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De novo engineering of protein agonists and antagonists

蛋白质激动剂和拮抗剂的从头工程

基本信息

批准号：
8690801
负责人：
Casim Sarkar
金额：
$ 16.03万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8690801
关键词：
Active Sites Affinity Agonist Amino Acid Motifs Ankyrin Repeat Ankyrins Antibodies Binding Buffers C-terminal Cell Line Cell Surface Receptors Cell membrane Cell surface Cells Cysteine Disease Drug Formulations Drug usage Engineering Epithelial Evolution Extracellular Domain Family Generations Genes Goals HIV Hematological Disease Immune System Diseases Individual Interleukin 12 Receptor Beta Interleukin-12 Lead Libraries Ligands Malignant Neoplasms Mediating Medical Membrane Methodology Methods Modeling Patient Care Peptide Hydrolases Physiology Production Protein Engineering Proteins Randomized Ribosomes Role Serine Protease Signal Transduction Site Technology Terminal Repeat Sequences Testing Therapeutic Therapeutic antibodies Tumor-Derived biophysical properties cancer therapy crosslink cytokine design directed evolution disulfide bond improved inhibitor/antagonist insight interest interleukin-12 receptor matriptase mimetics mutant novel novel therapeutics pressure public health relevance receptor mediated endocytosis research study scaffold small molecule tumor

项目摘要

DESCRIPTION (provided by applicant): In addition to their vital roles in normal physiology, recombinantly produced cytokines and antibodies constitute a large, critical family of drugs used in the treatments for cancer, HIV, and a host of hematological and immune disorders. However, these molecules are often difficult to produce in mass quantities, suffer from poor folding and aggregation due to the presence of disulfide bonds, and are relatively unstable as therapeutic formulations. Recently, alternative scaffolds with superior biophysical properties to cytokines and antibodies have been used in directed evolution experiments to engineer high-affinity binders to targets of interest. However, cytokine and antibody therapeutics can serve as either agonists or antagonists, and current directed evolution methodologies only select for target binding, not consequent agonism or antagonism. Here, we propose to exploit the modular fold of an alternative scaffold - the designed ankyrin repeat protein - to develop novel directed evolution approaches for isolating new agonists and antagonists. The goals of this R21 proposal are to: 1) engineer anykrin agonists that are mimetics of interleukin-12, a potent anti-tumor cytokine, and 2) create site-specific ankyrin inhibitors of matriptase, a cell-surface protease tha is implicated in a variety of epithelial-derived tumors. More generally, these methods will not only enable the production of new therapeutics, but will also provide unique insights into the basic requirements for specific, high-affinity protein recognition.

描述（由申请人提供）：除了在正常生理学中的重要作用外，重组产生的细胞因子和抗体构成了用于治疗癌症、HIV和许多血液学和免疫疾病的药物的一个大的关键家族。然而，这些分子通常难以大量生产，由于二硫键的存在，折叠和聚集较差，并且作为治疗制剂相对不稳定。最近，具有比细胞因子和抗体更优越的上级生物物理特性的替代支架已被用于定向进化实验中，以工程化对感兴趣的靶的高亲和力结合剂。然而，细胞因子和抗体治疗剂可以用作激动剂或拮抗剂，并且目前的定向进化方法仅选择靶结合，而不是随后的激动或拮抗。在这里，我们建议利用模块化折叠的替代支架-设计锚蛋白重复蛋白-开发新的定向进化方法，用于分离新的激动剂和拮抗剂。该R21提案的目标是：1）工程化作为白细胞介素-12（一种有效的抗肿瘤细胞因子）的模拟物的anykrin激动剂，和2）产生间质蛋白酶（一种与多种上皮来源的肿瘤有关的细胞表面蛋白酶）的位点特异性锚蛋白抑制剂。更一般地说，这些方法不仅能够生产新的治疗药物，而且还将为特异性高亲和力蛋白质识别的基本要求提供独特的见解。