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: 10463613
• 负责人: Benjamin Hackel
• 金额: $ 44.37万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10463613
• 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; Industry; Inflammatory; Inflammatory Bowel Diseases; Interruption; Libraries; Ligand Binding; Ligands; Literature; Malignant Neoplasms; Mammalian Cell; Maps; Methods; Molecular; Pathology; Peptides; Persons; 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; Western Blotting; Work; Yeasts; antagonist; base; dimer; empowered; experience; high throughput screening; improved; inhibitor; 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.

摘要 肿瘤坏死因子（TNF）配体和肿瘤坏死因子受体（TNFR）是免疫的重要调节因子 反应TNF的失调在许多自身免疫性疾病的病理学中起作用， 超过2350万人在美国。TNFR 1信号传导的治疗靶向（例如，用于类风湿性关节炎） 关节炎和炎症性肠病）是一个价值数十亿美元的产业。然而，现有的抗TNF药物 会引起严重的副作用因此，迫切需要开发“抗TNFR”而不是“抗TNFR”。 TNF治疗慢性炎症和自身免疫性疾病。尽管最近在这方面取得了一些进展， 现有技术的小分子方法未能发现任何高亲和力的小分子抑制剂。 为了重新启动所需的治疗发现工作，我们一直在建立现有的 酵母展示/定向进化技术来工程化高亲和力TNFR配体，代替小分子。 蛋白质配体支架，具有高亲和力和大表面积的肽，通过调节氨基而被工程化。 在蛋白质的特定区域（称为互补位）中的氨基酸，同时保留稳定的底层支架。一 一个特别的例子，已经被co-PI Hackel广泛研究和改进的抗体域， 已被有效地用作许多靶点的配体支架，亲和力强至20 pM， 到诊断、分子成像和治疗。 然而，当我们向TNFR家族的高亲和力结合物发展时，我们遇到了一个熟悉的瓶颈 在该领域：如何指导粘合剂的发展，而不是基于亲和力，而是基于功能性。虽然许多 尽管存在用于发现和进化蛋白质结合的平台，但尚未建立用于蛋白质结合的稳健方法。 选择精确的生物活性（除了一般的生存筛选）。 因此，本提案的目的是开发一种基于活性的高通量的新技术。 蛋白质配体的筛选。在这样做的过程中，我们将发现新的，高亲和力的TNFRs抑制剂。目标1将 实现发现和发展一个广泛的面板强结合剂的TNFR，虽然频率的功能性 抑制剂预期相当低。Aim 2开发了一种技术， 功能性结合剂，其将对所有活性配体筛选具有广泛的用途，除了集中的益处之外， 目前TNFR拮抗剂的发展。