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gp 41抑制剂的发现和开发。融合抑制剂具有中断HIV传播和预防疾病进展的优异特性，因为它们阻断健康细胞的初始感染和感染的细胞间传播。然而，目前还没有融合的小分子抑制剂，并且肽融合抑制剂具有一些不期望的特征，例如高成本和对蛋白水解的敏感性。本申请的目的是开发小分子先导化合物，其以高亲和力结合gp 41，防止N-和C-螺旋结构域的缔合，这是融合反应的中心特征。该提案在三个具体目标中实施了融合抑制剂开发的系统性基于结构的方法：（1）通过开发荧光、NMR和生物测定来检测和定量结合，将检查gp 41 N-螺旋卷曲螺旋的模块化子部分集中在已知疏水口袋上或周围的小分子结合。(2)将开发用于获得明确的结构约束定义结合配体构象和取向的新型NMR方法，为计算机建模和合成改进的抑制剂提供定量结构-活性关系数据。(3)具有肽模拟物支架的化合物在疏水口袋中显示出有前途的结合特性，并将形成用于制备新型抑制剂的设计策略的基础，使用结合、结构和生物学测定进行评估和优化。在试验应用过程中获得的额外或相邻命中和结构数据将纳入电极导线优化过程。将应用细胞-细胞融合和病毒感染性试验，以提供结合研究的确证，并确保先导候选物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可能延伸到疏水口袋附近的凹槽中的小分子可以发展成与肽竞争的高效融合抑制剂。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gp 41复合物的结构信息。因此，他们将有助于gp 41抑制机制的知识的差距，因为目前没有实验数据描述的小分子gp 41复合物的细节。该提案的重要性在于其有可能特别有助于HIV-1融合抑制剂的开发，也有助于开发适用于广泛的1类病毒的技术，这些病毒使用与HIV-1相似的融合机制，这是与NIH的使命相关的更广泛的目标，即增强治疗人类疾病的基础知识。该项目的长期目标是产生对HIV-1融合有活性的药物样化合物，适用于治疗或杀微生物剂用途，可用于对抗或预防全球每年发生的数百万新感染。 公共卫生相关性：艾滋病毒感染的流行仍然是一个重大的公共卫生问题，这是由于病毒株的发展对目前的治疗产生了抗药性，以及由于病毒从不知道自己被感染的个人传播。该提案旨在应用结构辅助药物设计来开发低分子量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。这类药物可用于杀微生物剂以预防感染，或者可以口服治疗感染，作为目前使用的融合抑制剂Enfuvirtide（R）的替代品，Enfuvirtide（R）必须静脉注射。

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.