Structured Protein Scaffolds for Phage-Display

用于噬菌体展示的结构化蛋白质支架

基本信息

批准号：
7390336
负责人：
INDRANEEL GHOSH
金额：
$ 28.1万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7390336
关键词：
Affinity Alanine Antibodies Antiviral Therapy Attention Binding Binding Sites Biochemical Biological Assay CCR5 gene CD4 Antigens CD4-IgG(2)CXCR4 Receptors CXCR4 gene Cell Surface Receptors Chemicals Circular Dichroism Spectroscopy Clinical Trials Complex Computer Assisted Computers Disulfides Docking Engineering Enzyme-Linked Immunosorbent Assay Event Fab Immunoglobulins Face Fluorescence Fluorescence Polarization Goals HIV HIV Envelope Protein gp120 HIV Infections Half-Life Human Immunoglobulin Constant Region Immunoglobulin G Immunoglobulins Length Life Cycle Stages Ligands Measurement Mediating Membrane Proteins Methodology Methods Modality Modeling Mutagenesis Peptides Phage Display Phase II Clinical Trials Protein Binding Protein Engineering Proteins Protocols documentation Publishing Recruitment Activity Scaffolding Protein Scheme Serum Site Structure Surface Testing Therapeutic Toxin Variant Viral Viral Proteins Virion analog base beta pleated sheet design directed evolution experience gp-120 Antigen inhibitor/antagonist mimetics neutralizing antibody novel prevent protein structure research study scaffold sedimentation equilibrium success therapeutic protein

项目摘要

DESCRIPTION (provided by applicant): HIV infection depends upon the coordinated binding of gp120 to human CD4 receptors followed by significant conformational reorganization of gp120, which results in binding to CCR5 or CXCR4 receptor. Directly interfering with viral life cycle by targeting gp120 is a promising therapeutic approach, given the recent success of a multivalent CD4-lgG2 based gp120 inhibitor, which has progressed to phase II clinical trials. Moreover recently published crystal structures of gp120 complexed with CD4 suggest how a beta- sheet based inhibitor scaffold can be utilized for abrogating gp120/CD4 interactions and thereby viral entry. We have recently developed a novel method for presenting stable beta-sheets for recognition of protein surfaces. Our thermostable beta-sheet scaffolds can bind human Immunoglobulins through a helical face, while binding a target protein (gp120) with its beta-sheet face. Based on these observations and our experience, our long-term goals are to provide new methodologies for targeting viral entry by creating CD4 mimetics based upon our miniature protein scaffolds. Our specific objectives are: A) Utilize computer guided docking to graft gp120-binding residues from CD4 upon our beta-sheet scaffold and optimize the interactions by directed evolution. B) Construct multivalent analogues of our beta-sheet inhibitors that can engage gp120 trimers and possibly spikes with very high-affinity that may surpass binding constants of known inhibitors of the gp120/CD4 interaction. C) Finally, we will test the hypothesis that our CD4 mimetics and their multivalent analogs can recruit endogenous bystander antibodies, which will prevent CCR5 and CXCR4 mediated viral entry by a unique steric blockage mechanism. A battery of cellular, biochemical, and biophysical assays will verify the success of our three interconnected approaches towards viral entry inhibitors and in the long-term provide new mini-protein therapeutics for antiviral therapy.

描述（申请人提供）：HIV 感染取决于 gp120 与人 CD4 受体的协调结合，随后 gp120 发生显着的构象重组，从而导致与 CCR5 或 CXCR4 受体的结合。鉴于基于 gp120 的多价 CD4-IgG2 抑制剂最近取得成功，并已进入 II 期临床试验，通过靶向 gp120 直接干扰病毒生命周期是一种有前途的治疗方法。此外，最近发表的gp120与CD4复合的晶体结构表明如何利用基于β折叠的抑制剂支架来消除gp120/CD4相互作用并从而消除病毒进入。我们最近开发了一种新的方法来呈现稳定的β-折叠以识别蛋白质表面。我们的热稳定 β 片层支架可以通过螺旋面结合人类免疫球蛋白，同时用其 β 片层面结合靶蛋白 (gp120)。根据这些观察结果和我们的经验，我们的长期目标是通过基于我们的微型蛋白质支架创建 CD4 模拟物，提供针对病毒进入的新方法。我们的具体目标是：A) 利用计算机引导的对接将 CD4 上的 gp120 结合残基移植到我们的 β-折叠支架上，并通过定向进化优化相互作用。 B)构建我们的β-片层抑制剂的多价类似物，其可以以非常高的亲和力与gp120三聚体和可能的尖峰结合，该亲和力可能超过已知的gp120/CD4相互作用抑制剂的结合常数。 C) 最后，我们将测试我们的 CD4 模拟物及其多价类似物可以招募内源性旁观者抗体的假设，这将通过独特的空间阻断机制阻止 CCR5 和 CXCR4 介导的病毒进入。一系列细胞、生物化学和生物物理测定将验证我们针对病毒进入抑制剂的三种相互关联的方法的成功，并从长远来看为抗病毒治疗提供新的微型蛋白疗法。