Effect of Cdk inhibitor on HIV-1 replication

描述(申请人提供)：目前世界上有4000-1亿人感染了人类免疫缺陷病毒(HIV)，根据世界卫生组织的估计，全世界每天有近16,000例新感染病例。高效抗逆转录病毒疗法(HAART)的引入大大降低了艾滋病相关的发病率和死亡率。不幸的是，多达25%的患者因治疗失败(无法将艾滋病毒复制抑制到低于目前的检测极限，50拷贝/毫升)、毒副作用或在治疗的头几个月内没有依从性，而停止了最初的HAART方案。因此，今天人们普遍认为，艾滋病毒-1治疗的成功将需要靶向其他艾滋病毒和/或宿主细胞蛋白。HIV所致疾病的发病机制是复杂的、多因素的。一些关键的艾滋病毒和细胞蛋白被指定为感染过程中所必需的，包括反式激活因子TAT。缺乏TAT的病毒克隆不会在体外或体内复制到高滴度。此外，潜伏感染的细胞在各种刺激下的细胞激活(细胞从G0进入细胞周期的GI/S阶段的过程)会导致病毒表达，然后形成后代。在GO静止期细胞中，HIV-1长末端重复序列(LTR)的最小激活转录发生在细胞中。我们先前已经证明，在潜伏感染的细胞中，HIV-1可以下调天然的细胞周期蛋白依赖性激酶(CDK)抑制剂，进而能够控制CDK2/Cyclin E复合体等主要的CDK靶点。因此，抑制Gi/S激酶，可能是抑制HIV-1复制的一个可能的靶点。更重要的是，CDK2和Cyclin E对正常(如CDK2“”或Cyclin E‘a小鼠)、非癌细胞的生长不是必需的，因此，使用针对CDK2/Cyclin E复合体的抑制剂可能是在感染细胞中抑制HIV-1的可行选择。我们目前的初步数据表明，CDK2/Cyclin E可以磷酸化RNA PolIICTD，可以在HIV-1延伸复合体中发现，重组CDK2/Cyclin E在重组转录实验中刺激TAT依赖的HIV-1转录，免疫耗竭核提取液中的CDK2/Cyclin E阻止TAT依赖的转录。此外，RNAi实验表明，CDK2下调影响HIV-1，但不影响HTLV-1、CMV或IgH启动子。在这里，我们建议使用一种三磷酸腺苷类似物和一种新的衍生物来抑制CDK活性。这些药物在HIV-1感染细胞中的靶点是CDK2/细胞周期蛋白E复合体，其IC50值为0.009-0.15um。我们的具体目标包括：目标1：CyC202及其衍生物对HIV-1延伸因子占据和剪接的影响。为了确定抑制的机制，将使用芯片分析方法在体内测试转录延伸因子在药物存在的情况下的占据情况。使用HIV-RTTA(KWK和KYK)、PBMCs和TAT变体(野生型、C22G和L43A)，在体外和体内评估CyC202及其衍生物对HIV-1单个或多个剪接信息的影响。目的2：细胞色素C202及其衍生化合物#4对HIV-1变异株的影响及可能的细胞毒性。药物治疗对HIV-1变异体的影响将在野生型和重组病毒以及LTR和TAT转基因中进行测试。使用微阵列确定细胞基因表达在CyC202或衍生产品#4处理后的任何显著变化。因此，目前的提案旨在确定在体外和体内治疗HIV-1感染细胞的药物如ATP类似物是否是一个可行的选择。

DESCRIPTION (provided by applicant): There are currently 40-100 million individuals in the world infected with human immunodeficiency virus (HIV), and nearly 16,000 new infections occur worldwide each day based on World Health Organization estimates. The introduction of highly active anti-retroviral therapy (HAART) has led to a significant reduction in AIDS-related morbidity and mortality. Unfortunately, up to 25% of patients discontinue their initial HAART regimen because of treatment failure (inability to suppress HIV viral replication to below the current limit of detection, 50 copies/mL), toxic effects or non-compliance within the first few months of therapy. Therefore, today it is widely believed that the success in HIV-1 treatment will require targeting of other HIV and/or host cellular proteins. 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 liters. Also, cellular activation (a process where cells go from G0 to the Gi/S stage of the cell cycle) of latently infected cells by various stimuli leads to viral expression followed by progeny formation. Minimal activated transcription from the HIV-1 long terminal repeat (LTR) occurs in GO quiescent cells. We previously have shown that HIV-1 in latently infected cells can down modulate the natural cyclin dependent kinase (cdk) inhibitors, and in turn is able to control the primary cdk targets such as cdk2/cyclin E complex. Therefore, inhibition of the Gi/S kinases, could be a possible target for inhibition of HIV-1 replication. More importantly, cdk2 and cyclin E are not essential for the growth of normal (as seen in cdk2"" or cyclin E'A mice), non-cancerous cells, and therefore, use of inhibitors against the cdk2/cyclin E complex may pose a viable option to inhibit HIV-1 in infected cells. Our current preliminary data indicates that cdk2/cyclin E could phosphorylate the RNA Pol IICTD, can be found in the HIV- 1 elongation complexes, recombinant cdk2/cyclin E stimulates Tat-dependent HIV-1 transcription in a reconstituted transcription assay and immunodepletion of cdk2/cyclin E from nuclear extracts blocks Tat-dependent transcription. Also, RNAi experiments show that cdk2 downregulation affects HIV-1, but not HTLV-1, CMV, or IgH promoters. Here, we propose to use an ATP analog and a new derivative which can inhibit cdk activity. The target of these drugs in HIV-1 infected cells is the cdk2/cyclin E complex at an IC50 of 0.009-0.15 uM. Our specific aims include: Aim 1: Effect of CYC202 and its derivative on HIV-1 elongation factor occupancy and splicing. To define the mechanism of inhibition, transcription elongation factor occupancy in presence of drugs will be tested in vivo using ChIP assays. The effect of CYC202 and its derivative on HIV-1 singly or multiply spliced messages will be evaluated in vitro and in vivo using HIV-rtTA (KWK and KYK), PBMCs and Tat variants (wild type, C22G, and L43A). Aim 2: Effect of CYC202 and its derivative compound #4 on HIV-1 variants, and possible cell toxicity. Effect of drug treatment on HIV-1 variants will be tested in wild type and recombinant viruses as well as LTR and Tat transfections. Determine any significant alterations in cellular gene expression following CYC202 or derivative #4 treatments using Microarrays. Therefore, the current proposal is aimed at determining whether drugs such as ATP analogs are a viable option in treating HIV-1 infected cells both in vitro and in vivo.