RESUMO
The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.
Assuntos
Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Animais , Apoproteínas/antagonistas & inibidores , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2Y1/química , Transdução de Sinais , Anticorpos de Domínio Único/química , Especificidade da Espécie , Ácido Succínico/metabolismoRESUMO
Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.
Assuntos
Fator B do Complemento/antagonistas & inibidores , Via Alternativa do Complemento/efeitos dos fármacos , Descoberta de Drogas/métodos , Fatores Imunológicos/farmacologia , Animais , Modelos Animais de Doenças , Glomerulonefrite Membranosa/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos Sprague-DawleyRESUMO
Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory interleukin (IL)-1ß; it has recently become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1ß pathway in granuloma formation and sarcoidosis.19 sarcoid patients and 19 healthy volunteers were recruited into this pilot study. NLRP3 inflammasome activity was measured in bronchoalveolar lavage (BAL) cells and lung and skin biopsies using immunohistochemistry, Western blot, reverse-transcriptase PCR and ELISA. For in vivo experiments we used the trehalose 6,6'-dimycolate-granuloma mouse model and evaluated lung granuloma burden in miR-223 knockout and NLRP3 knockout mice, as well as the treatment effects of MCC950 and anti-IL-1ß antibody therapy.We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1ß in lung granuloma, and increased IL-1ß release of BAL cells from sarcoid patients compared to healthy volunteers (p=0.006). mRNA levels of miR-223, a micro-RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (p<0.005). NLRP3 knockout mice showed decreased and miR-223 knockout mice increased granuloma formation compared to wild-type mice. Pharmacological interference using NLRP3 pathway inhibitor MCC950 or an anti-IL-1ß antibody resulted in reduced granuloma formation (p<0.02).In conclusion, our data provide evidence of upregulated inflammasome and IL-1ß pathway activation in sarcoidosis and suggest both as valid therapeutic targets.
Assuntos
Granuloma , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Sarcoidose , Animais , Caspase 1 , Humanos , Interleucina-1beta , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Projetos PilotoRESUMO
GPR4, a G-protein coupled receptor, functions as a proton sensor being activated by extracellular acidic pH and has been implicated in playing a key role in acidosis associated with a variety of inflammatory conditions. An orally active GPR4 antagonist 39c was developed, starting from a high throughput screening hit 1. The compound shows potent cellular activity and is efficacious in animal models of angiogenesis, inflammation and pain.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Desenho de Fármacos , Inflamação/tratamento farmacológico , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/química , Artrite/tratamento farmacológico , Artrite/metabolismo , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Células HeLa , Humanos , Inflamação/metabolismo , Camundongos , Estrutura Molecular , Dor/tratamento farmacológico , Dor/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-AtividadeRESUMO
Cellular behaviors are governed by combinations of systemic and microenvironmental factors; together, these regulate cell signaling responses to growth factors. This contextual microenvironmental influence also determines drug sensitivity. Hence using in vitro systems that model contextual cellular behavior is highly beneficial for effective therapeutic development. Angiogenesis (formation of blood vessels) is driven by a series of dynamic endothelial cell signaling responses to growth factors under the influence of the vascular extracellular matrix and adjacent pericytes. In vitro primary human vascular cell co-cultures self-assemble into capillary-like structures through reciprocal heterotypic interactions that mimic angiogenic context dynamics. By using temporal live-cell imaging-based analysis, unique angiogenic microenvironments can be delineated to quantify the contextual activity of compound inhibitors. We used this in vitro organotypic contextual screening approach to conduct structure-activity relationship analysis on a combretastatin A-4 analogue series to identify novel compounds with potent vascular disrupting activity in vivo.
Assuntos
Inibidores da Angiogênese/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores da Angiogênese/química , Animais , Linhagem Celular , Técnicas de Cocultura/métodos , Células Endoteliais da Veia Umbilical Humana , Humanos , Artéria Pulmonar/citologia , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
The mechanism whereby IL-17 drives rheumatoid arthritis remains incompletely understood. We demonstrate that anti-IL-17 therapy in collagen-induced arthritis ameliorates bone damage by reducing the number of osteoclasts in joints. We found equal numbers of CD4(+) Th17 and IL-17 producing γδ T cells in the joints of arthritic mice, and in vitro, both populations similarly induced osteoclastogenesis. However, individual depletion and adoptive transfer studies revealed that in vivo, Th17 cells dominated with regard to bone destruction. Unlike γδ T cells, Th17 cells were found in apposition to tartrate-resistant acid phosphatase positive osteoclasts in subchondral areas of inflamed joints, a pattern reproduced in patient biopsies. This localization was caused by Ag-specific retention, because OVA-primed Th17 cells showed a γδ T cell-like diffuse distribution. Because IL-23, as produced by osteoclasts, enhanced T cell-mediated osteoclastogenesis, we propose that Ag-specific juxtaposition is key to foster the molecular cross talk of Th17 cells and osteoclasts, thus driving arthritic bone destruction.
Assuntos
Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Interleucina-17/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Células Th17/imunologia , Células Th17/patologia , Adulto , Idoso , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Cartilagem Articular/imunologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Comunicação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Técnicas de Cocultura , Colágeno Tipo II/administração & dosagem , Colágeno Tipo II/imunologia , Feminino , Humanos , Interleucina-17/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Osteoclastos/imunologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Células Th17/metabolismoRESUMO
Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1ß, or TLR agonists. BMX was required for phosphorylation of p38 MAPK and JNK, as well as activation of NF-κB. The following epistasis analysis indicated that BMX acts downstream of or at the same level as the complex TGF-ß activated kinase 1 (TAK1)-TAK1 binding protein. At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology domain of BMX, which could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter by in vitro BMX overexpression required its catalytic activity. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component of inflammatory cytokine signaling and that catalytic, as well as noncatalytic functions of BMX are involved.
Assuntos
Artrite/imunologia , Proteínas Tirosina Quinases/metabolismo , Animais , Artrite/metabolismo , Proteínas Sanguíneas , Linhagem Celular , Modelos Animais de Doenças , Células HeLa , Humanos , Immunoblotting , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , NF-kappa B/metabolismo , Fosfoproteínas , Fosforilação , Proteínas Tirosina Quinases/genética , Transdução de Sinais , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fatores de Necrose Tumoral/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In October 2019, Novartis launched brolucizumab, a single-chain variable fragment molecule targeting vascular endothelial growth factor A, for the treatment of neovascular age-related macular degeneration. In 2020, rare cases of retinal vasculitis and/or retinal vascular occlusion (RV/RO) were reported, often during the first few months after treatment initiation, consistent with a possible immunologic pathobiology. This finding was inconsistent with preclinical studies in cynomolgus monkeys that demonstrated no drug-related intraocular inflammation, or RV/RO, despite the presence of preexisting and treatment-emergent antidrug antibodies (ADAs) in some animals. In this study, the immune response against brolucizumab in humans was assessed using samples from clinical trials and clinical practice. In the brolucizumab-naïve population, anti-brolucizumab ADA responses were detected before any treatment, which was supported by the finding that healthy donors can harbor brolucizumab-specific B cells. This suggested prior exposure of the immune system to proteins with structural similarity. Experiments on samples showed that naïve and brolucizumab-treated ADA-positive patients developed a class-switched, high-affinity immune response, with several linear epitopes being recognized by ADAs. Only patients with RV/RO showed a meaningful T cell response upon recall with brolucizumab. Further studies in cynomolgus monkeys preimmunized against brolucizumab with adjuvant followed by intravitreal brolucizumab challenge demonstrated that high ADA titers were required to generate ocular inflammation and vasculitis/vascular thrombosis, comparable to RV/RO in humans. Immunogenicity therefore seems to be a prerequisite to develop RV/RO. However, because only 2.1% of patients with ADA develop RV/RO, additional factors must play a role in the development of RV/RO.
Assuntos
Vasculite Retiniana , Animais , Humanos , Adjuvantes Imunológicos , Inibidores da Angiogênese , Inflamação , Injeções Intravítreas , Macaca fascicularis , Fator A de Crescimento do Endotélio VascularRESUMO
Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.
Assuntos
Complexo Antígeno-Anticorpo , Análise de Causa Fundamental , Humanos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Inflamação , Inibidores da Angiogênese , Injeções IntravítreasRESUMO
IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1ß or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1ß-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.
Assuntos
Artrite Reumatoide , Quinases Associadas a Receptores de Interleucina-1 , Neutrófilos , Membrana Sinovial , Animais , Artrite Reumatoide/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-8/metabolismo , Camundongos , Neutrófilos/metabolismo , Membrana Sinovial/metabolismoRESUMO
Tumor necrosis factor (TNF) is a key driver of several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, in which affected tissues show an interferon-stimulated gene signature. Here, we demonstrate that TNF triggers a type-I interferon response that is dependent on the cyclic guanosine monophosphate-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. We show that TNF inhibits PINK1-mediated mitophagy and leads to altered mitochondrial function and to an increase in cytosolic mtDNA levels. Using cGAS-chromatin immunoprecipitation (ChIP), we demonstrate that cytosolic mtDNA binds to cGAS after TNF treatment. Furthermore, TNF induces a cGAS-STING-dependent transcriptional response that mimics that of macrophages from rheumatoid arthritis patients. Finally, in an inflammatory arthritis mouse model, cGAS deficiency blocked interferon responses and reduced inflammatory cell infiltration and joint swelling. These findings elucidate a molecular mechanism linking TNF to type-I interferon signaling and suggest a potential benefit for therapeutic targeting of cGAS/STING in TNF-driven diseases.
Assuntos
Artrite Experimental/imunologia , DNA Mitocondrial/metabolismo , Imunidade Inata , Inflamação/imunologia , Interferon Tipo I/farmacologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/genética , Artrite Experimental/metabolismo , DNA Mitocondrial/efeitos dos fármacos , Feminino , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Macrófagos/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MitofagiaRESUMO
The cytosolic metalloenzyme leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB4 generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.
Assuntos
Aminobutiratos/uso terapêutico , Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/uso terapêutico , Epóxido Hidrolases/antagonistas & inibidores , Piridinas/uso terapêutico , Aminobutiratos/síntese química , Aminobutiratos/farmacocinética , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacocinética , Artrite Experimental/tratamento farmacológico , Cães , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Feminino , Humanos , Inflamação/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Piridinas/síntese química , Piridinas/farmacocinética , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in multiple sclerosis (MS) is unclear. Central nervous system lesions from both MS and experimental autoimmune encephalomyelitis (EAE), the animal model of MS, contain T cells, macrophages and activated glia, which can produce pro-angiogenic factors. Previous EAE studies have demonstrated an increase in blood vessels, but differences between the different phases of disease have not been reported. Therefore we examined angiogenic promoting factors in MS and EAE lesions to determine if there were changes in blood vessel density at different stages of EAE. METHODS: In this series of experiments we used a combination of vascular casting, VEGF ELISA and immunohistochemistry to examine angiogenesis in experimental autoimmune encephalomyelitis (EAE). Using immunohistochemistry we also examined chronic active MS lesions for angiogenic factors. RESULTS: Vascular casting and histological examination of the spinal cord and brain of rats with EAE demonstrated that the density of patent blood vessels increased in the lumbar spinal cord during the relapse phase of the disease (p < 0.05). We found an increased expression of VEGF by inflammatory cells and a decrease in the recently described angiogenesis inhibitor meteorin. Examination of chronic active human MS tissues demonstrated glial expression of VEGF and glial and blood vessel expression of the pro-angiogenic receptor VEGFR2. There was a decreased expression of VEGFR1 in the lesions compared to normal white matter. CONCLUSIONS: These findings reveal that angiogenesis is intimately involved in the progression of EAE and may have a role in MS.
Assuntos
Indutores da Angiogênese/metabolismo , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/patologia , Neovascularização Patológica , Adulto , Idoso , Animais , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Cobaias , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Ratos , Ratos Endogâmicos Lew , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D-E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC(50)<3 nM and inhibit the release of TNFalpha (IC(50)<0.3 microM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells. The spirocyclopropane analogues are less potent against MK2 (IC(50)=0.05-0.23 microM), less potent in cells (IC(50)<1.1 microM), but show good oral absorption. Compound 13E (100mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.
Assuntos
Ácido Azetidinocarboxílico/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Administração Oral , Animais , Ácido Azetidinocarboxílico/síntese química , Ácido Azetidinocarboxílico/farmacologia , Azetidinas/química , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Ciclopropanos/química , Ciclopropanos/farmacologia , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Compostos de Espiro/química , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE: Comparison of the antiangiogenic/vascular properties of the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and the vascular endothelial growth factor receptor (VEGFR) inhibitor vatalanib (PTK/ZK). EXPERIMENTAL DESIGN: Antiproliferative activity against various tumor histotypes and downstream effects on the mTOR pathway were measured in vitro. In vivo, antitumor activity, plasma, and tumor RAD001 levels were measured. Activity in several different angiogenic/vascular assays in vitro and in vivo was assessed and compared with PTK/ZK. RESULTS: RAD001 inhibited proliferation in vitro (IC50 values<1 nmol/L to >1 micromol/L), and in sensitive and insensitive tumor cells, pS6 kinase and 4E-BP1 were inhibited. Activity in vitro did not correlate with activity in vivo and significant responses were seen in tumors with IC50 values>10-fold higher than tumor RAD001 concentrations. In vitro, RAD001 inhibited the proliferation of VEGF-stimulated and fibroblast growth factor-stimulated human endothelial cells but not dermal fibroblasts and impaired VEGF release from both sensitive and insensitive tumor cells but did not inhibit migration of human endothelial cells. In vivo, in tumor models derived from either sensitive or insensitive cells, RAD001 reduced Tie-2 levels, the amount of mature and immature vessels, total plasma, and tumor VEGF. RAD001 did not affect blood vessel leakiness in normal vasculature acutely exposed to VEGF nor did it affect tumor vascular permeability (Ktrans) as measured by dynamic contrast-enhanced magnetic resonance imaging. However, the pan-VEGFR inhibitor PTK/ZK inhibited endothelial cell migration and vascular permeability but had less effect on mature vessels compared with RAD001. CONCLUSIONS: VEGFR and mTOR inhibitors show similar but also distinct effects on tumor vascular biology, which has implications for their clinical activity alone or in combination.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Ftalazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Piridinas/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Sirolimo/análogos & derivados , Inibidores da Angiogênese/farmacocinética , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Everolimo , Feminino , Humanos , Técnicas Imunoenzimáticas , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Ftalazinas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Quinases/metabolismo , Piridinas/farmacocinética , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos WF , Receptor TIE-2/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Sirolimo/farmacocinética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Distribuição Tecidual , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Halting tumor growth by interfering with tumor-induced angiogenesis is an attractive therapeutic approach. Such treatments include humanized antibodies blocking the activity of vascular endothelial growth factor (VEGF)-A (bevacizumab), soluble VEGF receptor (VEGFR) constructs (VEGF-Trap), or small-molecule inhibitors of VEGFR signaling, including PTK787/ZK222584 (PTK/ZK), sorafenib, and sunitinib. PTK/ZK has been shown previously to specifically block VEGF-induced phosphorylation of VEGFR-1, -2 and -3 and thereby to inhibit endothelial cell proliferation, differentiation, and tumor angiogenesis. We have investigated the effect of PTK/ZK on tumor angiogenesis and tumor lymphangiogenesis using the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis. In Rip1Tag2 mice, tumor angiogenesis is predominantly mediated by VEGF-A, and as expected, PTK/ZK efficiently impaired tumor blood vessel angiogenesis and tumor growth. Double-transgenic Rip1Tag2;Rip1VEGF-C and Rip1Tag2;Rip1VEGF-D mice not only exhibit VEGF-A-dependent blood vessel angiogenesis but also tumor lymphangiogenesis induced by the transgenic expression of VEGF-C or -D. In these mouse models, PTK/ZK also repressed tumor blood vessel angiogenesis and tumor growth yet failed to affect tumor lymphangiogenesis and lymphogenic metastasis. Adenoviral delivery of soluble VEGFR-3 also did not prevent tumor lymphangiogenesis in these mice. In contrast, spontaneous tumor lymphangiogenesis, as observed by the stochastic expression of VEGF-C and -D in tumors of neural cell adhesion molecule-deficient Rip1Tag2 mice, was repressed by PTK/ZK and soluble VEGFR-3. The results indicate that the time of onset and the levels of VEGF-C/D expression may be critical variables in efficiently repressing tumor lymphangiogenesis and that pathways other than VEGFR signaling may be involved in tumor lymphangiogenesis.
Assuntos
Linfangiogênese/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Ftalazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Anti-inflammatory cytokines are a promising class of therapeutics for treatment of rheumatoid arthritis (RA), but their use is currently limited by a rapid clearance and systemic toxicity. Interleukin-4 is a small cytokine with potential for RA therapy. To increase its pharmacokinetic features, we engineered a murine IL4 conjugate by incorporating an unnatural amino acid through genetic code expansion to which PEG-folate, as a targeting moiety and PEG alone as control, were site-specifically bound. Both IL4 conjugates retained bioactivity and induced primary murine macrophage polarization into an alternatively activated (M2) related phenotype. The PEGylated conjugates had a terminal half-life of about four hours in healthy mice compared to unPEGylated IL4 (0.76 h). We showed that both conjugates successfully accumulated into arthritic joints in an antigen-induced arthritis (AIA) mouse model, as assessed by non-invasive fluorescence imaging. The modular nature of the IL4 conjugate chemistry presented herein facilitates easy adaption of PEG chain length and targeting moieties for further improvement of half-life and targeting function for future efficacy studies.
Assuntos
Artrite Experimental , Artrite Reumatoide , Interleucina-4/uso terapêutico , Aminoácidos , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Interleucina-4/administração & dosagem , Camundongos , PolietilenoglicóisRESUMO
G-protein-coupled receptor SUCNR1 (succinate receptor 1 or GPR91) senses the citric cycle intermediate succinate and is implicated in various pathological conditions such as rheumatoid arthritis, liver fibrosis, or obesity. Here, we describe a novel SUCNR1 antagonist scaffold discovered by high-throughput screening. The poor permeation and absorption properties of the most potent compounds, which were zwitterionic in nature, could be improved by the formation of an internal salt bridge, which helped in shielding the two opposite charges and thus also the high polarity of zwitterions with separated charges. The designed compounds containing such a salt bridge reached high oral bioavailability and oral exposure. We believe that this principle could find a broad interest in the medicinal chemistry field as it can be useful not only for the modulation of properties in zwitterionic compounds but also in acidic or basic compounds with poor permeation.
Assuntos
Benzamidas/farmacologia , Fenilacetatos/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Benzamidas/síntese química , Benzamidas/metabolismo , Benzamidas/farmacocinética , Linhagem Celular , Descoberta de Drogas , Humanos , Masculino , Camundongos Endogâmicos C57BL , Fenilacetatos/síntese química , Fenilacetatos/metabolismo , Fenilacetatos/farmacocinética , Ligação Proteica , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Eletricidade EstáticaRESUMO
Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase da Agamaglobulinemia/química , Animais , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cristalografia por Raios X/métodos , Cães , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Ratos Endogâmicos Lew , OvinosRESUMO
Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.