Discovery of Novel HIV-1 Reverse Transcriptase Inhibitors by Fragment Screening

描述(由申请人提供)：拟议研究的目标是应用结构、生化和生物物理分析，努力发现艾滋病毒逆转录酶核糖核酸酶H活性的新抑制剂。HIV-1逆转录酶(RT)是逆转录病毒的核心和特征酶，也是许多关键的抗艾滋病药物的靶标。RT具有DNA聚合酶和核糖核酸酶H(RNH)两种酶活性。虽然RT利用DNA聚合酶和RNH活性来进行合成，但到目前为止，所有已被批准用于临床的RT抑制剂都针对聚合酶活性，而不是RNH活性。鉴于病毒的变异能力和抗药性，探索RN抑制剂作为潜在的抗艾滋病治疗药物至关重要。虽然已经发现几种RNH抑制剂可以抑制RNH活性，但对抑制剂结合机制的可视化可能会推动基于结构的更有效抗病毒药物的设计。该项目的目标是提供X射线结晶学领域的培训，并利用这些技能发现和设计艾滋病毒-1逆转录酶的新抑制剂。第一个目标是使用X射线结晶学来确定与HIV-1RT结合的活性部位和变构RNH抑制剂的结构，并建立结构-活性关系和详细的结合模式。为了发现新的RNH抑制剂化学类别，将使用表面等离子共振和X射线结晶学分析1500个类药物片段的文库是否可能与HIV-1 RT结合，并使用基于荧光的高通量筛选专门针对RNA切割来分析RNH抑制作用。片段筛选方法允许有效地搜索化学空间，并应导致识别在活性部位和变构口袋中抑制HIV-1 RNH活性的新支架。

DESCRIPTION (provided by applicant): The goal of the proposed research is to apply structural, biochemical, and biophysical analysis in an effort to discover novel inhibitors of HIV- reverse transcriptase ribonuclease H activity. HIV-1 reverse transcriptase (RT) is a central and characteristic enzyme of retroviruses, and is the target of many key anti-AIDS drugs. RT possesses two enzymatic activities, a DNA polymerase and a ribonuclease H (RNH). Although RT utilizes both DNA polymerase and RNH activities to carry out synthesis, to date, all RT inhibitors that have been approved for clinical use target the polymerase activity, not the RNH activity. Given the virus' ability to mutate and become drug resistant, it is crucial to explore RN inhibitors as potential anti-AIDS therapeutic agents. Though several RNH inhibitors have been found to inhibit RNH activity, visualization of inhibitor binding mechanisms could drive structure-based design of more effective antiviral drugs. The project goals are designed in order to provide training in the field of X-ray crystallography and utilize those skills in discovery and design of novel inhibitors of HIV-1 RT. The first goal will be to use X-ray crystallography to determine the structures of both active site and allosteric RNH inhibitors bound to HIV-1 RT and establish structure-activity relationships and detailed binding modes. In an effort toward the discovery of novel chemical classes of RNH inhibitors, a library of 1500 drug-like fragments will be analyzed for potential binding to HIV-1 RT using surface plasmon resonance and X-ray crystallography, and for RNH inhibition using a fluorescence based high throughput screen that looks specifically for RNA cleavage. The fragment screening approach allows for efficient search of chemical space and should lead to the identification of novel scaffolds for inhibiting HIV-1 RNH activity at both the active site and allosteric pockets.