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

 描述（由申请方提供）：B型肝炎病毒（HBV）是一种嗜肝病毒，通过逆转录进行复制。它慢性感染约3.5亿人，每年杀死60多万人。治疗主要采用核苷（酸）类似物，其在大多数患者中将HBV驱动至检测限以下。然而，病毒复制并没有被根除，如果停药，病毒复制会重新出现，因此治疗是终身的。然而，目前的治疗确实清除了一些患者的HBV，因此更多的患者可以通过进一步抑制HBV而治愈。更大程度的抑制HBV将需要新的药物，这些药物可能与核苷（酸）类似物联合使用。 HBV逆转录需要病毒DNA聚合酶来合成新的DNA，并需要病毒核糖核酸酶H（RNaseH）来在RNA转化为DNA后破坏RNA。阻断任何一种活性都可以防止HBV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。我们最近生产了适合药物发现的HBV RNaseH。我们筛选了54种托品类化合物，重点是α-羟基托酚酮（α HT），基于它们抑制HIV RNaseH和β-thujaplicinol抑制HBV RNaseH的能力。13种化合物抑制HBV RNaseH，最好的IC 50值为2.3 µM。六种化合物通过阻断病毒RNaseH活性抑制培养物中的HBV复制。最好的化合物的EC 50为0.34 µM，治疗指数为94。初步的结构-活性关系（SAR）的推导，药物化学优化提供指导。 在这里，Tavis博士（病毒学家）和Murelli和Meyers博士（合成和药物化学家）将合作开始将α HT开发成有效的抗HBV药物。 目标1.生产HBV复制的最佳αHT抑制剂。我们将在初步SAR的基础上合成~320 α HT。将在生化、病毒复制和细胞毒性试验中以迭代方式检测这些化合物的抗HBV RNaseH活性。 目标2.在HBV复制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。将在HBV复制小鼠模型中表征主要抑制剂的疗效。 目标3.评价αHT化合物与核苷（酸）类似物联合使用的潜力。我们将评估优化的αHT化合物对常见核苷（酸）类似物耐药HBV毒株的疗效。我们还将评估αHT抑制剂是否与核苷（酸）类似物协同作用，如根据它们与HBV逆转录酶上物理上不同的活性位点的结合所预测的那样。 该项目将生产首个针对HBV RNaseH的αHT抑制剂，并将确定其用于抗HBV治疗的潜力。它还可能产生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 However, viral replication is not eradicated and viral replication resurges if drug is withdrawn, so therapy is life-long.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.