High throughput screening to find inhibitors of pathogenic pemphigus antibodies

描述（由申请人提供）：寻常型天疱疮（Pemphigus vulgaris， 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，由针对角化细胞桥粒钙粘蛋白、桥粒蛋白（Dsg） 3的循环抗体介导，该抗体介导细胞粘附。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。尽管目前针对总抗体库的免疫抑制方案已经降低了患者死亡率，但与治疗副作用相关的发病率是显著的，并且强调了专门针对致病性抗dsg抗体的治疗的必要性。这些观察结果表明，一种新的治疗方法是使用小分子试剂来阻断致病性单抗与Dsg的结合。Dsg3的胞外部分显示在蛋白G珠上，病原单克隆抗体表达为单链可变片段抗体（scFv）增强的绿色荧光蛋白（EGFP）构建物。384孔板格式的均质流式细胞术检测已经实施，Prestwick化学库的初始筛选已经完成。该分析通常在0.5 - 0.8的范围内执行Z值。我们将使用该试验筛选Molecular Libraries Small Molecule Repository (MLSMR)，以识别干扰致病性scFv anti-Dsg3与Dsg3疾病重要表位结合的分子。基于流式细胞术的二次分析将用于验证在初级筛选中鉴定的活性化合物。鉴定出的化合物将在人体皮肤器官培养的第三次后续生物测定中用于天疱疮抗体致病性的抑制试验。这些系统在PI的实验室中已经很好地建立起来。这一建议预计将导致新的先导化合物具有革命性的治疗天疱疮的潜力。

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.