Development of a High Throughput FRET-based screening assay for the identificatio

描述（由申请人提供）：HIV-1/艾滋病流行病继续在全世界蔓延，尽管为控制它做出了相当大的努力。2006年，约有4000万人感染艾滋病毒，比2004年多260万人，撒哈拉以南非洲和印度次大陆是全球流行病的重灾区。然而，在北美和西欧，抗逆转录病毒药物复方疗法的出现已证明在控制HIV-1感染方面非常成功，并导致艾滋病发病率和死亡率大幅下降。尽管取得了这些成功，但抑制HIV-1复制的新目标仍是非常可取的，而且需要新的药物来解决日益严重的抗逆转录病毒药物耐药性问题。慢病毒，包括HIV-1，具有比其他逆转录病毒长得多的胞质结构域的跨膜糖蛋白是不寻常的，这表明在病毒的生命周期中起着重要作用。几项研究表明，gp 41胞质（gp 41 c）结构域对Env掺入病毒体产生影响，其中gp 41 c结构域与Gag的基质（p17）部分的相互作用对于有效掺入至关重要。这种相互作用由宿主细胞因子，47 kDa的尾相互作用蛋白（TIP 47）介导。TIP 47的细胞水平沉默，或a？已经发现在用于产生病毒的细胞中的半乳糖苷酶-gp 41 c融合蛋白对Env掺入具有负面影响，产生感染性大大降低的病毒体。鉴于这些观察结果，我们认为破坏HIV-1 gp 41 c结构域-TIP 47和TIP 47-基质相互作用中的一种或两种将导致产生感染性较低的病毒，并且这些相互作用是治疗干预的可行靶标。此外，参与这些相互作用的基序在HIV-1亚型中广泛保守，这表明靶向这些以前未开发的相互作用可以产生广谱抗逆转录病毒药物，该药物可能能够抵抗HIV-1的突变性。因此，我们建议进行试点研究，以调查这一假设。提出了一种两层研究方法，包括通过表面等离子体共振（SPR）进行诱变和相互作用分析，以确定相互作用组分之间的界面（具体目标1），然后基于该表位鉴定，开发基于FRET的测定法，以允许具有成本效益的高通量筛选破坏这些相互作用的化合物（具体目标2）。除了产生可用于TIP 47-Matrix和TIP 47-gp 41 c结构域复合物的结构表征的试剂之外，所提出的研究将为抑制性化合物鉴定提供基础。我们希望，这些结果和后续研究的结果将共同将这些相互作用定义为可行的治疗靶点，并将推动针对这些复合物的新型抑制剂的设计。在西方世界，抗逆转录病毒药物联合疗法已被证明在控制HIV-1感染方面非常成功。尽管取得了这一成功，但抑制HIV-1复制的新靶点是非常可取的，需要新的药物来解决日益严重的耐药性问题。本申请中提出的实验将作为一个平台，探索HIV-1生命周期中以前未开发的过程的治疗潜力。

DESCRIPTION (provided by applicant): The HIV-1/AIDS epidemic continues to spread worldwide, despite considerable efforts to control it. In 2006 approximately 40 million people were living with HIV, 2.6 million more than in 2004, with sub-Saharan Africa and the Indian subcontinent bearing the brunt of the global epidemic. In North America and Western Europe, however, the advent of combination antiretroviral drug regimens have proven remarkably successful at controlling HIV-1 infection and have resulted in significant reductions in morbidity and mortality from AIDS. Despite these successes, new targets for inhibition of HIV-1 replication are highly desirable, and new drugs will be needed to combat the rising problem of anti-retroviral drug resistance. Lentiviruses, including HIV-1, are unusual in having transmembrane glycoproteins with much longer cytoplasmic domains than other retroviruses, suggesting an essential role in the lifecycle of the virus. Several studies have demonstrated that the gp41 cytoplasmic (gp41c) domain exerts an effect on the incorporation of Env into virions, with the interaction of the gp41c domain with the Matrix (p17) portion of Gag being essential for effective incorporation. This interaction is mediated by a host cell factor, Tail Interacting Protein of 47kDa (TIP47). Silencing of cellular levels of TIP47, or overproduction of a ?-galactosidase-gp41c fusion protein in cells used to produce virus have been found to have a negative effect on Env incorporation, producing virions with greatly reduced infectivity. Given these observations, we believe that disruption of either or both the HIV-1 gp41c domain- TIP47 and TIP47-Matrix interactions will result in the production of less infectious virus and that these interactions are viable targets for therapeutic intervention. Moreover, motifs involved in these interactions are broadly conserved across HIV-1 subtypes, suggesting that targeting of these previously unexploited interactions could yield a broad-spectrum antiretroviral agent that may be able to withstand the mutability of HIV-1. Therefore, we propose pilot studies to allow the investigation of this hypothesis. A two-tiered research approach is proposed involving mutagenesis and interaction analysis by surface plasmon resonance (SPR) to determine interfaces between interacting components (Specific Aim 1), and then based upon this epitope identification, development of a FRET-based assay to allow the cost-effective, high-throughput screening of compounds that disrupt these interactions (Specific Aim 2). The proposed studies will provide the foundation for inhibitory compound identification, in addition to generating reagents that could be used for the structural characterization of the TIP47-Matrix and TIP47-gp41c domain complexes. It is our hope that, collectively, these results and the results from subsequent studies will define these interactions as viable therapeutic targets and will drive the design of novel inhibitors targeting these complexes. Combination antiretroviral drug regimens have proven remarkably successful at controlling HIV-1 infection in the Western world. Despite this success, new targets for inhibition of HIV-1 replication are highly desirable, and new drugs are needed to combat the rising problem of drug resistance. The experiments proposed in this application will serve as a platform to explore the therapeutic potential of a previously unexploited process in the HIV-1 lifecycle.