ASSAY DEVELOPMENT AND HIGH THROUGHPUT SCREENING FOR INHIBITORS OF KAPOSIS SARCOMA-ASSOCIATED HERPESVIRUS N-TERMINAL LANA BINDING TO NUCLEOSOMES

摘要

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 binds histones H2A/H2B to attach to chromosomes. Currently, there are no specific inhibitors of KSHV latent infection. To enable high throughput screening 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 ELISA to assess N-LANA binding to nucleosomes in the absence of fluorescence. High throughput screening of libraries containing more than 350,000 compounds identified multiple compounds that inhibited N-LANA binding to nucleosomes. However, no compounds survived all counterscreens. More complex small molecule libraries will likely be necessary to identify specific inhibitors of N-LANA binding to histones H2A/H2B; these assays should prove useful for future screens.