Assay Development for HT Molecular Screening for Inhibitors of Integrin Signaling

整合素信号传导抑制剂 HT 分子筛选的检测方法开发

• 批准号: 7290706
• 负责人: Mark HOWARD Ginsberg
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7290706
• 关键词: Antibodies; Autoimmune Process; Binding; Biochemistry; Biological Assay; Biotinylation; Cellular biology; Chemicals; Collaborations; Comparative Study; Congenital Heart Defects; Cytoplasmic Tail; Detection; Development; Disease; Enzyme-Linked Immunosorbent Assay; Focal Adhesions; Genomics; Health; Hematopoiesis; Inflammation; Inflammatory; Institutes; Integrins; Lead; Ligands; Malignant Neoplasms; Modeling; Molecular; Molecular Bank; Morphologic artifacts; Multiple Sclerosis; Mus; Myocardial Infarction; Placenta; Poison; Positioning Attribute; Proteins; Recombinants; Reproducibility; Research Project Grants; Screening procedure; Signal Transduction; Specificity; Stroke; Tail; Therapeutic; Toxic effect; Translating; Tysabri; Variant; assay development; base; follow-up; high throughput screening; in vivo; inhibitor/antagonist; leupaxin; natalizumab; novel strategies; paxillin; small molecule; therapeutic target

DESCRIPTION (provided by applicant): Support is requested to develop and validate a high-throughput screening assay to identify small molecule inhibitors of the integrin a4-paxillin interaction. Inhibition of a4 integrin function by antibodies, such as Tysabri (Natalizumab) is of proven therapeutic value in diseases such as Multiple Sclerosis and may be beneficial in other autoimmune and inflammatory diseases. Nevertheless, mechanism-based toxicities involving developmental abnormalities of the heart and placenta and of hematopoiesis may accompany complete blockade of a4 integrin function. The applicants' studies using biochemistry, cell biology, and genetically-modified mice show that blocking a4 integrin signaling by inhibiting the binding of paxillin to the a4 cytoplasmic domain can inhibit inflammation, yet avert some mechanism- based toxicity. Furthermore, a low throughput screen has identified small molecule inhibitors of the interaction, thus establishing the feasibility of this approach. He proposes to adapt the existing ELISA to a high throughput mode by modifying the a4 integrin ligand for in vivo biotinylation and purifying paxillin's two paralogues, HIC-5 and leupaxin, for comparative studies. The assay will be adapted to chemiluminescence-based detection and a 384 well format, variation and stability will be established and a small pilot screen will be conducted. Secondary screens will eliminate false positives and toxic compounds and will position the applicant to screen up to 500,000 compounds provided by the Molecular Libraries Screening Centers Network (MLSCN) through collaboration with Dr. Cosford, director of the Center for Chemical Genomics at the Burnham Institute. These studies may lead to new treatments of inflammatory diseases such as Multiple Sclerosis. Furthermore, integrins other than a4 are important therapeutic targets in diseases such as heart attack, stroke, and cancer; these studies will advance a novel approach to anti-integrin therapy, blockade of integrin signaling. Health Relevance: Inhibition of a4 integrin function is of proven therapeutic value, but mechanism-based toxicities may limit therapy. The applicant has shown that blocking a4 integrin-paxillin interaction can block inflammation with less toxicity. The applicant proposes to develop screens that may lead to small molecule inhibitors of the a4-paxillin interaction and thus serve as new treatments of inflammatory diseases such as Multiple Sclerosis

描述（由申请方提供）：要求支持开发和验证高通量筛选试验，以鉴定整合素α 4-桩蛋白相互作用的小分子抑制剂。通过抗体如Tysabri（那他珠单抗）抑制α 4整联蛋白功能在疾病如多发性硬化中具有经证实的治疗价值，并且在其他自身免疫性和炎性疾病中可能是有益的。然而，涉及心脏和胎盘以及造血发育异常的基于机制的毒性可能伴随α 4整联蛋白功能的完全阻断。申请人使用生物化学、细胞生物学和遗传修饰的小鼠的研究表明，通过抑制桩蛋白与α 4胞质结构域的结合来阻断α 4整联蛋白信号传导可以抑制炎症，但避免一些基于机制的毒性。此外，低通量筛选已经鉴定了相互作用的小分子抑制剂，从而确立了这种方法的可行性。他提出通过修饰α 4整联蛋白配体用于体内生物素化和纯化桩蛋白的两种旁系同源物HIC-5和亮帕辛用于比较研究来使现有的ELISA适应于高通量模式。该试验将适用于基于荧光的检测，并将建立384孔格式、变异性和稳定性，并将进行小型中试筛选。二次筛选将消除假阳性和有毒化合物，并将使申请人能够通过与伯纳姆研究所化学基因组学中心主任Cosford博士合作，筛选分子库筛选中心网络（MLSCN）提供的多达50万种化合物。这些研究可能会导致新的治疗炎症性疾病，如多发性硬化症。此外，除α 4以外的整合素是心脏病、中风和癌症等疾病的重要治疗靶点;这些研究将推进抗整合素治疗的新方法，阻断整合素信号传导。健康相关性：α 4整联蛋白功能的抑制已被证实具有治疗价值，但基于机制的毒性可能限制治疗。申请人已经表明，阻断α 4整联蛋白-桩蛋白相互作用可以以较小的毒性阻断炎症。申请人提出开发筛选，其可导致α 4-桩蛋白相互作用的小分子抑制剂，并因此用作炎性疾病如多发性硬化的新治疗。