Isolation and Characterization of Aptamers Targeted to Inhibit HIV Protease Matur

描述（由申请人提供）：HIV蛋白酶是一种很好的抑制病毒复制的靶标，并且有少数FDA批准的药物被用于降低HIV蛋白酶的活性。然而，这些药物仅针对HIV蛋白酶的催化位点，并且在接受治疗的患者中不断出现耐药菌株，这表明迫切需要发现和开发具有新型作用机制的治疗方法。在这里，我们试图探索一种旨在干扰蛋白酶成熟的非常规方法，蛋白酶成熟是一种负责产生活性蛋白酶的病毒特异性自动加工反应。在被感染的细胞中，HIV蛋白酶最初作为Gag-Pol前体的一部分被翻译。这种内嵌的未成熟蛋白酶具有内在的但非常有限的活性，完全的蛋白水解活性仅在成熟蛋白酶从前体释放后才与之相关。广泛的研究表明成熟的蛋白酶以稳定的二聚体形式存在，而蛋白酶的前体以单体为主，前体二聚与释放氨基末端的裂解对蛋白酶的成熟至关重要。我们最近发现，在大肠杆菌和转染的哺乳动物细胞中，将带正电的表面残基（H69）改变为带负电的氨基酸也可以阻止蛋白酶的成熟。因此，我假设，通过使用高亲和力分子来掩盖对自动加工反应至关重要的区域的前体，将干扰蛋白酶成熟并抑制充分活性蛋白酶的产生。为了测试这一概念的可行性，我们计划分离蛋白酶前体特异性的RNA适体（Aim 1）。其中，高亲和力（Kd < 1 <M）的适体将被进一步评估，以确定在体外和转染细胞中干扰蛋白酶成熟的候选适体（目的2）。公共卫生相关性：蛋白酶成熟是一种病毒特异性过程，负责产生活跃的HIV蛋白酶，这是HIV复制所绝对需要的一种不可或缺的酶。目前，还没有药物可以阻断或抑制这一过程。我们的目标是建立一种有效的干扰蛋白酶成熟的方法，用于开发新的抗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.