Structured Protein Scaffolds for Phage-Display

描述(申请人提供)：HIV感染依赖于gp120与人的CD4受体的协调结合，随后gp120的显著构象重组，导致与CCR5或CXCR4受体的结合。靶向gp120直接干扰病毒生命周期是一种很有前途的治疗方法，考虑到最近一种基于多价CD4-lgG2的gp120抑制剂的成功，该抑制剂已进入II期临床试验。此外，最近发表的gp120与CD4复合的晶体结构表明，基于Beta-Sheet的抑制物支架可以用于消除gp120/CD4相互作用，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，用于识别蛋白质表面。我们的耐热β-折叠支架可以通过螺旋面结合人类免疫球蛋白，同时将目标蛋白(Gp120)与其β-折叠表面结合。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模拟分子，为靶向病毒进入提供新的方法。我们的具体目标是：a)利用计算机引导的对接将gp120结合残基从CD4嫁接到我们的Beta-Sheet支架上，并通过定向进化优化相互作用。B)构建我们的Beta-Sheet抑制剂的多价类似物，这些类似物可以与gp120三聚体结合，并可能以非常高的亲和力产生尖峰信号，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，这将通过一种独特的空间阻断机制来阻止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三种相互关联的方法在病毒进入抑制剂方面的成功，并从长远来看，为抗病毒治疗提供新的微型蛋白质疗法。

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.