RESUMO
OBJECTIVES: Primary SS (pSS) is a chronic autoimmune disorder characterized by mucosal dryness and systemic symptoms. We tested the effects of inhibition of cathepsin S using the potent and selective inhibitor RO5459072 on disease activity and symptoms of pSS. METHODS: This was a randomized, double-blind, placebo-controlled, parallel-group, Phase IIA study to investigate the effects of RO5459072 (100 mg twice daily; 200 mg per day). Seventy-five patients with pSS were randomized 1:1 to receive either RO5459072 or placebo for 12 weeks. The primary outcome was the proportion of patients with a ≥3 point reduction from baseline in EULAR SS Disease Activity Index (ESSDAI) score. We also investigated the effects of RO5459072 on quality of life, exocrine gland function, biomarkers related to SS, and safety and tolerability. RESULTS: The proportion of patients showing an improvement in ESSDAI score was not significantly different between the RO5459072 and placebo arms. No clinically meaningful treatment effects were observed in favour of RO5459072 for all secondary outcomes. Analysis of soluble biomarkers indicated target engagement between RO5459072 and cathepsin S. There were modest decreases in the number of circulating B cells and T cells in the RO5459072 group, although these did not reach significance. RO5459072 was safe and well-tolerated. CONCLUSIONS: There was no clinically relevant improvement in ESSDAI score (primary endpoint), and no apparent benefit in favour of RO5459072 in any of the secondary clinical endpoints. Further work is needed in order to understand the mechanisms of MHC-II-mediated immune stimulation in pSS. TRIAL REGISTRATION: ClinicalTrials.gov; NCT02701985.
Assuntos
Síndrome de Sjogren , Humanos , Síndrome de Sjogren/complicações , Qualidade de Vida , Resultado do Tratamento , Método Duplo-Cego , Catepsinas/uso terapêutico , BiomarcadoresRESUMO
OBJECTIVE: Primary Sjögren syndrome (pSS) is characterized by T and B cell infiltration of exocrine glands. The cysteine protease cathepsin S (CatS) is crucially involved in MHCII processing and T cell stimulation, and elevated levels have been found in patients with RA, psoriasis and pSS. However, little is known about the functional characteristics and mechanisms of SS-A- and SS-B-specific T cells in pSS patients. We herein investigated the inhibition of CatS activity in different biocompartments of pSS patients including antigen-specific T cell responses. METHODS: Ex vivo CatS activity was assessed in tears, plasma and saliva of 15 pSS patients and 13 healthy controls (HC) and in the presence or absence of the specific CatS inhibitor RO5459072. In addition, antigen (SS-A (60kD), SS-B, influenza H3N2, tetanus toxoid and SEB)-specific T cell responses were examined using ex vivo IFN-γ/IL-17 Dual ELISPOT and Bromdesoxyuridin (BrdU) proliferation assays in the presence or absence of RO5459072. Supernatants were analysed for IL-1ß, IL-6, IL-10, TNF-α, IL-21, IL-22 and IL-23, using conventional ELISA. RESULTS: CatS activity was significantly elevated in tear fluid, but not other biocompartments, was inversely associated with exocrinic function in pSS patients and could significantly be suppressed by RO5459072. Moreover, CatS inhibition by RO5459072 led to strong and dose-dependent suppression of SS-A/SS-B-specific T cell effector functions and cytokine secretion by CD14+ monocytes. However, RO5459072 was incapable of suppressing SS-A/SS-B-induced secretion of cytokines in CD14+ monocytes when T cells were absent, confirming a CatS/MHCII-mediated mechanism of suppression. CONCLUSION: CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. Conversely, CatS inhibition diminishes T cell and associated monokine responses towards relevant autoantigens in pSS. Thus, CatS inhibition may represent a promising novel treatment strategy in pSS.
Assuntos
Catepsinas/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Pirazóis/farmacologia , Pirrolidinas/farmacologia , Saliva/imunologia , Síndrome de Sjogren/imunologia , Lágrimas/imunologia , Adulto , Idoso , Autoantígenos/imunologia , Autoantígenos/metabolismo , Catepsinas/imunologia , Catepsinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Ribonucleoproteínas/imunologia , Ribonucleoproteínas/metabolismo , Saliva/enzimologia , Síndrome de Sjogren/sangue , Síndrome de Sjogren/enzimologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Lágrimas/enzimologia , Antígeno SS-BRESUMO
In several types of antigen-presenting cells (APCs), Cathepsin S (CatS) plays a crucial role in the regulation of MHC class II surface expression and consequently influences antigen (Ag) presentation of APCs to CD4+ T cells. During the assembly of MHC class II-Ag peptide complexes, CatS cleaves the invariant chain p10 (Lip10) - a fragment of the MHC class II-associated invariant chain peptide. In this report, we used a selective, high-affinity CatS inhibitor to suppress the proteolytic activity of CatS in lymphoid and myeloid cells. CatS inhibition resulted in a concentration-dependent Lip10 accumulation in B cells from both healthy donors and patients with systemic lupus erythematosus (SLE). Furthermore, CatS inhibition led to a decreased MHC class II expression on B cells, monocytes, and proinflammatory macrophages. In SLE patient-derived peripheral blood mononuclear cells, CatS inhibition led to a suppressed secretion of IL-6, TNFα, and IL-10. In a second step, we tested the effect of CatS inhibition on macrophages being exposed to patient-derived autoantibodies against C1q (anti-C1q) that are known to be associated with severe lupus nephritis. As shown previously, those SLE patient-derived high-affinity anti-C1q bound to immobilized C1q induce a proinflammatory phenotype in macrophages. Using this human in vitro model of autoimmunity, we found that CatS inhibition reduces the inflammatory responses of macrophages as demonstrated by a decreased secretion of proinflammatory cytokines, the downregulation of MHC class II and CD80. In summary, we can show that the used CatS inhibitor is able to block Lip10 degradation in healthy donor- and SLE patient-derived B cells and inhibits the induction of proinflammatory macrophages. Thus, CatS inhibition seems to be a promising future treatment of SLE.
Assuntos
Catepsinas/antagonistas & inibidores , Inflamação/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/metabolismo , Adulto , Idoso , Antígenos CD20/genética , Antígenos CD20/metabolismo , Antígenos de Superfície , Linfócitos B , Células Cultivadas , Citocinas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.
Assuntos
Fator D do Complemento/antagonistas & inibidores , Via Alternativa do Complemento/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Fator D do Complemento/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Microarrays to examine the global expression levels of microRNAs (miRNAs) in a systematic in-parallel manner have become important tools to help unravel the functions of miRNAs and to understand their roles in RNA-based regulation and their implications in human diseases. We have established a novel miRNA-specific microarray platform that enables the simultaneous expression analysis of both known and predicted miRNAs obtained from human or mouse origin. Chemically modified 2'-O-(2-methoxyethyl)-(MOE) oligoribonucleotide probes were arrayed onto Evanescent Resonance (ER) microchips by robotic spotting. Supplementing the complementary probes against miRNAs with carefully designed mismatch controls allowed for accurate sequence-specific determination of miRNA expression profiles obtained from a panel of mouse tissues. This revealed new expression signatures of known miRNAs as well as of novel miRNAs previously predicted using bioinformatic methods. Systematic confirmation of the array data with northern blotting and, in particular, real-time PCR suggests that the described microarray platform is a powerful tool to analyze miRNA expression patterns with rapid throughput and high fidelity.
Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Células HeLa , Humanos , Camundongos , Sondas de Oligonucleotídeos/química , RNA/química , Distribuição TecidualRESUMO
Recent years have seen a rapid increase in our understanding of how double-stranded RNA (dsRNA) and 21- to 25-nucleotide small RNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), control gene expression in eukaryotes. This RNA-mediated regulation generally results in sequence-specific inhibition of gene expression; this can occur at levels as different as chromatin modification and silencing, translational repression and mRNA degradation. Many details of the biogenesis and function of miRNAs and siRNAs, and of the effector complexes with which they associate have been elucidated. The first structural information on protein components of the RNA interference (RNAi) and miRNA machineries is emerging, and provides some insight into the mechanism of RNA-silencing reactions.
Assuntos
Regulação da Expressão Gênica/genética , MicroRNAs/química , MicroRNAs/genética , Modelos Moleculares , Interferência de RNA/fisiologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Animais , Sítios de Ligação , Humanos , Conformação de Ácido Nucleico , Ribonuclease III/metabolismo , Relação Estrutura-AtividadeRESUMO
Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21-nt small interfering RNAs (siRNAs) during RNA interference and excises microRNAs (miRNAs) from precursor hairpins. PAZ and PIWI domain (PPD) proteins, also involved in RNAi and miRNA function, are the best-characterized proteins known to interact with Dicer. PPD proteins are the core constituents of effector complexes, RISCs and miRNPs, mediating siRNA and miRNA function. In this chapter we describe overexpression and purification of recombinant human Dicer, its biochemical properties, and mapping of domains responsible for Dicer-PPD protein interactions.
Assuntos
Proteínas/metabolismo , Ribonuclease III/isolamento & purificação , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. We have generated mutations in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and studied their effect on RNA processing. Our results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, we propose that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.
Assuntos
Endorribonucleases/química , Endorribonucleases/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , RNA Helicases DEAD-box , Dimerização , Endorribonucleases/genética , Endorribonucleases/isolamento & purificação , Escherichia coli/enzimologia , Humanos , Manganês/metabolismo , MicroRNAs/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Helicases/genética , RNA Helicases/isolamento & purificação , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
PAZ PIWI domain (PPD) proteins, together with the RNA cleavage products of Dicer, form ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs). RISCs mediate gene silencing through targeted messenger RNA cleavage and translational suppression. The PAZ domains of PPD and Dicer proteins were originally thought to mediate binding between PPD proteins and Dicer, although no evidence exists to support this theory. Here we show that PAZ domains are not required for PPD protein-Dicer interactions. Rather, a subregion of the PIWI domain in PPD proteins, the PIWI-box, binds directly to the Dicer RNase III domain. Stable binding between PPD proteins and Dicer was dependent on the activity of Hsp90. Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro. Lastly, we show that PPD proteins and Dicer are present in soluble and membrane-associated fractions, indicating that interactions between these two types of proteins may occur in multiple compartments.
Assuntos
Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Ribonuclease III/metabolismo , Animais , Proteínas Argonautas , Sítios de Ligação , Fator de Iniciação 2 em Eucariotos , Glutationa Transferase/análise , Glutationa Transferase/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Estrutura Terciária de Proteína/genética , Proteínas/genética , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Ratos , Ribonuclease III/química , Ribonuclease III/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.