? The objective of the proposed research is to evaluate the antiviral potential of synthetic small molecule compounds known as polyamides for their ability to specifically inhibit Epstein-Barr virus (EBV) latency-gene expression and functions that contribute to EBV-associated lymphomas and lymphoproliferative disease in immune compromised individuals. Polyamides bind within the minor groove of a DNA double helix in a sequence-specific manner with affinities comparable to many sequence-specific DNA-binding proteins. Consequently, these compounds have the ability to compete with proteins (e.g., transcription factors) that target the same or an overlapping binding site within dsDNA, thus disrupting their function. This and the generally favorable pharmacological properties of polyamides make them attractive as antiviral drugs to inhibit the expression or DNA-binding functions of viral proteins critical for pathogenesis. Such agents targeting latent EBV infection are particularly attractive, since current anti-herpesvirus drugs are designed to inhibit virus replication and are ineffective against the latent or transforming form of EBV infection.
Three specific aims are proposed.
Under Aim 1 we will evaluate the in vivo inhibitory potential of hairpin polyamides designed to target EBV latency gene promoters and the viral origin of DNA replication, oriP. Specifically, we will seek to block transcription of the EBV genes encoding the essential genome maintenance protein EBNA-1 and the oncoprotein LMP-1 by inhibition of the binding of transcription factors to critical cis-regulatory elements of the EBNA- 1 and LMP- 1 promoters. Additionally, we will test whether polyamides designed to inhibit binding of EBNA- 1 to oriP are capable of enforcing loss of the EBV genome from EBV-positive lymphoma cells.
Under Aim 2 we propose to develop DNA-binding and whole-cell reporter based assays that are compatible with high throughput screening of chemical libraries so that the most effective inhibitors of EBNA-1 binding to oriP and of EBNA-1 and LMP-1 expression can be identified.
Under Aim 3, we will take a combinatorial approach to identify polyamide inhibitors of EBNA-1 DNA-binding and of EBNA-1 and LMP-1 expression. Specifically, we propose to generate a combinatorial library of polyamide-based compounds that will be screened, using the bioassays developed under Aim 2, to identify lead compounds with anti-EBV properties. By taking the complementing approaches of rational and combinatorial drug design in Aims 1 and 3, respectively, we hope to effectively evaluate the anti-EBV potential of these compounds, and identify lead compounds with potential therapeutic value. ? ? ?
