Directed evolution to diversify HIV protease function

定向进化使HIV蛋白酶功能多样化

• 批准号: 7228921
• 负责人: ICHIRO MATSUMURA
• 金额: $ 27.13万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7228921
• 关键词: Active Sites; Affinity; Antibodies; Antibody Affinity; Ants; Caring; Cleaved cell; Communicable Diseases; Disease; Drug Delivery Systems; ERBB2 gene; Endopeptidases; Engineering; Enzymes; Evolution; Goals; HIV Protease; HIV-1 protease; Immune; In Vitro; Kinetics; Label; Light; Malignant Neoplasms; Modeling; Mutagenesis; Mutation; Natural Selections; Nature; Numbers; Oncogenic; Outcome; Peptide Hydrolases; Personal Satisfaction; Pharmaceutical Preparations; Pharmacologic Substance; Process; Property; Protease Inhibitor; Protein Engineering; Proteins; Purpose; Range; Research Personnel; Resistance; Rheumatoid Arthritis; Screening procedure; Site; Site-Directed Mutagenesis; Specificity; Standards of Weights and Measures; Surgical Flaps; Therapeutic; Therapeutic antibodies; Tumor Necrosis Factor-alpha; Variant; Work; design; directed evolution; enzyme therapy; high throughput screening; human TNF protein; improved; inhibitor/antagonist; molecular recognition; neoplastic cell; novel strategies; pathogen; programs; research study; response; skills; small molecule; theories

DESCRIPTION (provided by applicant): Therapeutic antibodies are the standard of care for certain cancers, auto-immune and infectious diseases, but their efficacy decreases when the number of target molecules is too large. The engineering of proteases that specifically catalyze the cleavage of disease-promoting proteins would enable enzyme therapies for a wider range of diseases. HIV protease is a nearly ideal starting point for this purpose because three lines of evidence suggest that this enzyme is unusually evolvable. First, HIV protease evolves rapidly in nature in response to synthetic protease inhibitors. Second, this observed evolvability is consistent with evolutionary theory. Third, the investigator has already employed random mutagenesis and screening to create a variant that is specific for TNF-alpha (target protein implicated in rheumatoid arthritis). He now proposes studies with the following specific aims: 1. to improve the activity and TNF-alpha specificity of the HIV protease variant through a directed evolution process that mimics antibody affinity maturation. 2. to direct the evolution of HIV protease variants specific for the oncogenic protein HER2. 3. to discover the structural mechanisms of adaptive evolution by purifying the wild-type and variant HIV proteases and characterizing their biophysical and kinetic properties in vitro. These experiments are a first step toward a new approach to enzyme therapy. We will demonstrate the unusual evolvability of HIV protease, and better understand why some enzymes are more amenable to protein engineering than others. The high throughput screens developed for these studies could also be used to identify small molecule protease inhibitors.

描述（由申请人提供）： 治疗性抗体是某些癌症、自身免疫性疾病和传染病的标准治疗方法，但当靶分子数量过多时，其疗效就会降低。专门催化促进疾病的蛋白质裂解的蛋白酶工程将使酶疗法能够治疗更广泛的疾病。 HIV蛋白酶是实现这一目的近乎理想的起点，因为三线证据表明这种酶具有异常的进化能力。首先，HIV蛋白酶在自然界中快速进化以响应合成蛋白酶抑制剂。其次，这种观察到的进化性与进化理论是一致的。第三，研究人员已经采用随机诱变和筛选来创建一种针对 TNF-α（与类风湿性关节炎有关的靶蛋白）特异的变体。他现在提出了以下具体目标的研究： 1.通过模拟抗体亲和力成熟的定向进化过程来提高HIV蛋白酶变体的活性和TNF-α特异性。 2. 指导针对致癌蛋白 HER2 的 HIV 蛋白酶变体的进化。 3. 通过纯化野生型和变异型HIV蛋白酶并在体外表征其生物物理和动力学特性，发现适应性进化的结构机制。 这些实验是迈向酶疗法新方法的第一步。我们将展示 HIV 蛋白酶不同寻常的进化能力，并更好地理解为什么某些酶比其他酶更适合蛋白质工程。为这些研究开发的高通量筛选也可用于鉴定小分子蛋白酶抑制剂。