Lead optimization of Hepatitis B Virus ribonuclease H inhibitors

乙肝病毒是一种嗜肝的DNA病毒，通过逆转录进行复制。它慢性感染全球2.5亿人，每年导致约87万人死亡。治疗主要使用核(T)类似物来对抗病毒DNA合成，病毒DNA合成经常使病毒血症低于检测下限。然而，复制并没有被消除，如果停用药物，乙肝病毒会卷土重来。然而，治疗可以治愈高达6%的患者，所以更多的患者可以通过进一步抑制乙肝病毒来治愈。反转录需要病毒核糖核酸酶H(RNAseH)，在RNA复制到DNA中后，它会破坏RNA。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形式和病毒粒子中的DNA。目前还没有针对RNAseH的药物设计。我们开发了第一个筛选HBVRNAseH抑制剂的管道，并发现了RNAseH&GT；150抑制剂可以阻断乙肝病毒的复制。最有效的是N-羟基吡啶二酮，其EC_(50)为0.11μM，治疗指数(TI，CC_(50)/EC_(50))为300。一种Hpd Hit化合物抑制了人源化肝脏小鼠的乙肝病毒血症。我们还发现，由真正的萘甲酮(HNO)和密切相关的羟基吡啶酮(HPP)组成的NPTH[NPTH]抑制乙肝病毒复制，EC50低至0.95μM，TIS高达83。在仅评估51个HPD后，EC50低至0.11μM，TIS高达350，再加上相关NPTH化学型的良好活性，导致Tavis博士(乙肝病毒学家)、Meyers、Jan和Zoidis博士以及(药物化学家)联手将HPDs和NPTHs开发为乙肝新药的临床前线索。目的1.HpD-HBVi抑制剂的主导优化。我们将基于我们现有的结构-活性关系(SAR)合成多达125个HPD，并评估它们的有效性、细胞毒性和溶解性。目的2.NPTH乙肝病毒抑制剂的Hit-to-Lead开发。我们将合成多达100个HNO和100个HPP，并评估它们的有效性、细胞毒性和溶解性。目的3.评价新型抑制剂对乙肝病毒的特异性。我们将评估HPDs和NPTHs在诱导HBV衣壳异源双链形成和抑制cccDNA形成方面的作用。将评估与其他乙肝病毒药物的协同作用。选择性将针对微生物病原体和包括RNaseH1在内的人类金属酶进行衡量。目的4.评价HPD和NPTH乙肝病毒抑制剂的药理参数。我们将评估关键抑制剂的稳定性、细胞通透性、血浆蛋白结合、药代动力学和毒理学。最好的化合物的有效性将在携带人源化肝脏的乙肝病毒感染小鼠身上进行测试。这些研究将使HBVRNAseH抑制剂作为一流的机制和一流的HBV药先导药物。最终的抗HBVRNAseH药物有望与核苷(T)类似物结合使用，以抑制足以在更多患者中清除HBV病毒的复制，而不是目前的治疗方法。

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 However, replication is not eliminated, and HBV resurges if drugs are withdrawn.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.