Structure-based discovery and development of HIV-1 gp41 fusion inhibitors

描述(由申请人提供)：本提案描述了有效对抗人类免疫缺陷病毒(HIV-1)融合的低分子量gp41抑制剂的发现和开发。融合抑制剂在阻断艾滋病毒传播和防止疾病进展方面具有良好的特性，因为它们阻止健康细胞的初始感染和感染的细胞间传播。然而，目前还没有小分子的融合抑制剂，而多肽融合抑制剂具有成本高、易发生蛋白降解等缺点。这一应用的目的是开发与gp41高亲和力结合的小分子先导化合物，防止N-螺旋结构域和C-螺旋结构域的结合，这是融合反应的中心特征。该提案实施了一种基于结构的系统方法来开发融合抑制剂，具体目标有三：(1)将通过开发荧光、核磁共振和生物分析来检测和定量结合，检查聚焦于已知疏水口袋或其周围的gp41 N-螺旋线圈的模块区段是否有小分子结合。(2)将开发新的核磁共振方法来获得定义结合配体构象和取向的显式结构约束，为计算机模拟和合成改进的缓蚀剂提供定量的构效关系数据。(3)具有模拟肽骨架的化合物在疏水口袋中表现出良好的结合特性，并将成为制备新型抑制剂的设计策略的基础，使用结合、结构和生物测试进行评估和优化。在分析应用过程中获得的其他或相邻的命中和结构数据将被合并到前导优化过程中。细胞-细胞融合和病毒感染性分析将用于提供结合研究的确证，并确保候选铅具有所需的体外特性。这种方法将允许测试中心假设，即结合到疏水口袋并潜在地延伸到疏水口袋附近凹槽中的小分子可以发展成高度有效的融合抑制剂，与多肽相媲美。所描述的研究将提供有效的方法来检测新一代的抑制剂，并获得其与gp41络合物的结构信息。因此，它们将有助于对gp41抑制机制的了解空白，因为目前还没有描述小分子-gp41复合体细节的实验数据。这项建议的意义在于，它有可能特别有助于HIV-1融合抑制剂的开发，并有助于开发适用于使用与HIV-1类似的融合机制的广泛的1类病毒的技术，这是与NIH加强治疗人类疾病的基础知识的使命相关的更广泛的目标。该项目的长期目标是产生抗HIV-1融合的活性药物类化合物，适合用于治疗或杀微生物剂，可用于对抗或预防每年在全球发生的数百万新感染。 与公共卫生相关：艾滋病毒感染的流行仍然是一个重大的公共卫生问题，这是由于对当前治疗方法产生抗药性的病毒株的发展，以及由于病毒从不知道自己被感染的个人身上传播。这项提议寻求应用结构辅助药物设计来开发低分子HIV融合抑制剂，以防止病毒进入和导致免疫缺陷的细胞间传播。这类药物将在杀菌剂中用于预防感染，或者可以口服治疗感染，作为目前使用的融合抑制剂Enfuvitid(R)的替代药物，后者必须静脉给药。

DESCRIPTION (provided by applicant): This proposal describes the discovery and development of low molecular weight gp41 inhibitors effective against Human Immunodeficiency Virus (HIV-1) fusion. Fusion inhibitors possess excellent characteristics for interrupting HIV transmission and preventing disease progression, since they block initial infection of healthy cells and cell-to-cell spread of infection. Yet there are currently no small molecule inhibitors of fusion, and peptide fusion inhibitors possess some undesirable characteristics such as high cost and susceptibility to proteolysis. The objective of this application is to develop small molecule lead compounds which bind to gp41 with high affinity, preventing the association of N- and C-helical domains that is a central feature of the fusion reaction. The proposal implements a systematic structure-based approach to fusion inhibitor development in three Specific Aims: (1) Modular subsections of the gp41 N-helical coiled coil focused on or around a known hydrophobic pocket will be examined for small molecule binding, through the development of fluorescence, NMR and biological assays to detect and quantitate binding. (2) Novel NMR methods for obtaining explicit structural constraints defining bound ligand conformation and orientation will be developed, providing quantitative structure-activity relationship data for computer modeling and synthesis of improved inhibitors. (3) Compounds with peptidomimetic scaffolds have shown promising binding characteristics in the hydrophobic pocket and will form the basis of design strategies for preparing novel inhibitors, using binding, structural and biological assays for evaluation and optimization. Additional or adjacent hits and structural data obtained during assay application will be incorporated into the lead optimization process. Cell-cell fusion and viral infectivity assays will be applied to provide corroboration of binding studies and to ensure that lead candidates have the desired in vitro properties. This approach will allow testing of the central hypothesis that small molecules binding to the hydrophobic pocket and potentially extending into grooves adjacent to the hydrophobic pocket can be developed into highly potent fusion inhibitors, rivaling peptides. The studies described will provide efficient methods to detect new generations of inhibitors and obtain structural information on their complexes with gp41. Thus they will contribute to a gap in knowledge of the mechanism of gp41 inhibition, since there are currently no experimental data describing the details of small-molecule - gp41 complexes. The significance of this proposal lies in its potential to contribute specifically to HIV-1 fusion inhibitor development and also to the development of techniques applicable to a wide range of Class 1 viruses which use a similar fusion mechanism to HIV-1, a broader goal relevant to NIH's mission of enhancing fundamental knowledge to treat human disease. The long-term goal of the project is to generate drug-like compounds active against HIV-1 fusion, suitable for therapeutic or microbicide use, which could be used to counter or prevent the millions of new infections that occur annually worldwide. PUBLIC HEALTH RELEVANCE: The prevalence of HIV infection remains a significant public health problem due to the development of viral strains resistant to current treatments, and due to spread of the virus from individuals who are unaware they are infected. This proposal seeks to apply structure-aided drug design to develop low molecular weight HIV fusion inhibitors which prevent viral entry and the cell-to-cell spread that causes immune deficiency. Drugs in this class would be useful in microbicides to prevent infection, or could be given orally to treat infection as an alternative to the currently used fusion inhibitor Enfuvirtide(R), which must be given intravenously.