Isolation and Characterization of Aptamers Targeted to Inhibit HIV Protease Matur

抑制 HIV 蛋白酶成熟的适体的分离和表征

基本信息

批准号：
7860304
负责人：
CHAOPING CHEN
金额：
$ 18.27万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-05 至 2012-05-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): HIV protease is a well established target for the inhibition of viral replication and there are a handful of FDA- approved drugs that are being used to weigh down HIV protease activity. However, these drugs are exclusively targeted to the catalytic site of HIV protease and constant emergence of drug-resistant strains in patients under the therapy underscores the urgent need for the discovery and development of therapeutics with novel mechanisms of action. Here, we seek to explore an unconventional approach that is aimed to interfere with protease maturation, a viral specific autoprocessing reaction responsible for the production of active protease. In the infected cell, HIV protease is initially translated as part of the Gag-Pol precursor. This embedded immature protease has intrinsic but very limited activity and the full proteolytic activity is only associated with the mature protease after it is released from the precursor. Extensive research has established that mature protease exists as stable dimers while protease precursors are predominantly monomer, and that precursor dimerization coupled with the cleavages releasing the amino terminus is critical for protease maturation. We recently discovered that changing a positively charged surface residue (H69) to negatively charged amino acids also abolished protease maturation in E. coli and transfected mammalian cells. Therefore, I hypothesize that masking the precursor at regions that are critical for the autoprocessing reaction through the use of high affinity molecules would interfere with protease maturation and inhibit the production of fully active protease. In order to test the feasibility of this concept, we plan to isolate RNA aptamers specific to the protease precursor (Aim 1). Among these, high affinity (Kd < 1 <M) aptamers will be further evaluated in order to identify candidate aptamers that interfere with protease maturation in vitro and in transfected cells (Aim 2). PUBLIC HEALTH RELEVANCE: Protease maturation is a virus specific process that is responsible for the production of active HIV protease, an indispensable enzyme that is absolutely required for HIV replication. Currently, there is no drug that is targeted to block or inhibit this process. Our goal here is to establish an effective way to interfere with protease maturation for the development of novel anti-HIV medicines.

描述（由申请人提供）：HIV 蛋白酶是抑制病毒复制的公认靶标，并且有少数 FDA 批准的药物可用于抑制 HIV 蛋白酶活性。然而，这些药物专门针对 HIV 蛋白酶的催化位点，并且接受治疗的患者中不断出现的耐药菌株强调了迫切需要发现和开发具有新作用机制的治疗方法。在这里，我们寻求探索一种非常规方法，旨在干扰蛋白酶成熟，这是一种负责产生活性蛋白酶的病毒特异性自动加工反应。在受感染的细胞中，HIV 蛋白酶最初被翻译为 Gag-Pol 前体的一部分。这种嵌入的未成熟蛋白酶具有内在但非常有限的活性，并且完整的蛋白水解活性仅在其从前体释放后与成熟蛋白酶相关。广泛的研究已经证实，成熟的蛋白酶以稳定的二聚体形式存在，而蛋白酶前体主要是单体，前体二聚化与释放氨基末端的裂解相结合对于蛋白酶的成熟至关重要。我们最近发现，将带正电的表面残基 (H69) 更改为带负电的氨基酸也能消除大肠杆菌和转染的哺乳动物细胞中的蛋白酶成熟。因此，我假设通过使用高亲和力分子在对自动处理反应至关重要的区域掩蔽前体会干扰蛋白酶成熟并抑制完全活性蛋白酶的产生。为了测试这个概念的可行性，我们计划分离针对蛋白酶前体的RNA适体（目标1）。其中，将进一步评估高亲和力（Kd < 1 < M）适体，以鉴定在体外和转染细胞中干扰蛋白酶成熟的候选适体（目标2）。公共卫生相关性：蛋白酶成熟是一种病毒特异性过程，负责产生活性 HIV 蛋白酶，这是 HIV 复制绝对必需的一种不可或缺的酶。目前，还没有药物能够靶向阻断或抑制这一过程。我们的目标是建立一种干扰蛋白酶成熟的有效方法，以开发新型抗艾滋病毒药物。