Structured Protein Scaffolds for Phage-Display

用于噬菌体展示的结构化蛋白质支架

• 批准号: 7209048
• 负责人: INDRANEEL GHOSH
• 金额: $ 28.66万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 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/7209048
• 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

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（由申请人提供）：HIV感染依赖于gp 120与人CD 4受体的协调结合，随后是gp 120的显著构象重组，导致与CCR 5或CXCR 4受体结合。通过靶向gp 120直接干扰病毒生命周期是一种有前途的治疗方法，鉴于最近基于多价CD 4-IgG 2的gp 120抑制剂的成功，其已进展到II期临床试验。此外，最近公布的与CD 4复合的gp 120的晶体结构表明了如何利用基于β折叠的抑制剂支架来消除gp 120/CD 4相互作用，从而消除病毒进入。我们最近开发了一种新方法，用于呈现稳定的β折叠以识别蛋白质表面。我们的热稳定β折叠支架可以通过螺旋面结合人免疫球蛋白，同时用其β折叠面结合靶蛋白（gp 120）。基于这些观察和我们的经验，我们的长期目标是通过基于我们的微型蛋白质支架创建CD 4模拟物来提供靶向病毒进入的新方法。我们的具体目标是：A）利用计算机引导的对接将来自CD 4的gp 120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折叠抑制剂的多价类似物，其可以接合gp 120三聚体并可能以非常高的亲和力尖峰，这可能超过已知的gp 120/CD 4相互作用抑制剂的结合常数。C）最后，我们将测试我们的CD 4模拟物及其多价类似物可以募集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CCR 5和CXCR 4介导的病毒进入。一组细胞、生物化学和生物物理学检测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质疗法。