Mode of action of a new Tat HIV-1 inhibitor

描述(申请人提供)：本项目的目标是阐明皮质抑素A抑制HIV-1转录的机制。人类免疫缺陷病毒I型(HIV-1)是艾滋病(艾滋病)的病原体。艾滋病毒抗逆转录病毒疗法的基础是联合用药，以最大限度地减少可能导致对病毒产生单一耐药性的突变的发展。病毒蛋白Tat是HIV基因表达的有效激活剂，是一个潜在的抗病毒靶点。我们发现皮质抑素A是一种很有前途的抗TAT药物候选药物。皮质酮A是从海绵中分离得到的一种甾体生物碱。我们已经发现二氢皮质酮A(简称CA)是一种非常有效的抑制HIV-1前病毒TAT激活的转录的药物。CA在减少急性感染细胞以及慢性感染培养细胞或新鲜分离的外周血淋巴细胞(PBL)的病毒输出方面非常有效。CA对慢性感染细胞抑制HIV的半数最大有效浓度(EC50)小于0.1 nM，对培养细胞或新鲜分离的PBL的半数细胞毒浓度(CC50)大于20.5M，为CA提供了一个非常有前景的治疗指标。我们已经确定了CA和TAT之间以及与DNA依赖的蛋白激酶(DNA-PK)催化亚基之间的相互作用。在HIV感染后，DNA-PK与TAT紧密结合，通过HIV-1启动子核心的三个Sp1结合位点，促进转录因子Sp1的磷酸化，导致HIV-1转录启动增加。我们的假设是，CA可能通过两种途径抑制HIV的复制：通过抑制TAT-TAR相互作用，从而抑制HIV启动子的RNA聚合酶I(Pol I)的延伸；和/或通过抑制转录因子Sp1的DNA-PK激活和Pol II的转录起始/延伸。这两种途径都会抑制HIV-1的转录激活。我们的具体目标包括：目的1.确定CA是否在体内外抑制HIV-1Tat-TAR相互作用，以及CA是否抑制病毒启动子的转录延伸。评估CA是否抑制TAT介导的Sp1的DNA-PK磷酸化。目的2.对CA的体内毒性进行深入评价。目的3.在人源化小鼠模型中检测CA对HIV-1复制的影响。因此，我们将阐明CA抑制HIV-1转录的机制，并评估其在人源化小鼠中抑制HIV-1复制的潜力。为了生成备用化合物，我们将评估在本项目过程中生成的其他类似物。这些类比随后将在人源化的小鼠模型中进行测试。

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the mechanism by which Cortistatin A inhibits HIV-1 transcription. Human immunodeficiency virus type I (HIV-1) is the causative agent of AIDS (Acquired Immunodeficiency Syndrome). HIV anti-retroviral therapy is based on the administration of drugs in combination, in order to minimize development of mutations that can confer single-drug resistance to the virus. The viral protein Tat, a potent activator of HIV gene expression, is a potential antiviral target. We have discovered that Cortistatin A is a promising anti-Tat drug candidate. Cortistatin A is a steroidal alkaloid isolated from the marine sponge Corticium simplex. We have found that didehydro-Cortistatin A (CA for short), is a very potent inhibitor of Tat- activated transcription of the HIV-1 provirus. CA is extremely efficient at reducing viral output from acutely infected cells as well as from chronically infected cultured cells or freshly isolated peripheral blood lymphocytes (PBLs). The half maximal effective concentration (EC50) of CA for inhibiting HIV in chronic infected cells is less than 0.1 nM and the half maximal cytotoxic concentration (CC50) for CA in cultured cell lines or freshly isolated PBLs is more than 20 5M, which confers to CA a very promising therapeutic index. We have identified an interaction between CA and Tat and with the DNA-dependent protein kinase (DNA-PK) catalytic subunit. Upon HIV infection, DNA-PK tightly associates with Tat to promote phosphorylation of the transcription factor Sp1, which results in increased HIV-1 transcription initiation, via three Sp1 binding sites in the core of the HIV-1 promoter. Our hypothesis is that CA might repress HIV replication in two ways: by inhibiting Tat-TAR interaction, thereby inhibiting elongation by RNA Polymerase I (Pol I) from the HIV promoter; and/or by inhibiting DNA-PK activation of the transcription factor Sp1 and initiation/elongation of transcription by Pol II. Both routes would result in repression of HIV-1 transcriptional activation. Our specific aims include: Aim 1.Determine whether CA inhibits HIV-1 Tat-TAR interaction in vitro and in vivo and if CA inhibits transcription elongation from the viral promoter. Assess whether CA inhibits Tat mediated DNA-PK phosphorylation of Sp1. Aim 2. In-depth assessment of CA toxicity in vivo. Aim 3. Determine CA effect on HIV-1 replication in humanized mice models. We will thus elucidate the mechanism by which CA inhibits HIV-1 transcription and evaluate its potential to inhibit HIV-1 replication in humanized mice. In order to generate a backup compound we will evaluate additional analogs generated during the course of this project. These analogs will be subsequently tested in the humanized mouse model.