Defining approaches for improving HID and HPD compounds as HBV RNaseH antagonists

乙型肝炎病毒（HBV）是一种嗜肝性DNA病毒，通过逆转录进行复制。它每年慢性感染3.5亿人，造成60万人死亡。治疗采用核苷类似物，通常使HBV达到或低于临床检测极限。然而，如果停药，病毒复制不会被消除，HBV会复发，因此治疗是终身的。然而，治疗治愈率高达6%，因此进一步抑制HBV可以帮助更多的患者。HBV逆转录需要病毒核糖核酸酶H （RNaseH）在RNA被复制成DNA后将其破坏。抑制RNaseH可阻断病毒复制，但由于技术限制，抗RNaseH药物尚未设计出来。我们最近开发了第一个HBV RNaseH抑制剂筛选管道。我们筛选了bbb280种含有抑制HIV RNaseH的药物团的化合物，以阻止HBV RNaseH和HBV复制。41个化合物为N-羟基异喹啉二酮（HID）、N-羟基吡啶二酮（HPD）或相关的多氧杂环化合物。其中13种化合物抑制了病毒在细胞中的复制。最有效的HPD/HID化合物在培养中对HBV复制的EC50为0.57µM（治疗指数= 26）。这些化合物在RNaseH活性位点螯合Mg++，正如对HIV RNaseH的研究所期望的那样。在这里,Drs。Tavis（病毒学家）和Meyers（药物化学家）将开展一个跨学科的探索性R21项目，以回答产生抗hbv药物开发所需的HPD/HID先导物的两个问题。目的1。对HPD/HID药效团进行哪些类型的修改可以提高疗效？我们将合成一系列不同的HPD/HID支架衍生物，包括设计中的预测特征（CLogP， ALOGpS等），并评估它们对病毒复制的功效。我们将评估药物样性质，如在微粒体提取物和肝细胞中的稳定性，以寻找最佳化合物。化合物的合成将是迭代的，从最初的化合物的结果为后来的设计提供信息。目标2。最大化HPD/HIDs对HBV RNaseH选择性的最佳方法是什么？治疗性抗HBV治疗可能持续数月，因此对HBV酶的高选择性和低细胞毒性将是关键。我们将对所有化合物对人类RNaseH1进行反筛选，并仔细评估最佳化合物的细胞毒性。目标1和目标2将同时进行，以便在迭代化合物设计期间解决选择性和细胞毒性问题。这些R21研究将产生HPD/HID药效团的结构-活性关系（SAR），指导药物化学优化，从而产生候选抗hbv药物。RNaseH药物有望与核苷(t)类似物联合使用，以抑制HBV复制，从而在比目前单一疗法所能达到的更多患者中清除HBV。

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects 350 million people and kills >600,000 annually. Therapy employs nucleos(t)ide analogs that usually drive HBV to at or below the clinical limit of detection. However, viral replication is not ablated and HBV resurges if drug is withdrawn, so therapy is life-long. Nevertheless, treatment cures up to 6% of patients, so many more patients could be helped by suppressing HBV further. HBV reverse transcription requires the viral ribonuclease H (RNaseH) to destroy the RNA after it has been copied into DNA. Inhibiting the RNaseH blocks viral replication, but anti-RNaseH drugs have not been designed due to technical limitations. We recently developed the first HBV RNaseH inhibitor screening pipeline. We screened >280 compounds containing pharmacophores that inhibit the HIV RNaseH for the ability to block the HBV RNaseH and HBV replication. 41 compounds were N-hydroxyisoquinolinediones (HID), N- hydroxypyridinediones (HPD), or related polyoxygenated heterocycles. Thirteen of these compounds inhibited suppressed viral replication in cells. The most effective HPD/HID compound has an EC50 of 0.57 µM against HBV replication in culture (therapeutic index = 26). These compounds chelate Mg++ in the RNaseH active site, as expected from studies on the HIV RNaseH. Here, Drs. Tavis (virologist) and Meyers (medicinal chemist) will conduct an interdisciplinary exploratory R21 project to answer 2 questions needed to generate HPD/HID leads for anti-HBV drug development. Aim 1. What types of modifications to the HPD/HID pharmacophore improve efficacy? We will synthesize a diverse series of ~70 derivatives of the HPD/HID scaffold including predictive characteristics (CLogP, ALOGpS, etc.) in the design and evaluate their efficacy against viral replication. We will assess drug- like properties such as stability in microsomal extracts and hepatocytes for the best compounds. Compound synthesis will be iterative, with results from the initial compounds informing design of later ones. Aim 2. What are the best approaches to maximize selectivity of HPD/HIDs for the HBV RNaseH? Curative anti-HBV therapy is likely to last for many months, so high selectivity for the HBV enzyme and low cytotoxicity will be key. We will counter-screen all compounds against the human RNaseH1 and carefully assess cytotoxicity for the best compounds. Aims 1 and 2 will be conducted concurrently so selectivity and cytotoxicity issues can be addressed during iterative compound design. These R21 studies will generate a structure-activity relationships (SAR) for the HPD/HID pharmacophore that will guide medicinal chemistry optimization, leading to candidate anti-HBV drugs. RNaseH drugs are anticipated to be used in combination with the nucleos(t)ide analogs to suppress HBV replication far enough to clear HBV in many more patients than the current monotherapies can achieve.