Identification of Small Pharmacologic Inhibitors of HIV-1 Replication

描述(申请人提供)：HIV是爱滋病(获得性免疫缺陷综合症)的病原体。2005年，全世界艾滋病病毒携带者估计人数约为4030万。虽然一些国家的艾滋病毒感染率已开始下降，但感染总人数仍在继续增加。2005年有490万新感染者，310万人死于与艾滋病相关的疾病。降低病毒载量的首选治疗方法是高效抗逆转录病毒疗法(HAART)。HAART是一种治疗方案，它结合了直接针对至少两种病毒蛋白的药物。然而，与HAART相关的一个主要问题是新的艾滋病毒耐药株的出现和选择。这些药物直接针对病毒蛋白，因此当艾滋病毒发生突变时就会变得无效。因此，迫切需要开发具有新作用机制的药物。特别是，“间接目标”可能是解决这一问题的一种办法。“间接靶标”是细胞宿主蛋白，其功能对艾滋病毒复制至关重要。一个这样的“间接靶点”是细胞周期蛋白T1/CDK9复合体，它对于HIV TAT介导的反式激活是必不可少的。该实验室和其他实验室的初步研究表明，在各种细胞中抑制CDK9可以显著抑制艾滋病毒的复制，而不会影响细胞宿主的增殖和生存能力。根据我们的初步数据，我们推测Cyclin T1/CDK9复合体的高选择性抑制剂可能是毒性小、有效的HIV治疗药物。我们已经部分筛选了Onconova Treeutics的专有小分子文库，该文库富含具有选择性抑制激酶活性的化合物，用于体外抑制纯化的重组细胞周期蛋白T1/CDK9的活性。这一筛选导致了在体外对Cyclin T1/CDK9活性具有抑制作用的各种化合物的鉴定，IC50在低微摩尔范围内。因此，我们建议：(1)完成Onconova Treeutics公司专有的小分子细胞周期蛋白T1/CDK9活性抑制剂的筛选工作。(2)鉴定和扩展上述文库初步筛选中鉴定的主要细胞周期蛋白T1/CDK9抑制剂的子集以及预计将被鉴定的其他化合物的化学结构活性关系。(3)对该文库进行基于细胞的无偏性高通量HIV转录筛选，目的是寻找无细胞毒性的HIV转录抑制物。项目简介：艾滋病毒是获得性免疫缺陷综合症的病原体。降低病毒载量的首选治疗方法是高效抗逆转录病毒疗法(HAART)。然而，与HAART相关的一个主要问题是新的艾滋病毒耐药株的出现和选择。因此，迫切需要开发具有新作用机制的药物。我们的数据表明宿主细胞周期蛋白T1/CDK9是一个有效的药物开发靶点。我们已经与Onconova Treateutics合作，这是一家专门开发用于治疗干预的小分子的小企业，以识别和表征具有不同作用模式的艾滋病毒复制的药理抑制剂。

DESCRIPTION (provided by applicant): HIV is the causative agent of AIDS (Acquired Immunodeficiency Syndrome). In 2005 the estimated number of individuals living with AIDS worldwide was about 40.3 million. While the rate of HIV infection has started to decrease in several countries, the total number of infected individuals has continued to increase. There were 4.9 million new infections in 2005 and 3.1 million deaths from AIDS-associated illnesses. The therapeutic method of choice to reduce viral loads is Highly Active AntiRetroviral Therapy (HAART). HAART is a therapeutic regime that combines drugs that directly target at least two viral proteins. However, one major problem associated with HAART is the appearance and selection of novel HIV resistant strains. These drugs directly target viral proteins, and thus, become inefficient when HIV mutates. Therefore, there is an urgent need for development of drugs with novel mechanisms of action. In particular, "indirect targets" may represent a solution to this problem. "Indirect targets" are cellular host proteins, whose function is essential for HIV replication. One such "indirect target" is the cyclin T1/CDK9 complex, which is essential for HIV Tat-mediated transactivation. Preliminary studies by this and other laboratories show that CDK9 inhibition in a variety of cells dramatically inhibits HIV replication without affecting cell host proliferation and viability. Based on our preliminary data we hypothesized that highly selective inhibitors of the cyclin T1/CDK9 complex may be effective drugs for HIV therapy with little toxicity. We have partially screened an Onconova Therapeutics' proprietary library of small molecules enriched with compounds exhibiting selective inhibitory kinase activities, for inhibition of purified recombinant cyclin T1/CDK9 activity in vitro. This screening led to the identification of various compounds that inhibit cyclin T1/CDK9 activity in vitro with IC50s in the low micromolar range. Thus, we propose: (1) To complete an ongoing screening of the Onconova Therapeutics' proprietary library of small molecules for inhibitors of cyclin T1/CDK9 activity. (2) To characterize and expand the chemical structure activity relationship of the subset of leading cyclin T1/CDK9 inhibitors identified during the initial screening of the above-mentioned library as well as other compounds, which predictably will be identified. (3) To perform an unbiased high throughput-amenable cell-based HIV transcription screening of this library with the goal of identifying inhibitors of HIV-transcription with no cytotoxicity. Project Narrative: HIV is the causative agent of the Acquired Immunodeficiency Syndrome. The therapeutic method of choice to reduce viral loads is Highly Active AntiRetroviral Therapy (HAART). However, one major problem associated with HAART is the appearance and selection of novel HIV resistant strains. Thus, there is an urgent need for development of drugs with novel mechanisms of action. Our data suggest that the host cyclin T1/CDK9 kinase is a valid target for drug development. We have teamed with Onconova Therapeutics, a small business specialized in development of small molecules for therapeutic intervention, to identify and characterize pharmacological inhibitors of HIV replication with a different mode of action.