Engineering synthetic ligands with potent allosteric inhibition of tumornecrosis factor receptors

工程合成配体对肿瘤坏死因子受体具有有效的变构抑制作用

• 批准号: 10227074
• 负责人: Benjamin Hackel
• 金额: $ 43.49万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227074
• 关键词: Address; Affinity; Agonist; Amino Acids; Antibody Binding Sites; Area; Autoimmune Diseases; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Biological Response Modifiers; Biophysics; Cell Survival; Chronic; Clinical; Development; Diagnostic; Dimerization; Directed Molecular Evolution; Disease; Distant; Engineering; Epitopes; Evolution; Extracellular Domain; Failure; Family; Flow Cytometry; Frequencies; Goals; Gold; Immune response; Immunoblotting; Industry; Inflammatory; Inflammatory Bowel Diseases; Interruption; Libraries; Ligand Binding; Ligands; Literature; Malignant Neoplasms; Mammalian Cell; Maps; Methods; Molecular; Pathology; Peptides; Pharmaceutical Preparations; Play; Protein Engineering; Proteins; Psoriatic Arthritis; Receptor Cell; Receptor Signaling; Rheumatoid Arthritis; Role; Sequence Homology; Signal Transduction; Specificity; Structure; Structure-Activity Relationship; Surface; TNF gene; TNFRSF10B gene; TNFRSF1A gene; Technology; Testing; Therapeutic; Time; Treatment Factor; Tumor Necrosis Factor Receptor; Tumor Necrosis Factors; United States; Variant; Work; Yeasts; base; dimer; empowered; experience; high throughput screening; improved; inhibitor/antagonist; innovation; molecular imaging; nanomolar; new technology; novel; novel strategies; protein protein interaction; protein structure function; receptor; receptor expression; scaffold; screening; side effect; small molecule; small molecule inhibitor; success; synthetic protein; technology development; technology validation; therapeutic target

Abstract Tumor necrosis factor (TNF) ligands and TNF receptors (TNFRs) are essential regulators of the immune response. Dysregulation of TNF plays a role in the pathology of many autoimmune diseases that currently afflict more than 23.5 million people in the United States. Therapeutic targeting of TNFR1 signaling (e.g. for rheumatoid arthritis, and inflammatory bowel disease) is a billion-dollar industry. However, the available anti-TNF agents cause severe and adverse side effects. Thus, there is a desperate need to develop 'anti-TNFR' instead of 'anti- TNF' treatments in chronic inflammatory and autoimmune disorders. Despite some recent progress in this regard, state-of-the-art small molecule approaches have failed to uncover any high affinity small molecule inhibitors. In an attempt to jumpstart renewed and needed therapeutic discovery efforts, we have been building on existing yeast display/directed evolution technology to engineer high affinity TNFR ligands, in place of small molecules. Protein ligand scaffolds, peptides with high affinity and large surface area, are engineered by modulating amino acids in a select region, known as the paratope, of a protein while conserving a stable underlying scaffold. One particular example, the affibody domain, which has been extensively studied and improved by co-PI Hackel, has been effectively used as a ligand scaffold to numerous targets, with affinities as strong as 20 pM, and application to diagnostics, molecular imaging, and therapy. However, as we progressed towards high affinity binders to the TNFR family, we reached a familiar bottleneck in the field: how to direct the evolution of binders based not on affinity, but on functionality. While numerous platforms exist for discovery and evolution of protein binding, no robust methods have been established for the selection of precise biological activity (aside from general survival screens). Thus, the objective of this proposal is the development of a new technology for activity-based, high-throughput screening of protein ligands. In so-doing, we will discover novel, high-affinity inhibitors of TNFRs. Aim 1 will achieve discovery and evolution of a broad panel of strong binders to TNFRs, though the frequency of functional inhibitors is expected to be quite low. Aim 2 develops a technology to dramatically enhance the discovery of functional binders, which will have broad utility for all active ligand screening in addition to a focused benefit on the current TNFR antagonist development.

抽象的 肿瘤坏死因子（TNF）配体和TNF受体（TNFR）是免疫的必要调节剂 回复。 TNF的失调在许多自身免疫性疾病的病理学中起作用 在美国，超过2350万人。 TNFR1信号传导的治疗靶向（例如类风湿 关节炎和炎症性肠病）是一个十亿美元的行业。但是，可用的抗TNF代理 引起严重和不利的副作用。因此，迫切需要发展“反tnfr”，而不是“反” TNF慢性炎症和自身免疫性疾病的治疗方法。尽管在这方面取得了一些进展，但 最先进的小分子方法未能发现任何高亲和力小分子抑制剂。 为了尝试重新开始并需要进行治疗的发现工作，我们一直在基于现有 酵母展示/定向的进化技术，用于工程高亲和力TNFR配体，代替小分子。 蛋白质配体支架，具有高亲和力和较大表面积的肽是通过调节氨基的 在保存稳定的基础支架的同时，蛋白质的选择区域（称为角膜膜）中的酸。一 特定的例子，已通过Co-Pi Hackel进行了广泛研究和改进的Affibody域具有 有效地用作众多靶标的配体支架，亲和力高达20 pm，并且应用 进行诊断，分子成像和治疗。 但是，随着我们朝着TNFR家族的高亲和力粘合剂迈进，我们达到了一个熟悉的瓶颈 在现场：如何基于亲和力，而是基于功能来指导粘合剂的演变。虽然很多 存在用于发现和蛋白质结合的进化的平台，没有建立强大的方法 选择精确的生物学活性（除了一般生存筛选）。 因此，该提案的目的是开发基于活动的高通量的新技术 筛选蛋白质配体。在So-doing中，我们将发现TNFR的新型高亲和力抑制剂。目标1意志 尽管功能频率 预计抑制剂将很低。 AIM 2开发了一项技术，以极大地增强发现 功能性粘合剂，除了重点利益外，还将为所有活性配体筛选具有广泛的实用性 当前的TNFR拮抗剂发展。