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

开发基于高通量 FRET 的筛选测定法，用于鉴定

• 批准号: 7554833
• 负责人: Simon Cocklin
• 金额: $ 7.5万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7554833
• 关键词: Acquired Immunodeficiency Syndrome; Africa South of the Sahara; Anti-Retroviral Agents; Antiretroviral drug resistance; Binding; Biological Assay; Biology; Cations; Cell physiology; Cells; Chimeric Proteins; Complex; Condition; Coupled; Crystallization; Cytoplasmic Tail; Development; Disruption; Dissection; Drug resistance; Epidemic; Epitopes; Experimental Designs; Fluorescence Resonance Energy Transfer; Foundations; Future; Gagging; Galactosidase; Glycoproteins; Goals; HIV; HIV-1; IGF Type 2 Receptor; Infection; Investigation; Knowledge; Life; Life Cycle Stages; Mediating; Methodology; Morbidity - disease rate; Mutagenesis; North America; Pharmaceutical Preparations; Pilot Projects; Play; Positioning Attribute; Process; Production; Proteins; Reagent; Research; Retroviridae; Role; Screening procedure; Subfamily lentivirinae; Surface Plasmon Resonance; T-Lymphocyte; Tail; Testing; Therapeutic; Therapeutic Intervention; Treatment Protocols; Viral; Virion; Virus; Western Europe; Western World; base; cell motility; cost; design; high throughput screening; inhibitor/antagonist; mortality; novel; particle; research study; small molecule; success; therapeutic target; transmission process

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.

描述(申请人提供)：艾滋病毒-1/艾滋病疫情继续在世界范围内传播，尽管作出了相当大的努力来控制它。2006年，约有4000万艾滋病毒携带者，比2004年多260万人，其中撒哈拉以南非洲和印度次大陆首当其冲地受到全球流行病的影响。然而，在北美和西欧，联合抗逆转录病毒药物疗法的出现已证明在控制艾滋病毒-1感染方面非常成功，并大大降低了艾滋病的发病率和死亡率。尽管取得了这些成功，但抑制HIV-1复制的新目标是非常可取的，需要新的药物来应对日益严重的抗逆转录病毒耐药性问题。慢病毒，包括HIV-1，具有比其他逆转录病毒更长的跨膜糖蛋白结构域，这表明在病毒的生命周期中扮演着重要的角色。一些研究表明，gp41细胞质(Gp41c)结构域对Env进入病毒粒子具有影响，gp41c结构域与GAG的基质(P17)部分的相互作用是有效掺入病毒所必需的。这种相互作用是由宿主细胞因子、47 kDa的尾巴相互作用蛋白(TIP47)介导的。研究发现，抑制TIP47的细胞水平或在用于生产病毒的细胞中过量生产β-半乳糖苷酶-gp41c融合蛋白会对Env的掺入产生负面影响，从而产生极大降低感染性的病毒粒子。鉴于这些观察结果，我们认为，破坏HIV-1 gp41c结构域-TIP47和TIP47-Matrix的任何一个或两者的相互作用将导致传染性较低的病毒的产生，这些相互作用是治疗干预的可行靶点。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被利用的相互作用可能产生一种广谱抗逆转录病毒药物，可能能够抵抗HIV-1的突变。因此，我们建议进行先导性研究，以便对这一假设进行调查。提出了一种两级研究方法，包括通过表面等离子共振(SPR)进行突变和相互作用分析，以确定相互作用组分之间的界面(特定目标1)，然后在此表位鉴定的基础上，开发基于FRET的分析方法，以实现经济高效、高通量地筛选破坏这些相互作用的化合物(特定目标2)。所提出的研究将为抑制化合物的鉴定提供基础，此外还将产生可用于TIP47-基质和TIP47-gp41c结构域复合体结构表征的试剂。我们希望，总的来说，这些结果和后续研究的结果将把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体的新型抑制剂的设计。事实证明，在西方世界，联合抗逆转录病毒药物方案在控制艾滋病毒-1感染方面非常成功。尽管取得了这一成功，但抑制艾滋病毒-1复制的新目标是非常可取的，需要新的药物来应对日益严重的耐药性问题。本申请中提议的实验将作为一个平台，探索HIV-1生命周期中以前未被开发的过程的治疗潜力。

项目成果

Discovery of a small-molecule antiviral targeting the HIV-1 matrix protein.

• DOI: 10.1016/j.bmcl.2012.11.041
• 发表时间: 2013-02-15
• 期刊: BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
• 影响因子: 2.7
• 作者: Zentner, Isaac;Sierra, Luz-Jeannette;Maciunas, Lina;Vinnik, Andrei;Fedichev, Peter;Mankowski, Marie K.;Ptak, Roger G.;Martin-Garcia, Julio;Cocklin, Simon
• 通讯作者: Cocklin, Simon

Identification of a small-molecule inhibitor of HIV-1 assembly that targets the phosphatidylinositol (4,5)-bisphosphate binding site of the HIV-1 matrix protein.

• DOI: 10.1002/cmdc.201200577
• 发表时间: 2013-03
• 期刊: ChemMedChem
• 影响因子: 3.4
• 作者: Zentner I;Sierra LJ;Fraser AK;Maciunas L;Mankowski MK;Vinnik A;Fedichev P;Ptak RG;Martín-García J;Cocklin S
• 通讯作者: Cocklin S