Structural Biology of HIV-1 Nef with Host Effectors and Small Molecule Inhibitors

描述(由申请人提供)：HIV-1辅助蛋白Nef对于高效价病毒复制和艾滋病进展是必不可少的。NEF通过宿主细胞信号蛋白包括HCK和其他Src家族激酶(SFK)发挥作用，这表明Nef-SFK复合体存在于所有类型的HIV感染细胞中。我们最近发现了选择性靶向Nef-SFK复合体以及Nef蛋白本身的小分子。这些化合物还以Nef依赖的方式有效地抑制HIV-1的复制，表明它们以HIV相关的方式干扰Nef的功能。因此，这项提议的主要目标是首次解决全长SFK络合物中Nef的X射线晶体结构，并绘制这些络合物中的抑制剂结合位置图。这些结构对于充分理解复合作用的机制是至关重要的，这将在补充功能分析中进行探索。实现这些目标的具体目标如下：目标1.在选择性抑制剂存在的情况下，确定HIV-1Nef-SFK复合体的X射线晶体结构。我们以前的工作表明，Nef变构结合在HCK活性部位诱导了一种独特的构象，为小分子提供了一个特定的靶点。为了测试Nef结合对HCK激动域构象的影响，我们将解决4-氨基取代的二苯基呋喃并嘧啶(DFP)激酶抑制剂存在下Nef-HCK蛋白复合体的晶体结构，我们最近报道的DFP是Nef诱导的HCK活性和HCK活性的选择性抑制剂。 依赖NEF的HIV-1复制。目的：用新发现的Nef功能拮抗剂确定HIV-1 Nef的X射线晶体结构。将HIV-1 Nef与HCK激活相结合，使大规模全自动筛选和发现具有抗逆转录病毒活性的一流Nef拮抗剂成为可能。初步的生物物理研究表明，这些化合物与Nef有直接的纳米分子结合。在这里，我们将通过与HIV-1 Nef共结晶来鉴定这些化合物的结合位点，并通过结合位点突变体的生化研究来验证结构。目的3.测试Nef导向的抗逆转录病毒化合物在HIV复制中的结构模型。基于现有的数据，以及在前两个目标中将获得的新的结构信息，我们将创建预计将抵抗 我们的化合物。这些突变将被改造成HIV-1的主干，它们对HIV-1对每种化合物敏感性的影响将在复制分析中进行评估。此外，我们将根据结构数据生成和测试每个先导化合物的新类似物，以改善溶解性、效力和药理性质。这些研究将提供重要的机制证据，证明这些化合物以结构生物学预测的方式拮抗与HIV-1相关的Nef功能。

DESCRIPTION (provided by applicant): The HIV-1 accessory protein Nef is essential for high-titer viral replication and AIDS progression. Nef works via host cell signaling proteins including Hck and other Src-family kinases (SFKs), suggesting that Nef-SFK complexes are present in all HIV-infected cell types. We have recently identified small molecules that selectively target Nef-SFK complexes as well as the Nef protein itself. These compounds also potently inhibit HIV-1 replication in a Nef-dependent manner, indicating that they interfere with Nef function in an HIV-relevant manner. The major goals of this proposal, therefore, are to solve X-ray crystal structures of Nef in complexes with full-length SFKs for the first time, and to map inhibitor binding sites within these complexes. These structures are critical to a full understanding of the mechanisms of compound action, which will be explored in complementary functional assays. These goals will be pursued with the following Specific Aims: Aim 1. Determine the X-ray crystal structures of HIV-1 Nef-SFK complexes in the presence of selective inhibitors. Our previous work suggests that Nef binding allosterically induces a unique conformation at the Hck active site, providing a specific target for small molecules. To test the impact of Nef binding on the conformation of the Hck kinase domain, we will solve the crystal structures of Nef-Hck protein complexes in the presence of 4- amino-substituted diphenylfuranopyrimidine (DFP) kinase inhibitors, which we recently reported as selective inhibitors of both Nef-induced Hck activity and Nef-dependent HIV-1 replication. Aim 2. Determine the X-ray crystal structures of HIV-1 Nef with newly identified antagonists of Nef function. Coupling of HIV-1 Nef to Hck activation enabled a large-scale fully automated screen and discovery of first-in-class Nef antagonists with antiretroviral activity. Preliminary biophysical studies demonstrate the direct nanomolar binding of these compounds to Nef. Here we will identify the binding sites for these compounds by co-crystallizing them with HIV-1 Nef, and validate the structures through biochemical studies with binding site mutants. Aim 3. Test structural models of Nef-directed antiretroviral compounds in HIV replication. Based on existing data, as well as new structural information to be obtained in the first two Aims, we will create Nef mutants that are predicted to be resistant to the actions of our compounds. These mutations will be engineered into the HIV-1 backbone, and their impact on HIV-1 sensitivity to each compound will be assessed in replication assays. In addition, we will generate and test new analogs of each lead compound based on structural data to improve solubility, potency and pharmacological properties. These studies will provide important mechanistic evidence that the compounds antagonize Nef functions relevant to HIV-1 in a manner predicted by the structural biology.