HIV-1 Inhibition using Tat Peptide Derivatives

描述（由申请人提供）：人类免疫缺陷病毒（HIV）是艾滋病的病原。HIV诱发疾病的发病机制是复杂的、多因素的。几个关键的HIV和细胞蛋白被指定为感染过程所必需的，包括反激活子Tat。缺乏Tat的病毒克隆不能在体外或体内复制到高滴度。目前的建议旨在扩展我们之前的结果，这些结果是在过去四年中获得的，关于Tat肽衍生物对HIV-1的抑制作用。先前的特定目标及时成功地实现了，其中包括Tat肽抑制剂的靶标确定，Tat核心肽衍生物抑制活性的最低结构要求，以及确定对少数HIV-1分离株的抑制范围。更多的初步数据包括，以显示这些Tat肽抑制剂的目标，使用体外和体内的分析。这些肽也在小范围内进行了环化，并成功地用于动物的非特异性毒性试验和生物利用度试验。因此，我们建议继续我们最初的目标，即开发Tat/TAR复合物特异性的HIV-1 Tat抑制剂。我们将通过使用新的环化策略，包括使用cys4和cys2化合物，来优化短Tat肽的肽环化。Tat肽也将含有Tat或SV40 NLS序列，以实现高效递送。除生物抑制活性外，还将利用各种生化和病毒学方法。为了确定Tat肽的抑制作用，我们将测定Tat肽对包括耐药菌株在内的各种HIV- 1分离株的抑制活性。我们将评估Tat肽对复制t细胞和pbmc的细胞毒性，并确定这些化合物对不同倾向的各种HIV-1野生型、逆转录酶和蛋白酶抗性病毒的抑制活性。此外，我们将利用SCID-Hu Thy/Liv小鼠模型来测试体内毒性和病毒抑制作用。最后，为了确定这些抑制剂是否具有任何非特异性细胞毒性，我们将利用Tat肽在处理细胞中的基因表达谱。这将通过在PBMC感染细胞中使用聚类和数据分析方法来完成。数据验证最初将包括计算机预测算法、独立阵列数据集和使用概率理性模型、GenMAPP和Pubgene的功能分析。

DESCRIPTION (provided by applicant): Human Immunodeficiency virus (HIV) is the etiologic agent of AIDS. The pathogenesis of HIV -induced disease is complex and multifactoral. Several key HIV and cellular proteins have been assigned to be necessary for the course of infection including the transactivator Tat. Viral clones deficient in Tat will not replicate in vitro or in vivo to high titers. The current proposal aims to extend our previous results, which were obtained during the past four years on the effect of Tat peptide derivatives in inhibition of HIV-1. Previous specific aims were successfully met in a timely manner, and they included determination of the target(s) of the Tat peptide inhibitor, defining the minimum structural requirement for the inhibitoryactivity of the Tat core peptide derivative, and determining the range of inhibition on a few HIV-1 isolates. More preliminary data is included to show the target of these Tat peptide inhibitors using in vitro and in vivo assays. Tat peptides were also cyclized at small scale and used in non-specific toxicity assays as well as bioavailability assays in animals with great success. Therefore, we have proposed to continue our initial goal, which was to develop HIV-1 Tat inhibitors that are specific to the Tat/TAR complex. We will optimize peptide cyclization of the short Tat peptide by using new cyclization strategies includingthe use of Cys 4 and Cys 2 compounds. Tat peptides will also contain either Tat or SV40 NLS sequences for efficient delivery. Biological inhibitory activities in addition various biochemical and virological methods will be utilized. To define the breath of Tat peptide inhibition, we will determine the inhibitoryactivity of the Tat peptide on various HIV- 1 isolates including drug-resistant strains. We will assess the cytotoxicity of Tat peptides on replicating T-cells and PBMCs as well as determining the inhibitory activity of these compounds on various HIV-1 wild type, reverse transcriptase and protease resistant viruses with different tropisms. Also, we will utilize the SCID-Hu Thy/Liv mice model to test toxicity and viral inhibition in vivo. Finally, to define whether these inhibitors have any non-specific cellular toxicity, we will utilize gene expression profiling of Tat peptides in treated cells. This will be accomplished through the use of clustering and data analysis methods in PBMC infected cells. Validation of the data will initially include in silico prediction algorithms, independent array data sets, and functional assays using probabilistic rational models, GenMAPP, and Pubgene.