Probes to target the 3-way hotspot of IL1RacP to abolish aberrant interleukin inflammation

靶向 IL1RacP 3 路热点以消除异常白细胞介素炎症的探针

• 批准号: 8944833
• 负责人: Lance Allen Liotta
• 金额: $ 59.52万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8944833
• 关键词: Acute; Affinity; Agonist; American; Amino Acids; Animal Model; Arginine; Arthritis; Asthma; Autoimmune Process; Binding; Biodistribution; Biological Assay; Biological Availability; Cell Culture Techniques; Cell Line; Cell membrane; Cell surface; Chemistry; Clinical; Complex; Cytokine Activation; Degenerative polyarthritis; Deuterium; Disease; Dose; Drug Targeting; Engineering; Family; Family member; Hot Spot; In Vitro; Inflammation; Inflammatory; Inhibitory Concentration 50; Interleukin Therapy; Interleukin-1; Interleukin-1 beta; Interleukins; Intra-Articular Injections; Kineret; Lead; Ligand Binding; Ligands; Maps; Mediating; Methods; Modeling; Modification; Molecular; Monoclonal Antibodies; Neonatal; Obstructive Lung Diseases; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Play; Positioning Attribute; Protein Binding; Protein Chemistry; Proteins; Psoriasis; Publishing; Receptor Signaling; Recruitment Activity; Role; Signal Transduction; Site; Specificity; Surface; Surface Plasmon Resonance; Tail; Technology; Testing; Therapeutic; Transmembrane Domain; Traumatic Arthropathy; Validation; Work; anakinra; base; crosslink; cytokine; design; domain mapping; in vitro Assay; in vivo; inhibitor/antagonist; interleukin-1 receptor accessory protein; member; mouse model; neoplastic; new technology; next generation; novel; osmotic minipump; peptidomimetics; prevent; protein complex; protein folding; protein protein interaction; public health relevance; receptor; small molecule

 DESCRIPTION (provided by applicant): Aberrant interleukin family cytokine activation of inflammation characterizes a wide spectrum of autoimmune, inflammatory, neoplastic, and neonatal disorders, including arthritis, obstructive lung disease, psoriasis and asthma. We created a novel technology, protein painting, and used it to discover, and create, a novel peptide inhibitor, called Arg286p, that abolishes the necessary interaction of IL1-RAcP with the IL-1β-IL1Receptor complex. Arg286p constitutes a novel specific and potent approach to treat interleukin mediated inflammatory diseases. The binding interface contact points between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Employing a new class of small molecule affinity chemistry, protein painting has a positive hit specificity of 93% and yield up to ten fold higher compared to conventional cross linking or deuterium exchange methods. Using our technology to study the 3-way interaction of interleukin-1β (IL-1β, IL-1 receptor I (IL-1RI), and IL-1 receptor accessory protein (IL-1RAcP), we identified a highly conserved beta-loop Arg286 region of the IL-1RAcP protein that participates in a multivalent interaction with the ligand and the receptor. We validated this novel target to be necessary for IL1β-mediated signaling. We created a folded protein peptide Arg286p that corresponds to the Arg286 beta loop domain, and a monoclonal antibody that recognizes the IL-1RAcP Arg286 surface domain. Our Arg286 peptide compound abolished interleukin signaling in a cell culture model in a dose-dependent manner, compared to a control peptide lacking the arginine in the 286 position. The Arg286 peptide and the anti-Arg286 mAb also abolished formation of the IL-1β, IL-1RI, and IL-1RAcP 3-way complex, in vitro, in a dose-dependent manner. IL1-RAcP is an accessory protein that is recruited after the interleukin ligand binds to the receptor, and signaling requires all members of this three-way complex formation. Our new inhibitor Arg286p mimics a small key multivalent interaction region at the arginine 286 beta loop of IL1-RacP, thereby abolishing complex formation and preventing downstream inflammatory signaling. Since Arg286p acts downstream of receptor-ligand binding, it can massively amplify the potency for interleukin therapy even in the face of excess ligand, and may be superior to, or synergistic with, existing IL-1 therapies that competitively target the ligand. Under the Aims, we will create new structural modifications in this lead compound to optimize its affinity and stability for IL-1 and we will use it as a basis to create novel inhibitos specific for IL33 and IL36, for which clinical inhibitors do not exist. We will employ our protein painting technology in a novel iterative workflow to guide the design of peptide modulators (agonists and antagonists) that are either optimized in potency for IL-1 or specific for IL-33 and IL-36 in vitro, in three types of assays. The optimized version of our lead compound will be studied in a well-established animal model of osteoarthritis. Osteoarthritis causes suffering for 27 million Americans.

 描述(申请人提供)：炎症的白介素家族细胞因子的异常激活是一系列自身免疫、炎症、肿瘤和新生儿疾病的特征，包括关节炎、阻塞性肺疾病、牛皮癣和哮喘。我们创造了一种新的技术，蛋白质涂抹，并利用它发现并创造了一种名为Arg286P的新型多肽抑制剂，它取消了IL-1-RACP与IL-1β-IL 1受体复合体的必要相互作用。Arg286p构成了一种治疗白介素性炎症性疾病的新的特异而有效的方法。相互作用的蛋白质伙伴之间的结合界面接触点是阻止这种相互作用的下一代疗法的重要药物靶点。采用一种新的小分子亲和化学，蛋白质涂布具有93%的阳性命中特异度，与传统的交联法或氘交换法相比，产率高达10倍。利用我们的技术研究IL-1β(IL-1β、IL-1受体I(IL-1RI)和IL-1受体附件)的三向相互作用 IL-1RAcP蛋白(IL-1RAcP)，我们发现IL-1RAcP蛋白的一个高度保守的β-loop Arg286区域参与了与配体和受体的多价相互作用。我们验证了这个新的靶点对于IL1β介导的信号转导是必要的。我们创建了与Arg286β环区相对应的折叠蛋白多肽Arg286p，以及识别IL-1RAcP Arg286表面域的单抗。我们的Arg286多肽化合物以剂量依赖的方式在细胞培养模型中取消白细胞介素2信号，而在286位缺少精氨酸的对照多肽。Arg286多肽和抗Arg286单抗在体外也以剂量依赖的方式抑制IL-1β、IL-1RI和IL-1RAcP三向复合体的形成。IL1-RACP是一种辅助蛋白，在白介素配体与受体结合后被招募，信号需要这个三向复合体形成的所有成员。我们的新抑制剂Arg286p模拟了IL1-RACP精氨酸286β环的一个小的关键多价相互作用区域，从而取消了复合体的形成，防止了下游的炎症信号。由于Arg286p作用于受体-配体结合的下游，即使面对过剩的配体，它也可以大量放大白细胞介素性治疗的效力，并可能优于或协同现有的竞争性靶向该配体的IL-1疗法。在这些目标下，我们将在这一先导化合物中创建新的结构修饰，以优化其对IL-1的亲和力和稳定性，并将以此为基础创建针对IL33和IL36的新型抑制剂，而临床上尚不存在针对它们的抑制剂。我们将在一种新颖的迭代工作流程中使用我们的蛋白质涂抹技术来指导多肽调节剂(激动剂和拮抗剂)的设计，这些调节剂在三种类型的检测中对IL-1的效力进行了优化，或在体外对IL-33和IL-36具有特异性。我们的先导化合物的优化版本将在一个成熟的骨关节炎动物模型中进行研究。骨关节炎导致2700万美国人遭受痛苦。