METALLOPEPTIDES OF GP41 IN HIV-1 FUSION INHIBITION

描述（由申请人提供）：该提案的重点是开发针对人类免疫缺陷病毒1型的进入抑制剂，靶向病毒融合蛋白gp41。Gp41通过高度保守的线圈结构域介导与哺乳动物宿主细胞的融合。假设结合到该结构域的化合物将通过阻止融合所需的gp41构象重排而起到融合抑制剂的作用。由于该结构域在溶液中不容易接近，因此阻碍了gp41卷曲线圈抑制剂的发现，该结构域被埋在完整的gp41中，并且当从蛋白质的其余部分切除时不稳定。我们通过在gp41肽的n端共价连接的双齿联吡啶基开发了一种稳定的卷曲线圈结构。将过渡金属离子加入到联吡啶化肽中，通过形成八面体三联吡啶络合物，稳定了卷曲线圈的三聚体螺旋结构，并消除了疏水肽的非特异性聚集。本提案的目标是利用各种金属离子的探针特性来建立与线圈结合的抑制剂的筛选和结构分析。1)利用核磁共振结构测定技术，开发并分析了代表gp41内线圈的离散金属离子配位结构。2)构建体将用于开发和改进荧光结合试验，以检测与卷曲线圈结合的化合物。3)进行文库筛选，并通过体外合胞体和病毒感染性试验分析成功命中。在这些特定目标中使用的金属离子将根据需要选择具有抗磁性、顺磁性或荧光特性，以便在生物物理分析中使用。这项提议的长期目标是开发有前途的非肽药物候选HIV-1融合抑制。

DESCRIPTION (provided by applicant): This proposal focuses on the development of entry inhibitors against Human Immunodeficiency Virus Type-1, by targeting the viral fusion protein gp41. Gp41 mediates fusion to mammalian host cells via a highly conserved coiled coil domain. The hypothesis is that compounds that bind to this domain will act as fusion inhibitors by preventing the gp41 conformational rearrangement required for fusion. Discovery of inhibitors to the gp41 coiled coil is hampered by the fact that this domain is not readily accessible in solution, being buried in intact gp41 and unstable when excised from the rest of the protein. We have developed a stabilized coiled coil construct through the use of bidentate bipyridyl groups covalently attached at the N-termini of gp41 peptides. The addition of transition metal ion to the bipyridylated peptides stabilizes the trimeric helical structure of the coiled coil, through the formation of an octahedral tris-bipyridyl complex, and removes non-specific aggregation of the hydrophobic peptide. The goal of this proposal is to make use of the probe properties of various metal ions to establish a screening and structural assay for inhibitors which bind to the coiled coil. 1) Discrete metal ion - coordinated constructs which represent the gp41 inner coiled coil will be developed and analyzed by NMR structure determination. 2) Constructs will be used in the development and refinement of a fluorescence binding assay to detect compound binding to the coiled coil. 3) Library screening will be conducted and successful hits analyzed with in vitro syncytium and viral infectivity assays. The metal ion used in each of these specific aims will be selected to have diamagnetic, paramagnetic or fluorescent properties, as needed, to be utilized in the biophysical assays. The long term goal of this proposal is to develop promising non-peptide drug candidates for HIV-1 fusion inhibition.