High throughput screening to find inhibitors of pathogenic pemphigus antibodies

高通量筛选寻找致病性天疱疮抗体的抑制剂

• 批准号: 8138732
• 负责人: John R Stanley
• 金额: $ 4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8138732
• 关键词: Adverse effects; Affect; Antibodies; Autoantibodies; Autoimmune Diseases; Autoimmune Process; Bacteriophages; Binding; Biological Assay; Bulla; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Disease; Epithelial; Epitopes; Flow Cytometry; G-substrate; Human; Immune system; Immunoglobulin Fragments; Immunosuppressive Agents; Laboratories; Lead; Life; Link; Longitudinal Studies; Mediating; Modeling; Molecular Bank; Molecular Weight; Monoclonal Antibodies; Morbidity - disease rate; Mucous Membrane; Organ Culture Techniques; Pathogenicity; Patients; Pemphigus; Pemphigus Vulgaris; Performance; Phage Display; Reagent; Regimen; Screening procedure; Serum; Skin; System; Testing; Tissues; antigen binding; base; desmoglein; desmoglein 2; desmoglein III; enhanced green fluorescent protein; extracellular; follow-up; genetic analysis; high throughput screening; inhibitor/antagonist; keratinocyte; mortality; novel; novel strategies; repository; small molecule; small molecule libraries

DESCRIPTION (provided by applicant): Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease affecting skin and mucous membranes that is mediated by circulating antibodies against a keratinocyte desmosomal cadherin, desmoglein (Dsg) 3, which mediates cell adhesion. Recent studies using phage display indicate the presence of both pathogenic and non-pathogenic monoclonal autoantibodies (mAbs) in PV patient sera. The pathogenic antibodies directly cause blister formation by interfering with cell adhesion. Although current immunosuppressive regimens targeting the total antibody pool have led to reduced patient mortality, morbidity associated with the side effects of treatment is significant and underscores the need for therapies that specifically target pathogenic anti-Dsg antibodies. These observations suggest a novel approach to treatment would be to use small molecule reagents that block the binding of pathogenic mAbs to Dsg. The target has been established with the extracellular portion of Dsg3 displayed on protein G beads and the pathogenic monoclonal antibody expressed as a single chain variable fragment antibody (scFv)-enhanced green fluorescent protein (EGFP) construct. A homogeneous flow cytometry assay has been implemented in 384-well plate format and an initial screen of the Prestwick Chemical Library has been completed. The assay routinely performs with Z' values in the range of 0.5 - 0.8. We will use this assay to screen the Molecular Libraries Small Molecule Repository (MLSMR) to identify molecules that interfere with the binding of pathogenic scFv anti-Dsg3 to disease important epitopes of Dsg3. Secondary assays based on the flow cytometry assay will be used to validate active compounds identified in the primary screen. Identified compounds will be tested for inhibition of pathogenicity of pemphigus antibodies in tertiary follow-up biologic assays of human skin organ culture. These systems are well established in the PI's laboratory. This proposal is expected to result in novel lead compounds with the potential of revolutionizing the treatment of pemphigus. PUBLIC HEALTH RELEVANCE: Pemphigus vulgaris is a disfiguring and potentially fatal blistering autoimmune disease. Current therapy is to suppress the immune system, which results in many potential adverse effects. This proposal seeks therapy directly targeted only to the autoantibodies that actually cause the blisters in this disease.

描述（由申请人提供）：Pemphigus vulgaris（PV）是一种潜在的威胁生命的自身免疫性泡沫疾病，影响皮肤和粘膜膜，是通过循环抗体抗体抗体抗体抗体抗体抗体，对角质形成细胞细胞类细胞体脱发蛋白细胞体钙粘蛋白（DSG），向粘膜（DSG）3，Mediatiates 3，Mendiation。使用噬菌体显示的最新研究表明，PV患者血清中存在致病性和非致病性单克隆自身抗体（MAB）。致病性抗体通过干扰细胞粘附而直接引起泡沫形成。尽管针对总抗体池的当前免疫抑制方案导致患者死亡率降低，但与治疗的副作用相关的发病率很大，并且强调了对特定针对致病性抗DSG抗体的疗法的需求。这些观察结果表明，一种新的治疗方法是使用小分子试剂，以阻止致病性mAb与DSG的结合。该靶标是在蛋白质G珠上显示的DSG3的细胞外部分建立的，并且以单链可变片段抗体（SCFV）增强的绿色荧光蛋白（EGFP）结构表示的致病单克隆抗体。均匀的流式细胞仪测定已以384孔板格式实施，并且已经完成了Prestwick化学库的初始屏幕。该测定法通常在0.5-0.8的范围内执行Z'值。我们将使用该测定法来筛选分子库小分子存储库（MLSMR），以鉴定干扰致病性SCFV抗DSG3与DSG3重要表位的结合的分子。基于流式细胞仪测定法的次要测定将用于验证主屏幕中确定的活性化合物。将测试已鉴定的化合物，以抑制人类皮肤器官培养的第三纪随访生物学测定中的Pemphigus抗体的致病性。这些系统已在PI的实验室中良好确定。预计该建议将导致新型的铅化合物，并具有革新Pemphigus治疗的潜力。 公共卫生相关性：Pemphigus dulgaris是一种毁容且可能致命的自身免疫性疾病。当前的疗法是抑制免疫系统，从而导致许多潜在的不良反应。该提案寻求直接针对实际引起这种疾病水泡的自身抗体的治疗。