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

基本信息

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
项目类别：
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

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

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项目摘要

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.

对于九种人类疱疹病毒（HHVS）中，超过90％的人口是血清阳性的。具有建立可以重新激活的终身潜在感染的能力。 HHV的重新激活在免疫功能低下的患者中，感染尤其严重感染。尽管迄今为止的许多抗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出色的新型靶标。需要新代理以更好地防止病理重新激活的后遗症以及病毒脱落和向新宿主传播。需要新方式治疗HHV感染的疗法促进了对HHV的更全面了解的重要性 DNA复制。除了七种基本疱疹单纯疱疹病毒（HSV）复制蛋白我们和其他人，我们已经表明，病毒醇核酸酶UL12对于生产病毒也是必不可少的可以包装成传染病的DNA。 UL12与HSV ssDNA结合蛋白ICP8与形成可以促进单链退火（SSA）的两个组分重组酶（EXO/SSAP）。我们有建议HSV使用与DNA复制的异常机制，该机制与噬菌体比真核细胞或其他真核病毒。实际上，所有细菌，原生动物的DNA病毒，通过助剂形成复制的植物，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切除了DsDNA，留下3' 由ICP8识别的ssDNA悬垂。然后，ICP8将ssDNA退火为主动复制叉以促进病毒DNA聚合酶（UL30）促进DNA合成。 UL30完成了两个功能域：3至5'外切酶，Polexo，在校对和催化聚合酶结构域中起作用在病毒DNA复制过程中伸展引物所需的必需。虽然SSA复制模型是一致的有了可用的证据，关于UL12和聚合酶功能的两个活动仍然存在问题在DNA合成过程中。在平行研究中，赖特和惠勒实验室对An和PoLexo感兴趣及其来自其他HHV作为新型抗病毒疗法的靶标，并产生了聚焦的靶标库设计的库是为了能够参与AN和POLEXO活动地点的两级结合基序的能力。我们已经确定了几种潜在抗病毒药的铅化合物，并抑制一种或多种外切核酸酶活动和在某些情况下，针对聚合酶活性本身的其他活动。在此提案中，我们将使用我们的铅化合物是研究病毒DNA复制机制的分子问题，并继续我们为开发抑制AN，Polexo和/或POL的广谱抗病毒剂的努力。可以预期抑制这些靶标中的多个靶标会增加抗病毒耐药性的障碍。