Exploring herpesvirus exonucleases as potential antiviral targets

探索疱疹病毒核酸外切酶作为潜在的抗病毒靶点

• 批准号: 10825475
• 负责人: SANDRA K WELLER
• 金额: $ 60.53万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10825475
• 关键词: 3-Dimensional; Active Sites; Antiviral Agents; Antiviral Therapy; Bacteria; Bacteriophages; Binding; Binding Proteins; Biochemical; Biological Assay; Biophysics; Blindness; Chemicals; Child; Complex; Congenital herpes simplex; Contracts; Cytomegalovirus; DNA; DNA Viruses; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Development; Disease; Dose Limiting; Drug Targeting; Encephalitis; Enzymes; Eukaryotic Cell; Exonuclease; Family member; Genetic; Genital; Genitalia; HIV Integrase; Hepatitis; Herpes Labialis; Herpesviridae; Herpesviridae Infections; Herpesvirus 1; Homologous Gene; Human; Human Herpesvirus 2; Human Herpesvirus 8; Immunocompromised Host; Immunosuppression; Impairment; Infant; Infection; Influenza; Insecta; Ions; Laboratories; Lead; Libraries; Life; Mammals; Meningitis; Metals; Methods; Modality; Modeling; Modification; Molecular; Molecular Probes; Morbidity - disease rate; Mutation Analysis; Neuropathy; New Agents; Nucleosides; Oral; Pain; Pathologic; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphodiesterase I; Plants; Play; Pneumonia; Polymerase; Population; Production; Proteins; Protozoa; Psyche structure; Reaction; Reporting; Resected; Resistance; Resistance profile; Retinitis; Role; Series; Simplexvirus; Single Stranded DNA Virus; Single-Stranded DNA; Structure; Therapeutic; Toxic effect; United States National Institutes of Health; Viral; Viral Drug Resistance; Virus; Virus Replication; Virus Shedding; congenital infection; design; drug discovery; ds-DNA; genetic analysis; inhibitor; interest; latent infection; mortality; mutant; novel; nuclease; nucleoside analog; pathogen; permanent hearing loss; prevent; recombinase; seropositive; small molecule; small molecule inhibitor; synergism; targeted agent; targeted nucleases; tool; viral DNA; viral alkaline nuclease; viral transmission

Over 90% of the world’s population is seropositive for three or more of the nine human herpesviruses (HHVs), which possess the ability to establish lifelong latent infections that can be reactivated. Reactivation of HHV infections is particularly serious in immunocompromised patients leading to disseminated life-threatening infections. Although many of the anti HHV drug discovery efforts to date have focused on nucleoside/tide HHV polymerase inhibitors, the large number of essential replication proteins encoded by the herpesviruses provide excellent novel targets for antiviral therapy. New agents are needed to better prevent pathological sequelae of reactivation as well as viral shedding and transmission to new hosts. The need for new modalities of therapeutics to treat HHV infections underscores the importance of a more thorough understanding of HHV DNA replication. In addition to the seven essential herpes simplex virus (HSV) replication proteins identified by us and others, we have shown that the viral alkaline nuclease UL12 is also essential for the production of viral DNA that can be packaged into infectious virus. UL12 interacts with the HSV ssDNA binding protein ICP8 to form a two-component recombinase (exo/SSAP) that can promote single strand annealing (SSA). We have suggested that HSV uses an unusual mechanism of DNA replication that has more in common with bacteriophage than eukaryotic cells or other eukaryotic viruses. In fact, all DNA viruses of bacteria, protozoa, plants, insects and mammals that replicate through concatemer formation encode a similar exo/SSAP complex. We hypothesize that ICP8 and UL12 promote a series of reactions in which UL12 resects dsDNA leaving a 3’ ssDNA overhang that is recognized by ICP8. ICP8 then promotes annealing of the ssDNA to an active replication fork to promote DNA synthesis by the viral DNA polymerase (UL30). UL30 is comprised of two functional domains: a 3’ to 5’ exonuclease, PolExo, that plays a role in proofreading and the catalytic polymerase domain required for extending primers during viral DNA replication. While the SSA model for DNA replication is consistent with available evidence, questions remain about how UL12 and the two activities of the polymerase function during DNA synthesis. In parallel studies, the Wright and Weller labs have been interested in both AN and PolExo and their othologs from other HHVs as targets for novel antiviral therapeutics and have generated focused libraries designed for their ability to engage a two-metal binding motif found in both AN and PolExo active sites. We have identified several lead compounds that are potent antivirals and inhibit one or more of the exonuclease activities and in some cases additional activity against polymerase activity itself. In this proposal we will use our lead compounds as molecular probes to study mechanisms of viral DNA replication and to continue our efforts to develop broad spectrum antiviral agents that inhibit AN, PolExo and/or Pol. Agents that can inhibit more than one of these targets would be expected to increase the barrier to antiviral drug resistances.

世界上超过 90% 的人口对九种人类疱疹病毒 (HHV) 中的三种或三种以上呈血清反应阳性， 它具有建立可重新激活的终生潜伏感染的能力。 HHV 重新激活 感染在免疫功能低下的患者中尤其严重，导致播散性危及生命 感染。尽管迄今为止许多抗 HHV 药物发现工作都集中在核苷/潮汐 HHV 上 聚合酶抑制剂，由疱疹病毒编码的大量必需复制蛋白 为抗病毒治疗提供了优异的新靶点。需要新的药物来更好地预防病理性 重新激活以及病毒脱落和传播给新宿主的后遗症。需要新的模式 治疗 HHV 感染的疗法强调了更彻底了解 HHV 的重要性 DNA复制。除了 7 种必需的单纯疱疹病毒 (HSV) 复制蛋白外， 我们和其他人已经证明，病毒碱性核酸酶 UL12 对于病毒的生产也是必不可少的 可包装成传染性病毒的DNA。 UL12 与 HSV ssDNA 结合蛋白 ICP8 相互作用， 形成可以促进单链退火（SSA）的双组分重组酶（exo/SSAP）。我们有 表明 HSV 使用一种不寻常的 DNA 复制机制，该机制与 噬菌体比真核细胞或其他真核病毒更重要。事实上，细菌、原生动物、 通过多联体形成复制的植物、昆虫和哺乳动物编码类似的 exo/SSAP 复合物。 我们假设 ICP8 和 UL12 促进了一系列反应，其中 UL12 切除 dsDNA，留下 3’ ICP8 识别的 ssDNA 突出端。然后 ICP8 促进 ssDNA 退火至主动复制 叉促进病毒 DNA 聚合酶 (UL30) 的 DNA 合成。 UL30 由两个功能组成 结构域：一种 3' 至 5' 核酸外切酶 PolExo，在校对和催化聚合酶结构域中发挥作用 病毒 DNA 复制期间延伸引物所需的。虽然 DNA 复制的 SSA 模型是一致的 根据现有证据，关于 UL12 和聚合酶的两种活性如何发挥作用的问题仍然存在 DNA合成过程中。在并行研究中，Wright 和 Weller 实验室对 AN 和 PolExo 都感兴趣 及其来自其他 HHV 的同源物作为新型抗病毒疗法的靶标，并产生了集中的 文库的设计目的是能够接合 AN 和 PolExo 活性位点中发现的双金属结合基序。 我们已经鉴定出几种先导化合物，它们是有效的抗病毒药物并抑制一种或多种核酸外切酶 活性以及在某些情况下针对聚合酶活性本身的额外活性。在这个提案中我们将使用 我们的先导化合物作为分子探针来研究病毒 DNA 复制机制并继续 我们致力于开发抑制 AN、PolExo 和/或 Pol 的广谱抗病毒药物。代理可以 抑制这些靶标中的一个以上预计会增加抗病毒药物耐药性的障碍。