RESUMO
The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a multiprotein complex and component of the innate immune system that is activated by exogenous and endogenous danger signals to promote activation of caspase-1 and the maturation and release of the proinflammatory cytokines interleukin (IL)-1ß and IL-18. Inappropriate activation of NLRP3 has been implicated in the pathophysiology of multiple inflammatory and autoimmune diseases, including cardiovascular disease, neurodegenerative diseases, and nonalcoholic steatohepatitis (NASH), thus increasing the clinical interest of this target. We describe in this study the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel and highly specific NLRP3 inhibitor, JT001 (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonylurea). In cell-based assays, JT001 potently and selectively inhibited NLRP3 inflammasome assembly, resulting in the inhibition of cytokine release and the prevention of pyroptosis, a form of inflammatory cell death triggered by active caspase-1. Oral administration of JT001 to mice inhibited IL-1ß production in peritoneal lavage fluid at plasma concentrations that correlated with mouse in vitro whole blood potency. Orally administered JT001 was effective in reducing hepatic inflammation in three different murine models, including the Nlrp3A350V /+CreT model of Muckle-Wells syndrome (MWS), a diet-induced obesity NASH model, and a choline-deficient diet-induced NASH model. Significant reductions in hepatic fibrosis and cell damage were also observed in the MWS and choline-deficient models. Our findings demonstrate that blockade of NLRP3 attenuates hepatic inflammation and fibrosis and support the use of JT001 to investigate the role of NLRP3 in other inflammatory disease models. SIGNIFICANCE STATEMENT: Persistent inflammasome activation is the consequence of inherited mutations of NLRP3 and results in the development of cryopyrin-associated periodic syndromes associated with severe systemic inflammation. NLRP3 is also upregulated in nonalcoholic steatohepatitis, a metabolic chronic liver disease currently missing a cure. Selective and potent inhibitors of NLRP3 hold great promise and have the potential to overcome an urgent unmet need.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR , Domínio Pirina , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/prevenção & controle , Caspase 1/metabolismo , Inflamação , Colina/efeitos adversos , Interleucina-1beta/metabolismoRESUMO
BACKGROUND/AIMS: Retinoid-related orphan receptor gamma t (RORγt) is a master regulator of T helper 17 cells that plays a pivotal role in the production of inflammatory cytokines including interleukin (IL)-17. Therefore, RORγt has attracted much attention as a target receptor for the treatment of inflammatory diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, and psoriasis. This study aims to characterize TAK-828F, a potent and selective RORγt inverse agonist. METHODS: The biochemical properties of TAK-828F were evaluated using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay, surface plasmon resonance (SPR) biosensor assay, cofactor recruitment assay, reporter assay, and IL-17 expression assay. RESULTS: TR-FRET binding assay and SPR biosensor assay revealed rapid, reversible, and high affinity binding of TAK-828F to RORγt. The cofactor recruitment assay showed that TAK-828F inhibited the recruitment of steroid receptor coactivator-1 to RORγt. Furthermore, TAK-828F inhibited the transcriptional activity of human and mouse RORγt with selectivity against human RORα and RORß. TAK-828F also suppressed IL-17 production in Jurkat cells, overexpressing human RORγt. CONCLUSION: These favorable properties will be of advantage in the evaluation of TAK-828F in clinical studies for inflammatory diseases. Furthermore, these findings demonstrate that TAK-828F could serve as a pharmacological tool for further studies of RORγt and inflammatory diseases.
Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , Receptores Nucleares Órfãos/agonistas , Sulfonas/química , Sulfonas/farmacologia , Animais , Benzofuranos/farmacocinética , Cromatografia de Afinidade , Transferência Ressonante de Energia de Fluorescência , Humanos , Interleucina-17/metabolismo , Células Jurkat , Cinética , Camundongos , Receptores Nucleares Órfãos/metabolismo , Ligação Proteica , Sulfonas/farmacocinética , Ativação TranscricionalRESUMO
The NLRP3 inflammasome is an intracellular, multiprotein complex that promotes the auto-catalytic activation of caspase-1 and the subsequent maturation and secretion of the pro-inflammatory cytokines, IL-1ß and IL-18. Persistent activation of the NLRP3 inflammasome has been implicated in the pathophysiology of a number of inflammatory and autoimmune diseases, including neuroinflammation, cardiovascular disease, non-alcoholic steatohepatitis, lupus nephritis and severe asthma. Here we describe the preclinical profile of JT002, a novel small molecule inhibitor of the NLRP3 inflammasome. JT002 potently reduced NLRP3-dependent proinflammatory cytokine production across a number of cellular assays and prevented pyroptosis, an inflammatory form of cell death triggered by active caspase-1. JT002 demonstrated in vivo target engagement at therapeutically relevant concentrations when orally dosed in mice and prevented body weight loss and improved inflammatory and fibrotic endpoints in a model of Muckle-Wells syndrome (MWS). In two distinct models of neutrophilic airway inflammation, JT002 treatment significantly reduced airway hyperresponsiveness and airway neutrophilia. These results provide a rationale for the therapeutic targeting of the NLRP3 inflammasome in severe asthma and point to the use of JT002 in a variety of inflammatory disorders.
Assuntos
Doenças Cardiovasculares , Nefrite Lúpica , Animais , Camundongos , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Caspase 1RESUMO
Preeclampsia is a syndromic disease of the mother, fetus, and placenta. The main limitation in early and accurate diagnosis of preeclampsia is rooted in the heterogeneity of this syndrome as reflected by diverse molecular pathways, symptoms, and clinical outcomes. Gaps in our knowledge preclude successful early diagnosis, personalized treatment, and prevention. The advent of "omics" technologies and systems biology approaches addresses this problem by identifying the molecular pathways associated with the underlying mechanisms and clinical phenotypes of preeclampsia. Here, we provide a brief overview on how the field has progressed, focusing on studies utilizing state-of-the-art transcriptomics and proteomics methods. Moreover, we summarize our systems biology studies involving maternal blood proteomics and placental transcriptomics, which identified early maternal and placental disease pathways and showed that their interaction influences the clinical presentation of preeclampsia. We also present an analysis of maternal blood proteomics data which revealed distinct molecular subclasses of preeclampsia and their molecular mechanisms. Maternal and placental disease pathways behind these subclasses are similar to those recently reported in studies on the placental transcriptome. These findings may promote the development of novel diagnostic tools for the distinct subtypes of preeclampsia syndrome, enabling early detection and personalized follow-up and tailored care of patients.
Assuntos
Doenças Placentárias , Pré-Eclâmpsia , Biomarcadores , Feminino , Humanos , Placenta/metabolismo , Doenças Placentárias/patologia , Pré-Eclâmpsia/metabolismo , Gravidez , Biologia de SistemasRESUMO
Inappropriate activation of the NLRP3 inflammasome has been implicated in multiple inflammatory and autoimmune diseases. Herein, we aimed to develop novel NLRP3 inhibitors that could minimize the risk of drug-induced liver injury. Lipophilic ligand efficiency was used as a guiding metric to identify a series of 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazinesulfonylureas. A leading compound from this series was advanced into safety studies in cynomolgus monkeys, and renal toxicity, due to compound precipitation, was observed. To overcome this obstacle, we focused on improving the solubility of our compounds, specifically by introducing basic amine substituents into the scaffold. This led to the identification of GDC-2394, a potent and selective NLRP3 inhibitor, with an in vitro and in vivo safety profile suitable for advancement into human clinical trials.
Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Oxazinas , Animais , Humanos , Oxazinas/farmacologia , Oxazinas/uso terapêutico , Inflamassomos , Sulfonamidas/farmacologia , Macaca fascicularisRESUMO
Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.
Assuntos
Inibidores Enzimáticos/química , Indóis/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Tiofenos/química , Sítio Alostérico , Cristalografia por Raios X , Descoberta de Drogas , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Indóis/síntese química , Indóis/metabolismo , Estrutura Molecular , NAD/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Espermidina/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismoRESUMO
Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby, activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based design of allosteric inhibitors.
RESUMO
The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC50 of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse agonists.
Assuntos
Colesterol/metabolismo , Avaliação Pré-Clínica de Medicamentos , Hidrocarbonetos Fluorados/farmacologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Sulfonamidas/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Hidrocarbonetos Fluorados/química , Estrutura Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células Sf9 , Spodoptera , Sulfonamidas/química , Células Th17RESUMO
The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.