title: Assay Development and High-Throughput Screening for Inhibitors of Kaposi's Sarcoma-Associated Herpesvirus N-Terminal Latency-Associated Nuclear Antigen Binding to Nucleosomes.
abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies, especially in immunocompromised hosts. KSHV latently infects tumor cells and persists as an extrachromosomal episome (plasmid). KSHV latency-associated nuclear antigen (LANA) mediates KSHV episome persistence. LANA binds specific KSHV sequence to replicate viral DNA. In addition, LANA tethers KSHV genomes to mitotic chromosomes to efficiently segregate episomes to daughter nuclei after mitosis. N-terminal LANA (N-LANA) binds histones H2A and H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high-throughput screening (HTS) of inhibitors of N-LANA binding to nucleosomes, here we develop, miniaturize, and validate a fluorescence polarization (FP) assay that detects fluorophore-labeled N-LANA peptide binding to nucleosomes. We also miniaturize a counterscreen to identify DNA intercalators that nonspecifically inhibit N-LANA binding to nucleosomes, and also develop an enzyme-linked immunosorbent assay to assess N-LANA binding to nucleosomes in the absence of fluorescence. HTS of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. No compounds survived all counterscreens, however. More complex small-molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A and H2B; these assays should prove useful for future screens.
authors: Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors: Chantal
authors: C
authors: Beauchemin
authors: Department of Systems Biology, Harvard Medical School, Boston, MA, USA.
authors: Nathan J
authors: NJ
authors: Moerke
authors: Broad Institute, Center for the Science of Therapeutics, Therapeutics Platform, Cambridge, USA.
authors: Patrick
authors: P
authors: Faloon
authors: Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA kkaye@rics.bwh.harvard.edu.
authors: Kenneth M
authors: KM
authors: Kaye
mesh_terms: D000818:Animals; D000941:Antigens / Q000737:chemistry; D000956:Antigens, Viral / Q000737:chemistry; D000998:Antiviral Agents / Q000737:chemistry*; D002470:Cell Survival; D002645:Chickens; D015195:Drug Design; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000821:virology; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D019288:Herpesvirus 8, Human / Q000187:drug effects*; D057166:High-Throughput Screening Assays; D006657:Histones / Q000737:chemistry; D006801:Humans; D008938:Mitosis; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D009707:Nucleosomes / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry; D000072417:Protein Domains; D013050:Spectrometry, Fluorescence
chemical_list: D000941:Antigens; D000956:Antigens, Viral; D000998:Antiviral Agents; D005456:Fluorescent Dyes; D006657:Histones; D009687:Nuclear Proteins; D009707:Nucleosomes; C109540:latency-associated nuclear antigen; D005982:Glutathione Transferase
keywords: Kaposi’s sarcoma–associated herpesvirus (KSHV); episome; latency-associated nuclear antigen (LANA)