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，有一些FDA批准的药物用于降低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，并且在治疗下患者中不断出现耐药菌株，这突出了发现和开发具有新作用机制的治疗剂的迫切需要。在这里，我们试图探索一种非常规的方法，旨在干扰蛋白酶成熟，病毒特异性的自动加工反应，负责生产活性蛋白酶。在感染细胞中，HIV蛋白酶最初被翻译为Gag-Pol前体的一部分。这种包埋的未成熟蛋白酶具有内在的但非常有限的活性，并且完全的蛋白水解活性仅在成熟蛋白酶从前体释放后与成熟蛋白酶相关。广泛的研究已经确定，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，并且前体二聚化加上释放氨基末端的切割对于蛋白酶成熟至关重要。我们最近发现，将带正电荷的表面残基（H69）改变为带负电荷的氨基酸也可以消除E. coli和转染的哺乳动物细胞。因此，我假设通过使用高亲和力分子在对自动加工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。为了测试这一概念的可行性，我们计划分离蛋白酶前体（Aim 1）特异性的RNA适体。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，负责产生活性HIV蛋白酶，这是HIV复制绝对需要的不可或缺的酶。目前，还没有药物能够阻断或抑制这一过程。我们的目标是建立一种有效的方法来干扰蛋白酶成熟，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.