Lead optimization of Hepatitis B Virus ribonuclease H inhibitors

B型肝炎病毒（HBV）是一种嗜肝DNA病毒，通过逆转录进行复制。它在全世界慢性感染超过2.5亿人，每年杀死约87万人。治疗主要采用核苷（酸）类似物药物对抗病毒DNA合成，其通常使病毒血症低于检测限。然而，复制并没有被消除，如果停药，HBV会复发。尽管如此，治疗可以治愈高达6%的患者，因此更多的患者可以通过进一步抑制HBV来治愈。逆转录需要病毒核糖核酸酶H（RNaseH），它在RNA复制成DNA后将其破坏。阻断RNaseH可以阻止病毒基因组的合成，包括基因组的关键核cccDNA形式和病毒体中的DNA。目前还没有针对RNaseH的药物。我们开发了HBV RNaseH抑制剂的第一个筛选管道，并发现RNaseH >150抑制剂可阻断HBV复制。最有效的是N-羟基吡啶二酮（HPD），EC 50为0.11 μM，治疗指数（TI，CC 50/EC 50）为300。HPD命中化合物在具有人源化肝脏的小鼠中抑制HBV病毒血症。我们还发现，萘啶酮[NPTH，由真正的萘啶酮（HNO）和密切相关的羟基吡啶嘧啶酮（HPP）组成]抑制HBV复制，EC 50低至0.95 μM，TI高达83。在仅评估51种HPD后，EC 50低至0.11 μM，TI高达350，再加上相关NPTH化学型的良好活性，导致Tavis博士（HBV病毒学家），Meyers，Zhan和Zoppers和（药物化学家）联手开发HPD和NPTH成为新型HBV药物的临床前先导药物。目标1. HPD HBV抑制剂的先导优化。我们将根据现有的结构-活性关系（SAR）合成多达125种HPD，并评估其疗效、细胞毒性和溶解度。目标2. NPTH HBV抑制剂的开发。我们将合成多达100种HNO和100种HPP，并评估它们的功效、细胞毒性和溶解性。目标3。评估新型抑制剂对HBV的特异性。我们将评价HPD和NPTH诱导HBV衣壳中异源双链体形成和抑制cccDNA形成的情况。将评估与其他HBV药物的协同作用。将测量针对微生物病原体和人金属酶（包括RNaseH 1）的选择性。目标4。评价HPD和NPTH HBV抑制剂的药理学参数。我们将评估关键抑制剂的稳定性、细胞渗透性、血浆蛋白结合、药代动力学和毒理学。将在携带人源化肝脏的HBV感染小鼠中测试最佳化合物针对HBV复制的功效。这些研究将推进HBV RNaseH抑制剂作为首个机制和首个HBV药物先导。预期最终的抗HBV RNaseH药物将与核苷（酸）类似物联合使用，以充分抑制HBV复制，从而在比目前疗法实现的更多患者中清除HBV。

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >250 million people worldwide and kills ~870,000 annually. Therapy primarily employs nucleos(t)ide analog drugs against viral DNA synthesis that often drive viremia below the detection limit. However, replication is not eliminated, and HBV resurges if drugs are withdrawn. Nevertheless, treatment cures up to 6% of patients, so more patients could be cured by suppressing HBV further. Reverse transcription requires the viral ribonuclease H (RNaseH) that destroys the RNA after it has been copied into DNA. Blocking the RNaseH prevents synthesis of viral genomes, including both the key nuclear cccDNA form of the genome and the DNA in virions. Drugs have not yet been designed against the RNaseH. We developed the first screening pipeline for HBV RNaseH inhibitors and found RNaseH >150 inhibitors that block HBV replication. The most effective is an N-hydroxypyridinedione (HPD) with an EC50 of 0.11 μM and a therapeutic index (TI, CC50/EC50) of 300. An HPD hit compound suppressed HBV viremia in mice with humanized livers. We also found that napthyridinones [NPTH, comprised of true napthyridinones (HNOs) and the closely related hydroxypyridopyrimidinones (HPPs)] inhibit HBV replication with EC50s as low as 0.95 μM and TIs up to 83. Achieving EC50s as low as 0.11 μM and TIs up to 350 after assessing only 51 HPDs, combined with good activity of the related NPTH chemotype, led Drs. Tavis (HBV virologist), Meyers, Zhan, and Zoidis and (medicinal chemists) to join forces to develop the HPDs and NPTHs into pre-clinical leads for novel HBV drugs. Aim 1. Lead optimization of HPD HBV inhibitors. We will synthesize up to 125 HPDs based on our existing structure-activity relationships (SAR) and evaluate their efficacy, cytotoxicity, and solubility. Aim 2. Hit-to-lead development of NPTH HBV inhibitors. We will synthesize up to 100 HNOs and 100 HPPs and evaluate their efficacy, cytotoxicity, and solubility. Aim 3. Assess specificity for HBV of the novel inhibitors. We will evaluate HPDs and NPTHs for induction of heteroduplex formation in HBV capsids and suppression of cccDNA formation. Synergy with other HBV drugs will be assessed. Selectivity will be measured against microbial pathogens and human metalloenzymes including RNaseH1. Aim 4. Evaluate pharmacological parameters for HPD and NPTH HBV inhibitors. We will assess stability, cellular permeability, plasma protein binding, pharmacokinetics, and toxicology of key inhibitors. Efficacy of the best compounds will be tested against HBV replication in HBV-infected mice carrying humanized livers. These studies will advanced HBV RNaseH inhibitors as first-in-mechanism and first-in-class HBV drug leads. The eventual anti-HBV RNaseH drugs are anticipated to be used in combination with nucleos(t)ide analogs to suppress HBV replication enough to clear HBV in many more patients than current therapies achieve.