title: Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.
abstract: The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease.
  
  
  OBJECTIVE
  Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Mao
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Jayakanth
authors.initials: J
authors.lastname: Kankanala
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, USA gerag012@umn.edu.
authors.forename: Robert J
authors.identifier: http://orcid.org/0000-0002-0560-6509
authors.initials: RJ
authors.lastname: Geraghty
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D003587:Cytomegalovirus / Q000187:drug effects* / Q000502:physiology*; D004279:DNA, Viral / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D016679:Genome, Viral; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D021122:Protein Subunits / Q000037:antagonists & inhibitors* / Q000737:chemistry; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D004791:Enzyme Inhibitors; D021122:Protein Subunits; C436448:UL89 protein, Cytomegalovirus; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C030894:terminase
keywords: human cytomegalovirus; hydroxypyridonecarboxylic acid; metal chelation; pUL89 inhibitor; terminase