RESUMEN
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.
Asunto(s)
Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Humanos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Ubiquitina/genéticaRESUMEN
BACKGROUND: Immunity decreases with age, which leads to reactivation of varicella zoster virus (VZV). In human subjects age-associated immune changes are usually measured in blood leukocytes; however, this might not reflect alterations in tissue-specific immunity. OBJECTIVES: We used a VZV antigen challenge system in the skin to investigate changes in tissue-specific mechanisms involved in the decreased response to this virus during aging. METHODS: We assessed cutaneous immunity based on the extent of erythema and induration after intradermal VZV antigen injection. We also performed immune histology and transcriptomic analyses on skin biopsy specimens taken from the challenge site in young (<40 years) and old (>65 years) subjects. RESULTS: Old human subjects exhibited decreased erythema and induration, CD4+ and CD8+ T-cell infiltration, and attenuated global gene activation at the site of cutaneous VZV antigen challenge compared with young subjects. This was associated with increased sterile inflammation in the skin in the same subjects related to p38 mitogen-activated protein kinase-related proinflammatory cytokine production (P < .0007). We inhibited systemic inflammation in old subjects by means of pretreatment with an oral small-molecule p38 mitogen-activated protein kinase inhibitor (Losmapimod; GlaxoSmithKline, Brentford, United Kingdom), which reduced both serum C-reactive protein levels and peripheral blood monocyte secretion of IL-6 and TNF-α. In contrast, cutaneous responses to VZV antigen challenge were increased significantly in the same subjects (P < .0003). CONCLUSION: Excessive inflammation in the skin early after antigen challenge retards antigen-specific immunity. However, this can be reversed by inhibition of inflammatory cytokine production that can be used to promote vaccine efficacy and the treatment of infections and malignancy during aging.
Asunto(s)
Envejecimiento/inmunología , Antígenos Virales/inmunología , Herpesvirus Humano 3/inmunología , Piel/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/sangre , Proteína C-Reactiva/análisis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Humanos , Inflamación/sangre , Inflamación/inmunología , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/sangre , Adulto JovenRESUMEN
Cystic fibrosis (CF) is a major lethal genetic disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). This encodes a chloride ion channel on the apical surface of epithelial cells. The most common mutation in CFTR (F508del-CFTR) generates a protein that is misfolded and retained in the endoplasmic reticulum. Identifying small molecules that correct this CFTR trafficking defect is a promising approach in CF therapy. However, to date only modest efficacy has been reported for correctors in clinical trials. We identified the marine sponge metabolite latonduine as a corrector. We have now developed a series of latonduine derivatives that are more potent F508del-CFTR correctors with one (MCG315 [2,3-dihydro-1H-2-benzazepin-1-one]) having 10-fold increased corrector activity and an EC50 of 72.25 nM. We show that the latonduine analogs inhibit poly-ADP ribose polymerase (PARP) isozymes 1, 3, and 16. Further our molecular modeling studies point to the latonduine analogs binding to the PARP nicotinamide-binding domain. We established the relationship between the ability of the latonduine analogs to inhibit PARP-16 and their ability to correct F508del-CFTR trafficking. We show that latonduine can inhibit both PARP-3 and -16 and that this is necessary for CFTR correction. We demonstrate that latonduine triggers correction by regulating the activity of the unfolded protein response activator inositol-requiring enzyme (IRE-1) via modulation of the level of its ribosylation by PARP-16. These results establish latonduines novel site of action as well as its proteostatic mechanism of action.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , Proteínas de Ciclo Celular/química , Línea Celular , Endorribonucleasas/metabolismo , Técnicas de Silenciamiento del Gen , Glicoproteínas/metabolismo , Compuestos Heterocíclicos con 3 Anillos/química , Humanos , Modelos Moleculares , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/química , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP-Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ-/- liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.
Asunto(s)
Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Factores de Transcripción ARNTL/metabolismo , Animales , Proteínas CLOCK/metabolismo , Línea Celular , Cromatina/química , Cromatina/metabolismo , Péptidos y Proteínas de Señalización del Ritmo Circadiano/biosíntesis , Criptocromos/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes Neurológicos , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Elementos de Respuesta , Activación TranscripcionalRESUMEN
Background: Prinflammatory extracellular chromatin from neutrophil extracellular traps (NETs) and other cellular sources is found in COVID-19 patients and may promote pathology. We determined whether pulmonary administration of the endonuclease dornase alfa reduced systemic inflammation by clearing extracellular chromatin. Methods: Eligible patients were randomized (3:1) to the best available care including dexamethasone (R-BAC) or to BAC with twice-daily nebulized dornase alfa (R-BAC + DA) for seven days or until discharge. A 2:1 ratio of matched contemporary controls (CC-BAC) provided additional comparators. The primary endpoint was the improvement in C-reactive protein (CRP) over time, analyzed using a repeated-measures mixed model, adjusted for baseline factors. Results: We recruited 39 evaluable participants: 30 randomized to dornase alfa (R-BAC +DA), 9 randomized to BAC (R-BAC), and included 60 CC-BAC participants. Dornase alfa was well tolerated and reduced CRP by 33% compared to the combined BAC groups (T-BAC). Least squares (LS) mean post-dexamethasone CRP fell from 101.9 mg/L to 23.23 mg/L in R-BAC +DA participants versus a 99.5 mg/L to 34.82 mg/L reduction in the T-BAC group at 7 days; p=0.01. The anti-inflammatory effect of dornase alfa was further confirmed with subgroup and sensitivity analyses on randomised participants only, mitigating potential biases associated with the use of CC-BAC participants. Dornase alfa increased live discharge rates by 63% (HR 1.63, 95% CI 1.01-2.61, p=0.03), increased lymphocyte counts (LS mean: 1.08 vs 0.87, p=0.02) and reduced circulating cf-DNA and the coagulopathy marker D-dimer (LS mean: 570.78 vs 1656.96 µg/mL, p=0.004). Conclusions: Dornase alfa reduces pathogenic inflammation in COVID-19 pneumonia, demonstrating the benefit of cost-effective therapies that target extracellular chromatin. Funding: LifeArc, Breathing Matters, The Francis Crick Institute (CRUK, Medical Research Council, Wellcome Trust). Clinical trial number: NCT04359654.
Asunto(s)
Antiinflamatorios , Tratamiento Farmacológico de COVID-19 , COVID-19 , Desoxirribonucleasa I , Humanos , Masculino , Femenino , Desoxirribonucleasa I/administración & dosificación , Desoxirribonucleasa I/uso terapéutico , Persona de Mediana Edad , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Anciano , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Trampas Extracelulares/efectos de los fármacos , SARS-CoV-2 , Proteína C-Reactiva/análisis , Proteína C-Reactiva/metabolismo , Dexametasona/administración & dosificación , Dexametasona/uso terapéutico , Adulto , Nebulizadores y Vaporizadores , Resultado del Tratamiento , Administración por InhalaciónRESUMEN
MAPKAPK5 has been proposed to play a role in regulation of matrix metalloprotease expression and so to be a potential target for intervention in rheumatoid arthritis. We present here the identification of a series of compounds against this target which are effective in both biochemical and cell assays. The expansion of the series is described, along with early SAR and pharmacokinetics for some representative compounds.
Asunto(s)
Antirreumáticos/síntesis química , Imidazoles/síntesis química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/síntesis química , Animales , Antirreumáticos/farmacocinética , Antirreumáticos/farmacología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Sitios de Unión , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Imidazoles/farmacocinética , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/farmacocinética , Pirazoles/farmacología , Ratas , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
Most cases of cystic fibrosis (CF) are caused by class 2 mutations in the cystic fibrosis transmembrane regulator (CFTR). These proteins preserve some channel function but are retained in the endoplasmic reticulum (ER). Partial rescue of the most common CFTR class 2 mutant, F508del-CFTR, has been achieved through the development of pharmacological chaperones (Tezacaftor and Elexacaftor) that bind CFTR directly. However, it is not clear whether these drugs will rescue all class 2 CFTR mutants to a medically relevant level. We have previously shown that the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen can correct F508del-CFTR trafficking. Here, we utilized RNAi and pharmacological inhibitors to determine the mechanism of action of the NSAID glafenine. Using cellular thermal stability assays (CETSAs), we show that it is a proteostasis modulator. Using medicinal chemistry, we identified a derivative with a fourfold increase in CFTR corrector potency. Furthermore, we show that these novel arachidonic acid pathway inhibitors can rescue difficult-to-correct class 2 mutants, such as G85E-CFTR > 13%, that of non-CF cells in well-differentiated HBE cells. Thus, the results suggest that targeting the arachidonic acid pathway may be a profitable way of developing correctors of certain previously hard-to-correct class 2 CFTR mutations.
Asunto(s)
Fibrosis Quística , Glafenina , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Ácido Araquidónico , Ciclooxigenasa 2/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Glafenina/uso terapéutico , Humanos , MutaciónRESUMEN
The study of protein target families, as opposed to single targets, has become a very powerful tool in chemogenomics-led drug discovery. By integrating comprehensive chemoinformatics and bioinformatics databases with customised analytical tools, a 'Toolkit' approach for the target family is possible, thus allowing predictions of the ligand class, affinity, selectivity and likely off-target issues to be made for the guidance of the medicinal chemist. In this review, we highlight the development and application of the Toolkit approach to the protein kinase superfamily, drawing on examples from lead optimisation studies and the design of focused libraries for lead discovery.
Asunto(s)
Diseño de Fármacos , Genómica , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Proteínas Quinasas/química , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Psoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy. METHODS AND FINDINGS: The preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in ex vivo lesional psoriatic skin. CONCLUSIONS: Our work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.
Asunto(s)
Factores Inmunológicos/farmacología , Interleucina-17/antagonistas & inhibidores , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Psoriasis/tratamiento farmacológico , Piel/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Administración Cutánea , Aminoquinolinas , Animales , Evaluación Preclínica de Medicamentos , Femenino , Expresión Génica , Genes Reporteros , Humanos , Imiquimod , Factores Inmunológicos/síntesis química , Interleucina-17/genética , Interleucina-17/inmunología , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Permeabilidad , Cultivo Primario de Células , Psoriasis/inducido químicamente , Psoriasis/inmunología , Psoriasis/patología , Piel/inmunología , Piel/patología , Bibliotecas de Moléculas Pequeñas/síntesis química , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/patología , Investigación Biomédica TraslacionalRESUMEN
Cystic fibrosis (CF) is the most frequent lethal genetic disease and the most frequent mutation is F508del-cystic fibrosis transmembrane regulator (CFTR). In common with some other protein trafficking diseases the mutant protein is functional but recognized by the cellular quality control system retained in the endoplasmic reticulum (ER) and degraded. There have been some recent impressive advances in developing corrector compounds that restore the trafficking of the mutant protein to the plasma membrane. The targets of these correctors and possible mechanisms of action are discussed.