High throughput screening to find inhibitors of pathogenic pemphigus antibodies

描述（由申请方提供）：寻常天疱疮（PV）是一种影响皮肤和粘膜的潜在危及生命的自身免疫性起泡疾病，由针对介导细胞粘附的角质形成细胞桥粒钙粘蛋白桥粒芯蛋白（Dsg）3的循环抗体介导。最近使用噬菌体展示的研究表明，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直接引起水疱形成。尽管目前靶向总抗体库的免疫抑制方案已导致患者死亡率降低，但与治疗副作用相关的发病率是显著的，并强调了对特异性靶向致病性抗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阻断致病性mAb与Dsg结合的小分子试剂。已经用蛋白G珠上展示的Dsg 3的细胞外部分和表达为单链可变片段抗体（scFv）-增强的绿色荧光蛋白（EGFP）构建体的致病性单克隆抗体建立了靶标。已在384孔板格式中实施均相流式细胞术测定，并已完成Prestwick化学文库的初始筛选。该测定通常在Z'值为0.5 - 0.8的范围内进行。我们将使用该测定来筛选分子库小分子库（MLSMR）以鉴定干扰致病性scFv抗Dsg 3与Dsg 3的疾病重要表位结合的分子。将使用基于流式细胞术试验的二次试验验证初步筛选中鉴别的活性化合物。将在人皮肤器官培养物的第三次随访生物试验中检测已鉴别化合物对天疱疮抗体致病性的抑制作用。这些系统在PI的实验室中得到了很好的建立。这一提议有望产生新的先导化合物，具有彻底改变天疱疮治疗的潜力。 公共卫生相关性：寻常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自身抗体的治疗，这些抗体实际上导致了这种疾病的水泡。

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.