Protease Engineering

蛋白酶工程

• 批准号: 7994845
• 负责人: BRENT L IVERSON
• 金额: $ 31.52万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994845
• 关键词: Active Sites; Address; Affinity; Amino Acids; Anaphylatoxins; Antibodies; Area; Asthma; Back; Bacterial Proteins; Basic Science; Binding; Biological; Biological Assay; Biotechnology; Birds; C-terminal; Cardiovascular Diseases; Characteristics; Charge; Chicago; Cleaved cell; Clinical; Complement; Complement 3a; Complement Activation; Detergents; Disease; Drug Industry; Engineering; Enzymes; Exhibits; Experimental Designs; Eye; Face; Family; Flow Cytometry; Frequencies; Generations; Glutamine; Goals; Granzyme; Hand; Health; Human; Human Engineering; In Vitro; Individual; Industry; Inflammatory; Laboratories; Lead; Libraries; Ligands; Light; Location; Malignant Neoplasms; Marketing; Mediating; Medical; Methodology; Methods; Modification; Mutagenesis; Nature; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phosphorylation; Process; Property; Protein Engineering; Proteins; Proteomics; Randomized; Reporting; Research; Route; Science; Screening procedure; Sepsis; Serine; Signal Transduction; Site; Sorting - Cell Movement; Specificity; Staging; Structure; Substrate Specificity; Techniques; Technology; Testing; Therapeutic; Time; Translating; Trypsin; Universities; Variant; Work; angiogenesis; base; catalyst; design; direct application; directed evolution; effective therapy; experience; granzyme A; high throughput screening; immunogenicity; insight; interest; member; novel; pre-clinical; programs; protein aminoacid sequence; research study; success; therapeutic protein; tool; tyrosine O-sulfate; ward

DESCRIPTION (provided by applicant): The design of enzymes with tailored physical and catalytic properties is one of the fundamental thrusts of modern protein science, with the potential for profound technological and medical impact. Laboratory directed evolutionary approaches along with rational design principles have now been successfully applied to enhance protein properties and function. What remains is the important next step of "rolling up our sleeves" and attacking important problems with an eye toward details, which are likely to be enzyme specific. This proposal extends our enzyme directed evolution program in important enabling directions in the area of engineered proteases. Our most recent work with the OmpT protease represents by far the most general manipulation of P1 and P1' substrate specificity of a protease while retaining high overall levels of catalytic activity. Beyond their use in the detergent industry, engineered proteases have tremendous practical potential as either proteomic tools or catalytic therapeutics. In particular, proteases specific for substrates containing modifications such as phosphorylation or O-GlcNAc would represent useful new tools for identifying modified proteins in high throughput proteomics assays. Under Specific Aim 1, we will engineer OmpT variants specific for cleaving only substrates containing phosphorylated or O-GlcNAc serine. We will also investigate whether we can engineer "restriction-like" proteases that can very selectively recognize an extended sequence comprising residues well beyond P1 and P1'. Under Specific Aim 2, we will extend precise OmpT protease recognition to include P2, P3, P2', and P3'. In particular, we will target Gln-His-Ala-Arg-Ala-Ser (QHA?RAS), residues 68-73 of the C-terminus of the C3a anaphylatoxin peptide. C-terminal cleavage of C3a interferes with its biological effects in complement activation. We recognize that creating highly specific subsites in OmpT by mutagenesis and sorting is an exciting yet risky goal and that an engineered OmpT is an unlikely therapeutic clinical candidate because of its bacterial origin. Therefore, for Specific Aim 3, we will engineer precise C3a cleavage activity into the secreted human trypsin-like protease granzyme A in hopes of producing a clinical candidate. As a practical deliverable, following the functional assays of our best variants (Section D6) we will, for the first time, be able to validate the proteolytic approach to complement inhibition, applicable to a wide variety of inflammatory disease therapies. PUBLIC HEALTH RELEVANCE: In general terms, current therapies for almost every disease involve molecules that interact with specific disease targets in a "one-for-one" ratio, i.e. one drug molecule is required to interact with each disease molecule. We are proposing a route to the engineering of molecules, called proteases, that will catalytically destroy many disease target molecules (i.e. a new paradigm in which one drug molecule destroys hundreds, thousands or even more disease molecules)! In particular, we will be attempting to generate a potent catalyst capable of "zeroing in" on a target (called the C3a anaphylatoxin) that would allow effective treatment of a wide range of inflammatory diseases including asthma and sepsis.

描述（由申请人提供）：具有量身定制的物理和催化特性的酶的设计是现代蛋白质科学的基本推力之一，具有深远的技术和医学影响。现已成功地应用了实验室定向进化方法以及合理设计原理，以增强蛋白质特性和功能。剩下的是“滚动我们的袖子”和攻击重要问题的重要下一步的重要下一步，这些问题可能是特定于酶的细节。该建议将我们的酶定向进化计划扩展到工程蛋白酶领域的重要方向。我们与OMPT蛋白酶的最新工作代表了迄今为止对蛋白酶的P1和P1底物特异性的最普遍的操作，同时保留了较高的总体催化活性水平。除了在洗涤剂行业中使用，工程蛋白酶作为蛋白质组学工具或催化疗法具有巨大的实践潜力。特别是，针对含有修饰（例如磷酸化或O-GLCNAC）的底物的蛋白酶将代表有用的新工具，用于识别高通量蛋白质组学测定中的修饰蛋白。在特定的目标1下，我们将设计用于仅裂解含有磷酸化或O-GlCNAC丝氨酸的底物的特定于切割的变体。我们还将调查是否可以设计“限制”蛋白酶，这些蛋白酶可以非常有选择地识别包含P1和P1'的残基的扩展序列。在特定目标2下，我们将将精确的OMPT蛋白酶识别扩展到包括P2，P3，P2'和P3'。特别是，我们将瞄准gln-his-ala-arg-ala-ser（qha？ras），C3A过敏毒素肽的C-末端的残基68-73。 C3a的C末端切割会干扰其在补体激活中的生物学作用。我们认识到，通过诱变和排序在OMPT中创建高度特定的子站点是一个令人兴奋但有风险的目标，并且工程化的OMPT是不太可能的治疗性临床候选者，因为其细菌起源。因此，对于特定的目标3，我们将精确的C3A裂解活性在分泌的人类胰蛋白酶样蛋白酶颗粒酶A中，以期产生临床候选。作为一种实用的可交付，遵循我们最佳变体的功能测定（第D6节），我们将首次能够验证蛋白水解方法以补充抑制作用，适用于多种炎症性疾病疗法。公共卫生相关性：一般而言，几乎每种疾病的当前疗法都涉及与特定疾病靶标相互作用的分子以“一对一”比率相互作用，即需要一种药物分子与每个疾病分子相互作用。我们提出了一种通往分子工程（称为蛋白酶的工程）的途径，该途径将催化破坏许多疾病靶标分子（即一种新的范式，其中一种药物分子破坏了数百，数千甚至更多的疾病分子）！特别是，我们将尝试产生能够在靶标上“零”（称为C3a过敏毒素）的有效催化剂，该催化剂将有效治疗包括哮喘和败血症在内的广泛炎症性疾病。