RESUMO
The endogenous cannabinoid (endocannabinoid) anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH). Pharmacological blockade of FAAH has emerged as a potentially attractive strategy for augmenting endocannabinoid signaling and retaining the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as weight gain and impairments in cognition and motor control, observed with direct cannabinoid receptor 1 agonists. Here, we report the detailed mechanistic and pharmacological characterization of N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzylidene)piperidine-1-carboxamide (PF-04457845), a highly efficacious and selective FAAH inhibitor. Mechanistic studies confirm that PF-04457845 is a time-dependent, covalent FAAH inhibitor that carbamylates FAAH's catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (k(inact)/K(i) = 40,300 M(-1)s(-1); IC(50) = 7.2 nM) and is exquisitely selective in vivo as determined by activity-based protein profiling. Oral administration of PF-04457845 produced potent antinociceptive effects in both inflammatory [complete Freund's adjuvant (CFA)] and noninflammatory (monosodium iodoacetate) pain models in rats, with a minimum effective dose of 0.1 mg/kg (CFA model). PF-04457845 displayed a long duration of action as a single oral administration at 1 mg/kg showed in vivo efficacy for 24 h with a concomitant near-complete inhibition of FAAH activity and maximal sustained elevation of anandamide in brain. Significantly, PF-04457845-treated mice at 10 mg/kg elicited no effect in motility, catalepsy, and body temperature. Based on its exceptional selectivity and in vivo efficacy, combined with long duration of action and optimal pharmacokinetic properties, PF-04457845 is a clinical candidate for the treatment of pain and other nervous system disorders.
Assuntos
Amidoidrolases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Dor/tratamento farmacológico , Dor/enzimologia , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Ureia/análogos & derivados , Amidoidrolases/metabolismo , Animais , Inibidores Enzimáticos/química , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas/química , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Piridazinas/química , Ratos , Ratos Sprague-Dawley , Ureia/química , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
Elongation Factor P (EF-P) is an essential component of bacterial protein synthesis, enhancing the rate of translation by facilitating the addition of amino acids to the growing peptide chain. Using purified Staphylococcus aureus EF-P and a reconstituted Escherichia coli ribosomal system, an assay monitoring the addition of radiolabeled N-formyl methionine to biotinylated puromycin was developed. Reaction products were captured with streptavidin-coated scintillation proximity assay (SPA) beads and quantified by scintillation counting. Data from the assay were used to create a kinetic model of the reaction scheme. In this model, EF-P binding to the ribosome essentially doubled the rate of the ribosomal peptidyl transferase reaction. As described here, EF-P bound to the ribosomes with an apparent K(a) of 0.75 microM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent K(m)s of 19 microM and 0.5 microM, respectively. The assay was shown to be sensitive to a number of antibiotics known to target ribosomal peptide bond synthesis, such as chloramphenicol and puromycin, but not inhibitors that target other stages of protein synthesis, such as fusidic acid or thiostrepton.
Assuntos
Antibacterianos/análise , Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Sensibilidade Microbiana , Fatores de Alongamento de Peptídeos/antagonistas & inibidores , Peptidil Transferases/antagonistas & inibidores , Proteínas Ribossômicas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Cinética , Reprodutibilidade dos Testes , Ribossomos/metabolismo , Staphylococcus aureus/metabolismo , Fatores de TempoRESUMO
The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.
Assuntos
Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas RGS/metabolismo , Animais , Proteínas Culina/antagonistas & inibidores , Proteínas Culina/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Regulação da Expressão Gênica , Biblioteca Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/química , Miocárdio/metabolismo , Ligação Proteica , Proteólise , Proteínas RGS/antagonistas & inibidores , Proteínas RGS/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , UbiquitinaçãoRESUMO
Novel antimicrobials that effectively inhibit bacterial growth are essential to fight the growing threat of antibiotic resistance. A promising target is the bacterial ribosome, a 2.5 MDa organelle susceptible to several biorthogonal modes of action used by different classes of antibiotics. To promote the discovery of unique inhibitors, we have miniaturized a coupled transcription/translation assay using E. coli and applied it to screen a natural product library of ~30 000 extracts. We significantly reduced the scale of the assay to 2 µL in a 1536-well plate format and decreased the effective concentration of costly reagents. The improved assay returned 1327 hits (4.6% hit rate) with %CV and Z' values of 8.5% and 0.74, respectively. This assay represents a significant advance in molecular screening, both in miniaturization and its application to a natural product extract library, and we intend to apply it to a broad array of pathogenic microbes in the search for novel anti-infective agents.
Assuntos
Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Anti-Infecciosos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Luciferases/genética , Miniaturização/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Bibliotecas de Moléculas Pequenas , Transcrição Gênica/efeitos dos fármacosRESUMO
The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.
Assuntos
Mitocôndrias/efeitos dos fármacos , Oxazolidinonas/farmacologia , Peptidil Transferases/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico/efeitos dos fármacos , Software , Acetamidas , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Resistência a Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Humanos , Linezolida , Mitocôndrias/enzimologia , Modelos Moleculares , Estrutura Molecular , Mutação/genética , Oxazolidinonas/química , Peptidil Transferases/metabolismo , Inibidores da Síntese de Proteínas/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico 23S , Aminoacil-RNA de Transferência/antagonistas & inibidores , Aminoacil-RNA de Transferência/metabolismo , Coloração e Rotulagem , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologiaRESUMO
The key enzymatic activity of the ribosome is catalysis of peptide bond formation. This reaction is a target for many clinically important antibiotics. However, the molecular mechanisms of the peptidyl transfer reaction, the catalytic contribution of the ribosome, and the mechanisms of antibiotic action are still poorly understood. Here we describe a novel, simple, convenient, and sensitive method for monitoring peptidyl transferase activity (SPARK). In this method, the ribosomal peptidyl transferase forms a peptide bond between two ligands, one of which is tritiated whereas the other is biotin-tagged. Transpeptidation results in covalent attachment of the biotin moiety to a tritiated compound. The amount of the reaction product is then directly quantified using the scintillation proximity assay technology: binding of the tritiated radioligand to the commercially available streptavidin-coated beads causes excitation of the bead-embedded scintillant, resulting in detection of radioactivity. The reaction is readily inhibited by known antibiotics, inhibitors of peptide bond formation. The method we developed is amenable to simple automation which makes it useful for screening for new antibiotics. The method is useful for different types of ribosomal research. Using this method, we investigated the effect of mutations at a universally conserved nucleotide of the active site of 23S rRNA, A2602 (Escherichia coli numbering), on the peptidyl transferase activity of the ribosome. The activities of the in vitro reconstituted mutant subunits, though somewhat reduced, were comparable with those of the subunits assembled with the wild-type 23S rRNA, indicating that A2602 mutations do not abolish the ability of the ribosome to catalyze peptide bond formation. Similar results were obtained with double mutants carrying mutations at A2602 and another universally conserved nucleotide in the peptidyl transferase center, A2451. The obtained results agree with our previous conclusion that the ribosome accelerates peptide bond formation primarily through entropic rather than chemical catalysis.
Assuntos
Imunoensaio/métodos , Peptidil Transferases/química , Proteínas Ribossômicas/química , Antibacterianos/farmacologia , Biotina/química , Ativação Enzimática/efeitos dos fármacos , Cinética , Peptidil Transferases/antagonistas & inibidores , Subunidades Proteicas , Puromicina/química , RNA Ribossômico/química , RNA Ribossômico/genética , RNA de Transferência Aminoácido-Específico/química , Proteínas Ribossômicas/biossíntese , Ribossomos/efeitos dos fármacos , Ribossomos/enzimologia , Ribossomos/genética , Contagem de Cintilação/métodos , Sensibilidade e Especificidade , Thermus/enzimologia , Thermus/genéticaRESUMO
The oxazolidinones are a novel class of antibiotics that inhibit initiation of protein synthesis in bacteria. In order to investigate their novel mechanism of action, the interactions of several oxazolidinones with bacterial 70S ribosomes, 50S subunits, and 30S subunits have been characterized by (1)H nuclear magnetic resonance (NMR) line-broadening analyses and transferred nuclear Overhauser enhancement (TRNOE) experiments. PNU-177553 and PNU-100592 (eperezolid) and their corresponding enantiomers, PNU-184414 and PNU-107112, were studied. The dissociation constants were determined to be 94 +/- 44 microM and 195 +/- 40 microM for PNU-177553 and eperezolid, respectively. There was a approximately 4-fold decrease in affinity for their corresponding enantiomers. The NMR-derived dissociation constants are consistent with their antibacterial activity. PNU-177553 and eperezolid were found to bind only to the 50S subunit, with similar affinity as to the 70S ribosome, and to have no affinity for the 30S subunit. Specific binding of PNU-177553 was further confirmed in TRNOE experiments in which positive NOEs observed for the small molecule alone were changed to negative NOEs in the presence of bacterial 70S ribosomes. The observed NOEs indicated that PNU-177553 did not adopt a significantly different conformation when bound to the 70S ribosome, compared to the extended conformation that exists when free in solution. Since this is likeliest the case for each of the four compounds included in this study, the A ring C5 side chain may be positioned in the proper orientation for antibacterial activity in PNU-177553 and eperezolid but not in their inactive enantiomers.