Isolation and Characterization of Aptamers Targeted to Inhibit HIV Protease Matur

描述(由申请人提供)：HIV蛋白酶是一种公认的抑制病毒复制的靶点，有几种FDA批准的药物正在用于降低HIV蛋白酶的活性。然而，这些药物只针对HIV蛋白酶的催化部位，而且在接受治疗的患者中不断出现耐药菌株，这突显了发现和开发具有新作用机制的治疗药物的迫切需要。在这里，我们试图探索一种非传统的方法，旨在干扰蛋白酶成熟，这是一种病毒特异性的自动处理反应，负责产生活性蛋白酶。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水解酶具有固有的但非常有限的活性，并且只有在成熟的蛋白水解酶从前体中释放出来后，它才能完全与蛋白水解酶相关。广泛的研究表明，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，前体二聚体与释放氨基末端的裂解结合在一起是蛋白酶成熟的关键。我们最近发现，将表面带正电的残基(H69)改变为带负电的氨基酸，也会破坏大肠杆菌和转基因哺乳动物细胞中的蛋白酶成熟。因此，我推测，通过使用高亲和力分子来掩盖对自动处理反应至关重要的区域的前体，将干扰蛋白酶的成熟，并抑制完全活性的蛋白酶的产生。为了测试这一概念的可行性，我们计划分离蛋白酶前体的RNA适配子(目标1)。其中，高亲和力(Kd&lt；1&lt；M)适配子将被进一步评估，以确定干扰体外和转基因细胞中蛋白酶成熟的候选适配子(目标2)。与公共卫生相关：蛋白酶成熟是病毒特有的过程，负责产生活性的艾滋病毒蛋白酶，这是艾滋病毒复制绝对必需的一种不可或缺的酶。目前，还没有靶向阻断或抑制这一过程的药物。我们的目标是建立一种有效的方法来干扰蛋白水解酶的成熟，以开发新的抗HIV药物。

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.