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: 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
• 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

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gp120与人类CD4受体的协调结合，随后gp120发生重大构象重组，从而与CCR5或CXCR4受体结合。考虑到最近基于CD4-lgG2的多价gp120抑制剂的成功，该抑制剂已进入II期临床试验，通过靶向gp120直接干扰病毒生命周期是一种很有前景的治疗方法。此外，最近发表的gp120与CD4复合物的晶体结构表明，如何利用基于β -薄片的抑制剂支架来消除gp120/CD4相互作用，从而阻止病毒进入。我们最近开发了一种新的方法来提供稳定的β -片来识别蛋白质表面。我们的耐热β -片支架可以通过螺旋面结合人类免疫球蛋白，同时将靶蛋白（gp120）与其β -片面结合。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CD4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)利用计算机引导对接将CD4的gp120结合残基移植到我们的β -片支架上，并通过定向进化优化相互作用。B)构建我们的β片抑制剂的多价类似物，可以结合gp120三聚体和可能的尖峰，具有非常高的亲和力，可能超过已知的gp120/CD4相互作用抑制剂的结合常数。C)最后，我们将验证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，这些抗体将通过独特的位阻机制阻止CCR5和CXCR4介导的病毒进入。一系列细胞、生化和生物物理分析将验证我们的三种相互关联的病毒进入抑制剂方法的成功，并在长期内为抗病毒治疗提供新的微型蛋白治疗方法。