High throughput screening to find inhibitors of pathogenic pemphigus antibodies

描述(申请人提供)：寻常型天疱疮(PV)是一种潜在威胁生命的自身免疫性水疱病，影响皮肤和粘膜，由抗角质形成细胞桥粒钙粘附素(DSG)3的循环抗体介导，该抗体介导细胞黏附。最近利用噬菌体展示技术的研究表明，PV患者血清中同时存在致病性和非致病性的单抗。致病抗体通过干扰细胞黏附直接导致水泡的形成。虽然目前针对总抗体库的免疫抑制方案已经降低了患者的死亡率，但与治疗副作用相关的发病率是显著的，并强调了针对致病性抗DSG抗体的治疗的必要性。这些观察表明，一种新的治疗方法将是使用小分子试剂来阻断病原性单抗与DSG的结合。将Dsg3胞外部分展示在蛋白G小球上，将致病单抗表达为单链可变区抗体(ScFv)增强型绿色荧光蛋白(EGFP)。用384孔板进行了均相流式细胞术分析，并完成了Prestwick化学文库的初步筛选。该分析通常用Z‘值在0.5-0.8的范围内进行。我们将利用本实验筛选分子文库小分子资料库(MLSMR)，以确定干扰致病单链抗体抗Dsg3与疾病重要表位Dsg3结合的分子。基于流式细胞术分析的二次分析将用于验证初步筛选中确定的活性化合物。已确定的化合物将在第三次人体皮肤器官培养的生物检测中测试天疱疮抗体的致病性抑制作用。这些系统在PI的实验室中已经很好地建立起来了。这项提议有望产生新的先导化合物，有可能彻底改变天疱疮的治疗方法。 公共卫生相关性：寻常型天疱疮是一种毁容和潜在致命的起泡性自身免疫性疾病。目前的治疗方法是抑制免疫系统，这会导致许多潜在的不良反应。这项建议寻求直接针对实际上导致这种疾病水泡的自身抗体的治疗。

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.