Optimization of alpha-hydroxytropolones as novel inhibitors of the HBV RNaseH

 描述(申请人提供)：乙肝病毒(乙肝病毒)是一种嗜肝病毒，通过逆转录进行复制。它每年慢性感染约3.5亿人，导致60万人死亡。治疗主要使用核糖核酸(T)类似物，使大多数患者的乙肝病毒检测下限以下。然而，如果停药，病毒复制并不会被根除，病毒复制会卷土重来，因此治疗是终生的。然而，目前的治疗确实清除了一些患者的乙肝病毒，所以更多的患者可以通过进一步抑制乙肝病毒来治愈。更好地抑制乙肝病毒将需要新的药物，这些药物可能会与核(T)化物类似物结合使用。乙肝病毒的逆转录需要病毒DNA聚合酶来合成新的DNA，而病毒核糖核酸酶H(RNAseH)则需要在RNA转化为DNA后将其摧毁。阻断这两种活动中的任何一种都可以防止乙肝病毒复制。由于不能产生适合筛选的酶，因此不存在针对RNAseH的药物。我们最近生产出了适合于药物发现的HBVRNAseH。我们筛选了54个肌钙蛋白类化合物，重点是α-羟基肌醇(αHTs)，基于它们抑制HIV RNAseH的能力和β-hujaplicinol对HBVRNAseH的抑制作用。其中13个化合物对HBVRNAseH有抑制作用，IC50值最大为2.3µM。6个化合物通过阻断病毒RNAseH活性抑制病毒复制。最佳化合物的EC50为0.34微米，治疗指数为94。建立了初步的构效关系，为药物化学优化提供了指导。在这里，塔维斯博士(病毒学家)、穆雷利博士和迈耶斯博士(合成和药物化学家)将合作，开始将α高温超导药物开发成有效的抗乙肝病毒药物。目的1.合成最佳的乙肝病毒复制抑制剂α-HT。我们将在我们初步的合成孔径雷达的基础上合成~320α高温超导。这些化合物将在生化、病毒复制和细胞毒性测试中以迭代方式测试抗HBVRNAseH活性。目的2.在复制乙肝病毒的小鼠模型中优化细胞毒性、ADME、PK和疗效参数。将测量吸收、分布、代谢、排泄(ADME)、初始药代动力学(PK)和毒性，以指导化合物设计。主要的抑制剂将在小鼠模型中表征乙肝病毒复制的疗效。目的3.评估α-羟色胺化合物与核(T)化物类似物联合使用的可能性。我们将评估优化的α-HT化合物对耐类似物乙肝病毒株的疗效。我们还将评估HBVHT抑制剂是否如预测的那样与核(T)类似物协同作用，这是从它们与α逆转录酶上不同的物理活性部位结合而预测的。该项目将生产第一个针对HBVRNAseH设计的αHT抑制剂，并将确定它们对抗HBVc治疗的贡献潜力。它还可能生产用于药物开发的先导化合物。

 DESCRIPTION (provided by applicant): Hepatitis B virus (HBV) is a hepatotropic virus that replicates by reverse transcription. It chronically infects ~350 million people and kills >600,000 annually. Therapy primarily employs nucleos (t)ide analogs that drive HBV to below the limit of detection in most patients. However, viral replication is not eradicated and viral replication resurges if drug is withdrawn, so therapy is life-long. Nevertheless, current treatment does clear HBV in some patients, so more patients could be cured by suppressing HBV further. Greater suppression of HBV will require new drugs that will probably be used in combination with the nucleos (t)ide analogs. HBV reverse transcription requires a viral DNA polymerase to synthesize new DNA and a viral ribonuclease H (RNaseH) to destroy the RNA after its conversion into DNA. Blocking either activity prevents HBV replication. Drugs against the RNaseH do not exist due to an inability to produce enzyme suitable for screening. We recently produced HBV RNaseH suitable for drug discovery. We screened 54 troponoid compounds, focusing on the α-hydroxytropolones (αHTs), based on their ability to inhibit the HIV RNaseH and β-thujaplicinol's inhibition of the HBV RNaseH. Thirteen compounds inhibited the HBV RNaseH, and the best had an IC50 value of 2.3 µM. six compounds suppressed HBV replication in culture via blocking viral RNaseH activity. The best compound had an EC50 of 0.34 µM and a therapeutic index of 94. A preliminary structure-activity relationship (SAR) was derived that provides guidance for optimization by medicinal chemistry. Here, Dr. Tavis (virologist) and Drs. Murelli and Meyers (synthetic and medicinal chemists) will collaborate to begin development of the αHTs into potent anti-HBV drugs. Aim 1. Produce optimal αHT inhibitors of HBV replication. We will synthesize ~320 αHTs based on our preliminary SAR. These compounds will be tested in an iterative fashion for anti-HBV RNaseH activity in biochemical, viral replication, and cytotoxicity assays. Aim 2. Optimize cytotoxicity, ADME, PK, and efficacy parameters in a mouse model of HBV replication. Absorption, distribution, metabolism, excretion (ADME), initial pharmacokinetics (PK), and toxicity will be measured to guide compound design. Efficacy will be characterized in a mouse model of HBV replication for the leading inhibitors. Aim 3. Evaluate the potential for use of αHT compounds in combination with nucleos(t)ide analogs. We will assess efficacy of the optimized αHT compounds against common nucleos(t)ide analog- resistant HBV strains. We will also evaluate whether αHT inhibitors work synergistically with the nucleos(t)ide analogs as predicted from their binding to physically distinct active sites on the HBV reverse transcriptase. This project will produce the first αHT inhibitors designed against the HBV RNaseH and will define their potential to contribute to anti-HBV therapy. It may also produce lead compounds for drug development.