Engineering synthetic ligands with potent allosteric inhibition of tumornecrosis factor receptors

摘要 肿瘤坏死因子配体和肿瘤坏死因子受体是免疫的重要调节因子。 回应。肿瘤坏死因子调节失调在目前困扰许多自身免疫性疾病的病理中起作用 美国有超过2350万人。针对TNFR1信号的治疗靶点(例如，针对类风湿 关节炎和炎症性肠病)是一个价值数十亿美元的行业。然而，现有的抗肿瘤坏死因子药物 造成严重和不良的副作用。因此，迫切需要发展“抗肿瘤坏死因子受体”，而不是“抗- 肿瘤坏死因子在慢性炎症和自身免疫性疾病中的治疗。尽管最近在这方面取得了一些进展， 最先进的小分子方法未能发现任何高亲和力的小分子抑制剂。 为了尝试启动新的和需要的治疗发现努力，我们一直在现有的基础上 酵母展示/定向进化技术，以设计高亲和力的TNFR配体，取代小分子。 蛋白质配基支架是通过调节氨基来构建的具有高亲和力和大表面积的多肽。 蛋白质中特定区域的酸，称为副位，同时保存一个稳定的底层支架。一 具体来说，已经由合作者Pi Hackel广泛研究和改进的仿体结构域具有 被有效地用作众多目标的配基支架，亲和力高达20 pm，并应用于 到诊断、分子成像和治疗。 然而，随着我们向TNFR家族的高亲和力结合迈进，我们遇到了熟悉的瓶颈 在该领域：如何指导粘结剂的演变，不是基于亲和力，而是基于功能。虽然数量众多 蛋白质结合的发现和进化平台已经存在，还没有为蛋白质结合建立可靠的方法 选择精确的生物活性(除了一般的生存筛查)。 因此，这项提议的目标是开发一种基于活动的、高吞吐量的新技术 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TNFR抑制剂。目标1将 实现对TNFR的广泛的强结合组的发现和进化，尽管功能性的频率 预计抑制剂的含量将相当低。Aim 2开发了一种技术，可以极大地增强对 功能结合剂，它将在所有活性配体筛选中具有广泛的用途，此外还具有以下主要好处 目前肿瘤坏死因子受体拮抗剂的研究进展。

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.