RESUMO
TNFα is a homotrimeric pro-inflammatory cytokine, whose direct targeting by protein biotherapies has been an undeniable success for the treatment of chronic inflammatory diseases. Despite many efforts, no orally active drug targeting TNFα has been identified so far. In the present work, we identified through combined in silico/in vitro/in vivo approaches a TNFα direct inhibitor, compound 1, displaying nanomolar and micromolar range bindings to TNFα. Compound 1 inhibits the binding of TNFα with both its receptors TNFRI and TNFRII. Compound 1 inhibits the TNFα induced apoptosis on L929 cells and the TNFα induced NF-κB activation in HEK cells. In vivo, oral administration of compound 1 displays a significant protection in a murine TNFα-dependent hepatic shock model. This work illustrates the ability of low-cost combined in silico/in vitro/in vivo screening approaches to identify orally available small-molecules targeting challenging protein-protein interactions such as homotrimeric TNFα.
Assuntos
Anti-Inflamatórios/farmacologia , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Bibliotecas de Moléculas Pequenas/química , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Interleukin-6 (IL-6) overproduction has been involved in the pathogenesis of several chronic inflammatory diseases and the administration of an anti-IL-6 receptor monoclonal antibody has been proven clinically efficient to treat them. However, the drawbacks of monoclonal antibodies have led our group to develop an innovative anti-IL-6 strategy using a peptide-based active immunization. This approach has previously shown its efficacy in a mouse model of systemic sclerosis. Here the safety, immunogenicity, and efficacy of this strategy was assessed in non human primates. No unscheduled death and clinical signs of toxicity was observed during the study. Furthermore, the cynomolgus monkeys immunized against the IL-6 peptide produced high levels of anti-IL-6 antibodies as well as neutralizing antibodies compared to control groups. They also showed an important decrease of the cumulative inflammatory score following a delayed-type hypersensitivity reaction induced by the Tetanus vaccine compared to control groups (minus 57,9%, P = 0.014). These findings are highly significant because the immunizing IL-6 peptide used in this study is identical in humans and in monkeys and this novel anti-IL-6 strategy could thus represent a promising alternative to monoclonal antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Hipersensibilidade/imunologia , Interleucina-6/imunologia , Macaca fascicularis/imunologia , Peptídeos/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Humanos , Imunização/métodos , Receptores de Interleucina-6/imunologiaRESUMO
INTRODUCTION: Interleukin-6 (IL-6) is a pleiotropic cytokine for which preliminary data have suggested that it might contribute to systemic sclerosis (SSc). Our aims were to investigate, firstly, IL-6 expression in patients with SSc and, secondly, the efficacy of both passive and active immunization against IL-6 to reduce skin fibrosis in complementary mouse models of SSc. METHODS: Human serum levels and skin expression of IL-6 were determined by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. We first evaluated the antifibrotic properties of the monoclonal anti-IL-6R antibody, MR16-1, in the bleomycin-induced dermal fibrosis mouse model, reflecting early and inflammatory stages of SSc. Then, we assessed the efficacy of MR16-1 in tight skin-1 (Tsk-1) mice, an inflammation-independent model of skin fibrosis. Additionally, we have developed an innovative strategy using an anti-IL-6 peptide-based active immunization. Infiltrating leukocytes, T cells, and B cells were quantified, and IL-6 levels were measured in the serum and lesional skin of mice after passive or active immunization. RESULTS: Serum and skin levels of IL-6 were significantly increased in patients with early SSc. Treatment with MR16-1 led in the bleomycin mouse model to a 25% (P = 0.02) and 30% (P = 0.007) reduction of dermal thickness and hydroxyproline content, respectively. MR16-1 demonstrated no efficacy in Tsk-1 mice. Thereafter, mice were immunized against a small peptide derived from murine IL-6 and this strategy led in the bleomycin model to a 20% (P = 0.02) and 25% (P = 0.005) decrease of dermal thickness and hydroxyproline content, respectively. Passive and active immunization led to decreased T-cell infiltration in the lesional skin of mice challenged with bleomycin. Upon bleomycin injections, serum and skin IL-6 levels were increased after treatment with MR16-1 and were significantly reduced after anti-IL-6 active immunization. CONCLUSIONS: Our results support the relevance of targeting IL-6 in patients with early SSc since IL-6 is overexpressed in early stages of the disease. Targeting IL-6 by both passive and active immunization strategies prevented the development of bleomycin-induced dermal fibrosis in mice. Our results highlight the therapeutic potential of active immunization against IL-6, which is a seductive alternative to passive immunization.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunização/métodos , Interleucina-6/antagonistas & inibidores , Escleroderma Sistêmico/imunologia , Dermatopatias/prevenção & controle , Adulto , Idoso , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrose/induzido quimicamente , Fibrose/imunologia , Fibrose/prevenção & controle , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Dermatopatias/induzido quimicamente , Dermatopatias/imunologiaRESUMO
BACKGROUND: Genetic association studies aim at finding correlations between a disease state and genetic variations such as SNPs or combinations of SNPs, termed haplotypes. Some haplotypes have a particular biological meaning such as the ones derived from SNPs located in the promoters, or the ones derived from non synonymous SNPs. All these haplotypes are "subhaplotypes" because they refer only to a part of the SNPs found in the gene. Until now, subhaplotypes were directly computed from the very SNPs chosen to constitute them, without taking into account the rest of the information corresponding to the other SNPs located in the gene. In the present work, we describe an alternative approach, called the "global method", which takes into account all the SNPs known in the region and compare the efficacy of the two "direct" and "global" methods. RESULTS: We used empirical haplotypes data sets from the GH1 promoter and the APOE gene, and 10 simulated datasets, and randomly introduced in them missing information (from 0% up to 20%) to compare the 2 methods. For each method, we used the PHASE haplotyping software since it was described to be the best. We showed that the use of the "global method" for subhaplotyping leads always to a better error rate than the classical direct haplotyping. The advantage provided by this alternative method increases with the percentage of missing genotyping data (diminution of the average error rate from 25% to less than 10%). We applied the global method software on the GRIV cohort for AIDS genetic associations and some associations previously identified through direct subhaplotyping were found to be erroneous. CONCLUSION: The global method for subhaplotyping can reduce, sometimes dramatically, the error rate on patient resolutions and haplotypes frequencies. One should thus use this method in order to minimise the risk of a false interpretation in genetic studies involving subhaplotypes. In practice the global method is always more efficient than the direct method, but a combination method taking into account the level of missing information in each subject appears to be even more interesting when the level of missing information becomes larger (>10%).
Assuntos
Biologia Computacional/métodos , Haplótipos , Polimorfismo de Nucleotídeo Único , Software , Apolipoproteínas E/genética , Infecções por HIV/genética , Infecções por HIV/imunologia , Soropositividade para HIV/genética , Soropositividade para HIV/imunologia , HIV-1/imunologia , Hormônio do Crescimento Humano/genética , Humanos , Interleucina-6/genética , Receptores de Interleucina-10/genética , Receptores de Interleucina-4/genéticaRESUMO
Large-scale genomic studies in cohorts have been made possible for the last few years thanks to the progress of molecular biology and bioinformatics. This systematic approach allows a better understanding of the molecular mechanisms of disease development and as a consequence can contribute to the rational design of new diagnostic and therapeutic tools. We present here the exhaustive genotyping of a candidate gene, tumor necrosis factor receptor 1 (TNFR1), in the genetic of resistance to immunodeficiency virus (GRIV) AIDS cohort. This gene was chosen because it is likely to be involved in the apoptosis pathways of CD4+ and CD8+ T-cells during human immunodeficiency virus 1 (HIV-1) infection. Seven frequent polymorphisms were characterized in 319 HIV-1 seropositive patients from the GRIV cohort with extreme disease progression phenotypes, slow progression or rapid progression, and in 427 healthy controls. The TNFR1 gene locus does not appear to be part of any haploblock and contains only a small haploblock of two successive SNPs. One promoter SNP (TNFR1_17444594, position -581) and one intronic SNP (TNFR1_27223241, position +11511) gave weak positive signals of association (resp. P=0.03 and P=0.04) as well as two haplotypes. To our knowledge, this is the first genetic association study dealing with the TNFR1 gene in AIDS and the putative associations identified will need to be validated through other AIDS cohort analyses or by further biological experimentation.