RESUMO
Nuclear factor kappa B (NFκB) is a pathogenic factor in chronic lymphocytic leukemia (CLL) that is not addressed specifically by current therapies. NFκB is activated by inflammatory factors that stimulate toll-like receptors (TLRs) and receptors for interleukin-1 (IL-1) family members. IL-1 is considered a master regulator of inflammation, and IL-1 receptor signaling is inhibited by the IL-1 receptor antagonist anakinra. These considerations suggested that anakinra might have a role in the treatment of CLL. Consistent with this idea, anakinra inhibited spontaneous and TLR7-mediated activation of the canonical NFκB pathway in CLL cells in vitro. However, CLL cells exhibited only weak signaling responses to IL-1 itself, and anakinra was found to inhibit NFκB along with oxidative stress in an IL-1 receptor-independent manner. Anakinra was then administered with minimal toxicity to 11 previously untreated CLL patients in a phase I dose-escalation trial (NCT04691765). A stereotyped clinical response was observed in all patients. Anakinra lowered blood lymphocytes and lymph node sizes within the first month that were associated with downregulation of NFκB and oxidative stress in the leukemia cells. However, inhibition of NFκB was accompanied by upregulation of type 1 interferon (IFN) signaling, c-MYC-regulated genes and proteins, and loss of the initial clinical response. Anakinra increased IFN signaling and survival of CLL cells in vitro that were, respectively, phenocopied by mitochondrial antioxidants and reversed by IFN receptor blocking antibodies. These observations suggest that anakinra has activity in CLL and may be a useful adjunct for conventional therapies as long as compensatory IFN signaling is blocked at the same time.
Assuntos
Proteína Antagonista do Receptor de Interleucina 1 , Leucemia Linfocítica Crônica de Células B , NF-kappa B , Transdução de Sinais , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Interferons/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/antagonistas & inibidoresRESUMO
The aberrant secretion of proinflammatory cytokines by immune cells is the principal cause of inflammatory diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Toll-like receptor 7 (TLR7) and TLR9, sequestered to the endosomal compartment of dendritic cells and macrophages, are closely associated with the initiation and progression of these diseases. Therefore, the development of drugs targeting dysregulated endosomal TLRs is imperative to mitigate systemic inflammation. Here, we applied the principles of computer-aided drug discovery to identify a novel low-molecular-weight compound, TLR inhibitory compound 10 (TIC10), and its potent derivative (TIC10g), which demonstrated dual inhibition of TLR7 and TLR9 signaling pathways. Compared to TIC10, TIC10g exhibited a more pronounced inhibition of the TLR7- and TLR9-mediated secretion of the proinflammatory cytokine tumor necrosis factor-α in a mouse macrophage cell line and mouse bone marrow dendritic cells in a concentration-dependent manner. While TIC10g slightly prevented TLR3 and TLR8 activation, it had no impact on cell surface TLRs (TLR1/2, TLR2/6, TLR4, or TLR5), indicating its selectivity for TLR7 and TLR9. Additionally, mechanistic studies suggested that TIC10g interfered with TLR9 activation by CpG DNA and suppressed downstream pathways by directly binding to TLR9. Western blot analysis revealed that TIC10g downregulated the phosphorylation of the p65 subunit of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs), including extracellular-signal-regulated kinase, p38-MAPK, and c-Jun N-terminal kinase. These findings indicate that the novel ligand, TIC10g, is a specific dual inhibitor of endosomal TLRs (TLR7 and TLR9), disrupting MAPK- and NF-κB-mediated proinflammatory gene expression.
Assuntos
Bibliotecas de Moléculas Pequenas , Receptor 7 Toll-Like , Receptor Toll-Like 9 , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/metabolismo , Animais , Camundongos , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Descoberta de Drogas , Simulação de Acoplamento Molecular , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , HumanosRESUMO
Epstein-Barr virus (EBV), a Herpesviridae family member, is associated with an increased risk of autoimmune disease development in the host. We previously demonstrated that EBV DNA elevates levels of the pro-inflammatory cytokine IL-17A and that inhibiting Toll-like receptor (TLR) 3, 7, or 9 reduces its levels. Moreover, this DNA exacerbated colitis in a mouse model of inflammatory bowel disease (IBD). In the study at hand, we examined whether inhibition of TLR3, 7, or 9 alleviates this exacerbation. Mice were fed 1.5% dextran sulfate sodium (DSS) water and administered EBV DNA. Then, they were treated with a TLR3, 7, or 9 inhibitor or left untreated. We also assessed the additive impact of combined inhibition of all three receptors. Mice that received DSS, EBV DNA, and each inhibitor alone, or a combination of inhibitors, showed significant improvement. They also had a decrease in the numbers of the pathogenic colonic IL-17A+IFN-γ+ foci. Inhibition of all three endosomal TLR receptors offered no additive benefit over administering a single inhibitor. Therefore, inhibition of endosomal TLRs reduces EBV DNA exacerbation of mouse colitis, offering a potential approach for managing IBD patients infected with EBV.
Assuntos
DNA Viral , Herpesvirus Humano 4 , Doenças Inflamatórias Intestinais , Receptores Toll-Like , Animais , Feminino , Camundongos , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/virologia , Sulfato de Dextrana , Modelos Animais de Doenças , DNA Viral/efeitos adversos , DNA Viral/farmacologia , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/virologia , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 3 Toll-Like/metabolismo , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/metabolismoRESUMO
Toll-like receptor 7 (TLR7) is a class of pattern recognition receptors (PRRs) recognizing the pathogen-associated elements and damage and as such is a major player in the innate immune system. TLR7 triggers the release of pro-inflammatory cytokines or type-I interferons (IFN), which is essential for immunoregulation. Increasing reports also highlight that the abnormal activation of endosomal TLR7 is implicated in various immune-related diseases, carcinogenesis as well as the proliferation of human immunodeficiency virus (HIV). Hence, the design and development of potent and selective TLR7 antagonists based on small molecules or oligonucleotides may offer new tools for the prevention and management of such diseases. In this review, we offer an updated overview of the main structural features and therapeutic potential of small-molecule antagonists of TLR7. Various heterocyclic scaffolds targeting TLR7 binding sites are presented: pyrazoloquinoxaline, quinazoline, purine, imidazopyridine, pyridone, benzanilide, pyrazolopyrimidine/pyridine, benzoxazole, indazole, indole, and quinoline. Additionally, their structure-activity relationships (SAR) studies associated with biological activities and protein binding modes are introduced.
Assuntos
Interferon Tipo I , Receptor 7 Toll-Like , Humanos , Citocinas/metabolismo , Relação Estrutura-Atividade , Receptor 7 Toll-Like/antagonistas & inibidoresRESUMO
Toll-like receptors (TLRs) 7 and 8 are key targets in the development of immunomodulatory drugs for treating infectious disease, cancer, and autoimmune disorders. These receptors can adopt both agonist and antagonist binding conformations that switch the receptor signal on or off to the downstream production of cytokines. In this study, we examined the effect of simple isomeric substitutions to the C2-butyl group of two imidazoquinoline agonists and evaluated the activity of these analogs using both TLR7 and TLR8 reporter cells and cytokine induction assays. Results are presented showing the C2-isobutyl and C2-cyclopropylmethyl isomers are both mixed TLR7/8 competitive antagonists of the parent agonist [4-Amino-1-(4-(aminomethyl)benzyl)-2-butyl-7-methoxycarbonyl-1H-imidazo[4,5-c]quinoline], indicating the conformation of the dimeric receptor complex is highly sensitive to steric perturbations to the ligand binding pocket. This observation is consistent with prior work demonstrating TLR7 and TLR8 activity is directly correlated to C2-alkyl substitutions that project into a hydrophobic pocket at the dimer interface of the receptor. The close structural relationship of the agonist/antagonist pairs identified here highlights the importance of this pocket in tipping the balance between the agonist and antagonist binding states of the receptor which may have significant ramifications to the design of imidazoquinoline-based immunomodulatory agents.
Assuntos
Imidazóis/farmacologia , Quinolinas/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Imidazóis/síntese química , Imidazóis/química , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and multiple organ damage. Toll-like receptor 7 (TLR7), an innate immune RNA sensor expressed in monocytes/macrophages, dendritic cells (DCs), and B cells, promotes disease progression. However, little is known about the cellular mechanisms through which TLR7 drives lupus nephritis. Here, we show that the anti-mouse TLR7 mAb, but not anti-TLR9 mAb, protected lupus-prone NZBWF1 mice from nephritis. The anti-TLR7 mAb reduced IgG deposition in glomeruli by inhibiting the production of autoantibodies to the RNA-associated antigens. We found a disease-associated increase in Ly6Clow patrolling monocytes that expressed high levels of TLR7 and had upregulated expression of lupus-associated IL-10, CD115, CD31, and TNFSF15 in NZBWF1 mice. Anti-TLR7 mAb abolished this lupus-associated increase in patrolling monocytes in the circulation, spleen, and glomeruli. These results suggested that TLR7 drives autoantibody production and lupus-associated monocytosis in NZBWF1 mice and, that anti-TLR7 mAb is a promising therapeutic tool targeting B cells and monocytes/macrophages.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Nefrite Lúpica/etiologia , Nefrite Lúpica/metabolismo , Monócitos/imunologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade , Linfócitos B/metabolismo , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Regulação da Expressão Gênica , Imunoglobulina G/imunologia , Imuno-Histoquímica , Imunofenotipagem , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/patologia , Camundongos , Monócitos/metabolismoRESUMO
This study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple oral doses of enpatoran (formerly named M5049), a new toll-like receptor (TLR) 7 and 8 dual antagonist, and the effect of food on a single dose in healthy participants. In this single phase 1, randomized (3:1), double-blind, placebo-controlled study, 96 participants received single and multiple ascending oral doses of enpatoran. Participants in single-dose cohorts received one dose of enpatoran (1, 3, 9, 25, 50, 100, or 200 mg) or placebo using a sentinel dosing strategy. Multiple-dose cohorts received enpatoran (9, 25, or 200 mg once daily, or 25 or 50 mg twice daily) or placebo for 14 days. Safety, tolerability, PK, and PD (ex vivo-stimulated cytokine secretion) were assessed in both parts. The effect of food was assessed in an open-label, one-way crossover study in the 25 mg single-dose cohort. Single- and multiple-oral doses of enpatoran up to 200 mg were well tolerated and no significant dose-limiting adverse events or safety signals were observed under fasting or fed conditions. PK parameters were linear and dose-proportional across the dose range evaluated, with a slightly delayed absorption and lower peak concentration observed at 25 mg with food. Exposure-dependent inhibition of ex vivo-stimulated interleukin-6 secretion was observed, with maximum inhibition at 200 mg. Enpatoran was well tolerated at doses up to 200 mg. Further investigation of enpatoran is warranted as a potential treatment for diseases driven by TLR7/8 overactivation, such as systemic lupus erythematosus and COVID-19 pneumonia.
Assuntos
Fatores Imunológicos/farmacologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/antagonistas & inibidores , Administração Oral , Adulto , COVID-19/imunologia , Método Duplo-Cego , Feminino , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Masculino , SARS-CoV-2 , Tratamento Farmacológico da COVID-19RESUMO
Several toll-like receptors (TLRs) reside inside endosomes of specific immune cells-among them, aberrant activation of TLR7 and TLR9 is implicated in myriad contexts of autoimmune diseases, making them promising therapeutic targets. However, small-molecule TLR7 and TLR9 antagonists are not yet available for clinical use. We illustrate here the importance of C2, C6, and N9 substitutions in the purine scaffold for antagonism to TLR7 and TLR9 through structure-activity relationship studies using cellular reporter assays and functional studies on primary human immune cells. Further in vitro and in vivo pharmacokinetic studies identified an orally bioavailable lead compound 29, with IC50 values of 0.08 and 2.66 µM against TLR9 and TLR7, respectively. Isothermal titration calorimetry excluded direct TLR ligand-antagonist interactions. In vivo antagonism efficacy against mouse TLR9 and therapeutic efficacy in a preclinical murine model of psoriasis highlighted the potential of compound 29 as a therapeutic candidate in relevant autoimmune contexts.
Assuntos
Purinas/farmacologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor Toll-Like 9/antagonistas & inibidores , Administração Oral , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Purinas/administração & dosagem , Purinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.
Assuntos
Nucleotidiltransferases/antagonistas & inibidores , Oligonucleotídeos Antissenso/química , Receptor Toll-Like 9/antagonistas & inibidores , Adulto , Animais , Sequência de Bases , Células Cultivadas , DNA , Humanos , Camundongos , Transdução de Sinais , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/antagonistas & inibidoresRESUMO
Although the cause of progressive neurodegeneration is often unclear, neuronal death can occur through several mechanisms. In conditions such as Alzheimer's or alcohol use disorder (AUD), Toll-like receptor (TLR) induction is observed with neurodegeneration. However, links between TLR activation and neurodegeneration are lacking. We report a role of apoptotic neuronal death in AUD through TLR7-mediated induction of death receptor signaling. In postmortem human cortex, a two-fold increase in apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in neurons was found in AUD versus controls. This occurred with the increased expression of TLR7 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors. Binge ethanol treatment in C57BL/6 mice increased TLR7 and induced neuronal apoptosis in cortical regions that was blocked by TLR7 antagonism. Mechanistic studies in primary organotypic brain slice culture (OBSC) found that the inhibition of TLR7 and its endogenous ligand let-7b blocked ethanol-induced neuronal cell death. Both IMQ and ethanol induced the expression of TRAIL and its death receptor. In addition, TRAIL-neutralizing monoclonal antibodies blocked both imiquimod (IMQ) and ethanol induced neuronal death. These findings implicate TRAIL as a mediator of neuronal apoptosis downstream of TLR7 activation. TLR7 and neuronal apoptosis are implicated in other neurodegenerative diseases, including Alzheimer's disease. Therefore, TRAIL may represent a therapeutic target to slow neurodegeneration in multiple diseases.
Assuntos
Alcoolismo/metabolismo , Alcoolismo/patologia , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Adulto , Animais , Apoptose , Consumo Excessivo de Bebidas Alcoólicas/genética , Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Consumo Excessivo de Bebidas Alcoólicas/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Caspase 3/metabolismo , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Modelos Neurológicos , Neurônios/metabolismo , Neurônios/patologia , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Transdução de Sinais , Técnicas de Cultura de Tecidos , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/metabolismo , Adulto JovemRESUMO
Objectives: To identify the importance of the Toll-like receptor (TLR) pathway using B cell high-throughput sequencing and to explore the participation of the TLR7 signaling pathway in primary Sjogren's syndrome (pSS)-associated thrombocytopenia in patient and mouse models. Methods: High-throughput gene sequencing and bioinformatic analyses were performed for 9 patients: 3 patients with pSS and normal platelet counts, 3 patients with pSS-associated thrombocytopenia, and 3 healthy controls. Twenty-four patients with pSS were recruited for validation. Twenty-four non-obese diabetic (NOD) mice were divided into the TLR7 pathway inhibition (CA-4948), activation (Resiquimod), and control groups. Serum, peripheral blood, bone marrow, and submandibular glands were collected for thrombocytopenia and TLR7 pathway analysis. Results: Seven hub genes enriched in the TLR pathway were identified. Compared to that in control patients, the expression of interleukin (IL)-8 and TLR7 pathway molecules in B-cells was higher in patients with pSS-associated thrombocytopenia. Platelet counts exhibited a negative correlation with serum IL-1ß and IL-8 levels. In NOD mice, CA-4948/Resiquimod treatment induced the downregulation/upregulation of the TLR7 pathway, leading to consistent elevation/reduction of platelet counts. Megakaryocyte counts in the bone marrow showed an increasing trend in the Resiquimod group, with more naked nuclei. The levels of IL-1ß and IL-8 in the serum and submandibular gland tissue increased in the Resiquimod group compared with that in CA-4948 and control groups. Conclusion: pSS-associated thrombocytopenia may be a subset of the systemic inflammatory state as the TLR7 signaling pathway was upregulated in B cells of patients with pSS-associated thrombocytopenia, and activation of the TLR7 pathway led to a thrombocytopenia phenotype in NOD mice.
Assuntos
Linfócitos B/imunologia , Interleucina-1beta/sangue , Interleucina-8/sangue , Síndrome de Sjogren/patologia , Trombocitopenia/imunologia , Receptor 7 Toll-Like/metabolismo , Animais , Plaquetas/citologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Neutrófilos/imunologia , Contagem de Plaquetas , Transdução de Sinais/fisiologia , Síndrome de Sjogren/imunologia , Trombocitopenia/patologia , Receptor 7 Toll-Like/antagonistas & inibidoresRESUMO
RO6870868 is an oral prodrug of the toll-like receptor 7 (TLR7) specific agonist, RO6871765. TLR7 agonists augment host immune activity and are in development to treat hepatitis B infection. We evaluated the safety, tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of RO6870868 in a first-in-human, phase I, randomized, single ascending oral dose study in 60 healthy volunteers at 6 dose levels (200-2000 mg). Single oral doses were generally well-tolerated with a predictable safety profile associated with dose-dependent increases in systemic interferon. No serious adverse events (AEs) were reported and no subject withdrew from the study due to an AE. No clinically significant changes were observed in vital signs, electrocardiograms, or laboratory parameters. Following oral RO6870868 doses, plasma RO6871765 concentrations increased rapidly, exhibiting mean terminal half-life ranging 2-6 h across all cohorts, with area under the plasma concentration versus time curve extrapolated to infinity (AUC0-∞ ) increasing proportionally with dose. A pattern of dose and time-dependent PD activity was demonstrated consistent with engagement of the TLR7 system. Single RO6870868 doses activated components of the TLR innate immune system in a dose-dependent manner with adequate safety and tolerability. Single-dose data in healthy volunteers are useful to evaluate safety, PK, and PD activity of TLR7 agonists and help to guide dose and regimen selection for further trials in patients with chronic hepatitis B.
Assuntos
Fatores Imunológicos/efeitos adversos , Receptor 7 Toll-Like/antagonistas & inibidores , Administração Oral , Adolescente , Adulto , Idoso , Área Sob a Curva , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Meia-Vida , Voluntários Saudáveis , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/farmacocinética , Interferons/sangue , Interferons/metabolismo , Masculino , Pessoa de Meia-Idade , Pró-Fármacos/administração & dosagem , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Adulto JovemRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Carvacrol, a monoterpene phenol from Mosla chinensis Maxim, which is a commonly Chinese herbal medicine. The most important pharmacology of it is dispelling exogenous evils by increasing perspiration. And it is the gentleman medicine in the Chinese herbal compound prescription of Xin-Jia-Xiang-Ru-Yin, mainly for the treatment of summer colds with dampness including influenza virus A infection. AIM OF THE STUDY: Our preliminary study verified that the Xin-Jia-Xiang-Ru-Yin could inhibit acute lung injury of mice with influenza virus A infection. And there have been some reports implicating the high antimicrobial activity of carvacrol for a wide range of product preservation, but little research including the effects of it on viral infection. The aim of this study was to reveal the antiviral effects of carvacrol, the main constituent in Mosla chinensis Maxim. MATERIALS AND METHODS: Initially, C57BL/6 mice were grouped and intranasally administered FM1 virus to construct viral infection models. After treatment with ribavirin and carvacrol for 5 days, all mice were euthanized, and specimens were immediately obtained. Histology, flow cytometry and Meso Scale Discovery (MSD) analysis were used to analyze pathological changes in lung tissue, the expression levels of cytokines and the differentiation and proportion of CD4+ T cells subsets, while Western blot and qRT-PCR were used to detect the expression of related proteins and mRNA. RESULTS: Carvacrol attenuated lung tissue damage, the proportions of Th1, Th2, Th17 and Treg in CD4+ T cells and the relative proportions of Th1/Th2 and Th17/Treg cells. Carvacrol inhibited the expression of inflammation-associated cytokines including IFN-γ, IL-2, IL-4, IL-5, IL-12 and TNF-É, IL-1, IL-10, IL-6. Decreased levels of TLR7, MyD88, IRAK4, TRAK6, NF-κB, RIG-I, IPS-I and IRF mRNA in carvacrol-treated mice were observed comparing to the mice in VC group. Further, the total expression of RIG-I, MyD88 and NF-κB proteins had increased significantly in the VC group but reduced obviously in the group treated with ribavirin or carvacrol. CONCLUSIONS: These results indicate that carvacrol is a potential alternative treatment for the excessive immune response induced by influenza virus A infection, the cold-fighting effect of Mosla chinensis Maxim may depend on the anti-virus of carvacrol.
Assuntos
Alphainfluenzavirus/efeitos dos fármacos , Cimenos/farmacologia , Proteína DEAD-box 58/antagonistas & inibidores , Imunidade Inata/efeitos dos fármacos , Glicoproteínas de Membrana/antagonistas & inibidores , Receptor 7 Toll-Like/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/metabolismo , Animais , Cimenos/uso terapêutico , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Feminino , Imunidade Inata/imunologia , Alphainfluenzavirus/imunologia , Alphainfluenzavirus/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo , Replicação Viral/imunologiaAssuntos
Fármacos Dermatológicos/uso terapêutico , Dermatomiosite/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/antagonistas & inibidores , Receptor Toll-Like 9/antagonistas & inibidores , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Toll-like receptor (TLR) 7 and TLR8 are transmembrane receptors that recognize single-stranded RNA. Activation of these receptors results in immune cell stimulation and inflammatory cytokine production, which is normally a protective host response. However, aberrant activation of TLR7/8 is potentially pathogenic and linked to progression of certain autoimmune diseases such as lupus. Thus, we hypothesize that an inhibitor that blocks TLR7/8 would be an effective therapeutic treatment. Prior efforts to develop inhibitors of TLR7/8 have been largely unsuccessful as a result of the challenge of producing a small-molecule inhibitor for these difficult targets. Here, we report the characterization of M5049 and compound 2, molecules which were discovered in a medicinal chemistry campaign to produce dual TLR7/8 inhibitors with drug-like properties. Both compounds showed potent and selective activity in a range of cellular assays for inhibition of TLR7/8 and block synthetic ligands and natural endogenous RNA ligands such as microRNA and Alu RNA. M5049 was found to be potent in vivo as TLR7/8 inhibition efficaciously treated disease in several murine lupus models and, interestingly, was efficacious in a disease context in which TLR7/8 activity has not previously been considered a primary disease driver. Furthermore, M5049 had greater potency in disease models than expected based on its in vitro potency and pharmacokinetic/pharmacodynamic properties. Because of its preferential accumulation in tissues, and ability to block multiple TLR7/8 RNA ligands, M5049 may be efficacious in treating autoimmunity and has the potential to provide benefit to a variety of patients with varying disease pathogenesis. SIGNIFICANCE STATEMENT: This study reports discovery of a novel toll-like receptor (TLR) 7 and TLR8 inhibitor (M5049); characterizes its binding mode, potency/selectivity, and pharmacokinetic and pharmacodynamic properties; and demonstrates its potential for treating autoimmune diseases in two mouse lupus models. TLR7/8 inhibition is unique in that it may block both innate and adaptive autoimmunity; thus, this study suggests that M5049 has the potential to benefit patients with autoimmune diseases.
Assuntos
Autoimunidade/efeitos dos fármacos , Descoberta de Drogas , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/antagonistas & inibidores , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Proteica , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/química , Receptor 8 Toll-Like/metabolismoRESUMO
GSK2245035, a small molecule Toll-like Receptor 7 (TLR7) agonist developed for immunomodulatory treatment for allergic airways disease, aimed to reduce Th2 and enhance Th1/Treg responses to aeroallergens via the local induction of type I interferons (IFNs). GSK2245035 demonstrated selectivity for potent release of type I IFNs compared to TNF-α and IL-6, with dose dependent increases in the interferon inducible chemokine, IP-10, in the nasal compartment. Implantation and parturition require pro-inflammatory processes including IFNs, Interferon Stimulated Genes, TNFα and IP-10 while pregnancy requires immune regulation to maintain maternal fetal immune tolerance, and recombinant type I IFNs induced abortions in monkeys. Due to its mechanism of action, GSK2245035 was studied at pharmacologically and clinically relevant doses in a monkey pregnancy model. Monkeys received 0, 3 or 30 ng/kg/week GSK2245035 intranasally once weekly, from Day 20 postcoitum through Day 63 postpartum. Although systemic IFN-α and IP-10 levels were approximately 14.8 or 40 -fold (respectively) above predose levels at 3 or 30 ng/kg/week, respectively, there were no effects on pregnancy and infant outcome. Non-adverse effects included increased incidence of nasal discharge, increased maternal body temperature at 30 ng/kg/week and dose-dependent increases in maternal IP-10 and IFN-α and decreased infant anti-KLH IgM and IgG titers following KLH immunization at ≥3 ng/kg/week, relative to controls. Potentially, lower IFN-α and IP-10 levels as well as once-weekly intranasal dosing vs daily subcutaneous or intramuscular dosing with recombinant type I IFNs could explain the lack of pregnancy effects; however, there was an undesired impact on offspring immune function.
Assuntos
Aborto Espontâneo/induzido quimicamente , Adenina/análogos & derivados , Asma/tratamento farmacológico , Piperidinas/efeitos adversos , Complicações na Gravidez/tratamento farmacológico , Receptor 7 Toll-Like/antagonistas & inibidores , Aborto Espontâneo/sangue , Aborto Espontâneo/imunologia , Adenina/efeitos adversos , Administração Intranasal , Animais , Asma/sangue , Asma/imunologia , Quimiocina CXCL10/sangue , Modelos Animais de Doenças , Feminino , Humanos , Interferon-alfa/sangue , Macaca fascicularis , Gravidez , Complicações na Gravidez/sangue , Complicações na Gravidez/imunologiaRESUMO
Aberrant activation of the endosomal Toll-like receptor 7 (TLR7) has been implicated in myriad autoimmune diseases and is an established therapeutic target in such conditions. Development of diverse TLR7 antagonists is mainly accomplished through random screening. To correlate human TLR7 (hTLR7) antagonistic activity with the structural features in different chemotypes, we derived a hypothetical binding model based on molecular docking analysis along with molecular dynamics (MD) simulations study. The binding hypothesis revealed different pockets, grooves and a central cavity where ligand-receptor interaction with specific residues through hydrophobic and hydrogen bond interactions take place, which correlate with TLR7 antagonistic activity thus paving the way for rational design using varied chemotypes. Based on the structural insight thus gained, TLR7 antagonists with quinazoline were designed to understand the effect of engagement of these pockets as well as boundaries of the chemical space associated with them. The newly synthesized most potent hTLR7 antagonist, i.e. compound 63, showed IC50 value of 1.03 ± 0.05 µM and was validated by performing primary assay in human plasmacytoid dendritic cells (pDC) (IC50pDC: 1.42 µM). The biological validation of the synthesized molecules was performed in TLR7-reporter HEK293 cells as well as in human plasmacytoid dendritic cells (pDCs). Our study provides a rational design approach thus facilitating further development of novel small molecule hTLR7 antagonists based on different chemical scaffolds.
Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Quinazolinas/farmacologia , Receptor 7 Toll-Like/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-Atividade , Receptor 7 Toll-Like/metabolismoRESUMO
Toll-like receptor 7 (TLR7) recognizes both microbial and endogenous RNAs and nucleosides. Aberrant activation of TLR7 has been implicated in several autoimmune diseases including systemic lupus erythematosus (SLE). Here, by modifying potent TLR7 agonists, we develop a series of TLR7-specific antagonists as promising therapeutic agents for SLE. These compounds protect mice against lethal autoimmunity. Combining crystallography and cryo-electron microscopy, we identify the open conformation of the receptor and reveal the structural equilibrium between open and closed conformations that underlies TLR7 antagonism, as well as the detailed mechanism by which TLR7-specific antagonists bind to their binding pocket in TLR7. Our work provides small-molecule TLR7-specific antagonists and suggests the TLR7-targeting strategy for treating autoimmune diseases.
Assuntos
Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/química , Animais , Doenças Autoimunes , Autoimunidade , Sítios de Ligação , Microscopia Crioeletrônica , Feminino , Ligantes , Lúpus Eritematoso Sistêmico , Camundongos , Camundongos Endogâmicos NZB , Modelos Moleculares , Conformação Proteica , Taxa de SobrevidaRESUMO
Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure-guided optimization of the pyridone hit 3 using this biochemical assay in combination with cellular and TLR8 cocrystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for optimization were (i) a core morph guided by a TLR7 sequence alignment to achieve a dual TLR7/8 antagonism profile and (ii) introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.
Assuntos
Lúpus Eritematoso Sistêmico/tratamento farmacológico , Piridonas/química , Piridonas/farmacologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/antagonistas & inibidores , Animais , Células Cultivadas , Descoberta de Drogas , Humanos , Indazóis/química , Indazóis/farmacocinética , Indazóis/farmacologia , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Piridonas/farmacocinética , Ratos Sprague-Dawley , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/química , Receptor 8 Toll-Like/metabolismoRESUMO
Antagonism of the Toll-like receptors (TLRs) 7 and TLR8 has been hypothesized to be beneficial to patients suffering from autoimmune conditions. A phenotypic screen for small molecule antagonists of TLR7/8 was carried out in a murine P4H1 cell line. Compound 1 was identified as a hit that showed antagonistic activity on TLR7 and TLR8 but not TLR9, as shown on human peripheral blood mononuclear cells (hPBMCs). It was functionally cross reactive with mouse TLR7 but lacked oral exposure and had only modest potency. Chemical optimization resulted in 2, which showed in vivo efficacy following intraperitoneal administration. Further optimization resulted in 8 which had excellent in vitro activity, exposure and in vivo activity. Additional work to improve physical properties resulted in 15, an advanced lead that had favorable in vitro and exposure properties. It was further demonstrated that activity of the series tracked with binding to the extracellular domain of TLR7 implicating that the target of this series are endosomal TLRs rather than downstream signaling pathways.