Múltiple , Flagelos/fisiología , Células HeLa , Humanos , Hidrazinas/administración & dosificación , Hidrazinas/efectos adversos , Hidrazinas/farmacología , Masculino , Ratones Endogámicos BALB C , Mutación , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Factor sigma/genética , Factor sigma/metabolismo , Análisis de Supervivencia , Transactivadores/genética , Transactivadores/metabolismo , Virulencia/efectos de los fármacos , Infección de Heridas/metabolismo , Infección de Heridas/microbiologíaRESUMEN
Neutrophils are the first line of defense against bacterial infections, and the generation of reactive oxygen species is a key part of their arsenal. Pathogens use detoxification systems to avoid the bactericidal effects of reactive oxygen species. Here we demonstrate that the Gram-negative pathogen Pseudomonas aeruginosa is susceptible to reactive oxygen species but actively blocks the reactive oxygen species burst using two type III secreted effector proteins, ExoS and ExoT. ExoS ADP-ribosylates Ras and prevents it from interacting with and activating phosphoinositol-3-kinase (PI3K), which is required to stimulate the phagocytic NADPH-oxidase that generates reactive oxygen species. ExoT also affects PI3K signaling via its ADP-ribosyltransferase activity but does not act directly on Ras. A non-ribosylatable version of Ras restores reactive oxygen species production and results in increased bacterial killing. These findings demonstrate that subversion of the host innate immune response requires ExoS-mediated ADP-ribosylation of Ras in neutrophils.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Neutrófilos/inmunología , Neutrófilos/metabolismo , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas ras/efectos de los fármacos , ADP Ribosa Transferasas/metabolismo , ADP-Ribosilación/efectos de los fármacos , Animales , Toxinas Bacterianas/inmunología , Recuento de Colonia Microbiana , Epitelio/patología , Ojo/patología , Femenino , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Humanos , Inmunidad Innata , Ratones Endogámicos C57BL , NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Fagocitosis , Fosfatidilinositol 3-Quinasas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/patogenicidad , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Sistemas de Secreción Tipo III/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/genética , Adenocarcinoma/patología , Neoplasias Colorrectales/patología , Técnicas de Silenciamiento del Gen/métodos , Proteínas Inhibidoras de la Apoptosis/genética , ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/metabolismo , Invasividad Neoplásica , ARN Interferente Pequeño/farmacología , Survivin , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , ADN de Cadena Simple/metabolismo , Mycobacterium tuberculosis/genética , ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/genética , Adenosina Difosfato/metabolismo , Secuencias de Aminoácidos , Antitoxinas/química , Antitoxinas/genética , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Timidina/metabolismoRESUMEN
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.
Asunto(s)
Biflavonoides/farmacología , Catequina/análogos & derivados , Toxina del Cólera/antagonistas & inhibidores , Fenoles/farmacología , Proantocianidinas/farmacología , ADP Ribosa Transferasas/antagonistas & inhibidores , Animales , Toxinas Bacterianas/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Células CHO , Catequina/farmacología , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Toxina del Cólera/metabolismo , Cricetulus , Toxina Diftérica/antagonistas & inhibidores , Exotoxinas/antagonistas & inhibidores , Frutas/química , Extracto de Semillas de Uva/farmacología , Simulación del Acoplamiento Molecular , Extractos Vegetales/farmacología , Ricina/antagonistas & inhibidores , Células Vero , Factores de Virulencia/antagonistas & inhibidores , Vitis/química , Exotoxina A de Pseudomonas aeruginosaRESUMEN
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.