Research Project
9909297
ABSTRACT Infection of the eye by Herpes Simplex Virus-1 (HSV-1) can result in Herpes Keratitis (HK), which is the leading cause of corneal blindness worldwide. Ocular herpes infections are often recurrent and culminate in progressive corneal scarring and loss of vision. The gold standard treatment is Acyclovir (ACV) that targets HSV-1 thymidine kinase (TK). Although ACV is highly effective against oral herpes with negligible drug failure, emergence of viral mutants resistant to TK in 7-14% of ocular HK patients is compelling. A new antiviral directed against a different HSV-1 target is needed to circumvent this dilemma. One novel class of antiviral targets is the processivity factors (PFs) that are essential for tethering their polymerases (Pols) to the template to enable continuous DNA synthesis. Our objective is to develop a topical drug that specifically targets the HSV-1 PF as a means of preventing HK. Initially, we identified small molecules that blocked processive DNA synthesis in vitro, but struck a roadblock in our attempts to improve upon potency and toxicity. We thus made a paradigm shift to focus on developing a stapled peptide that will mechanistically prevent the PF (UL42) of HSV-1 from functionally interacting with its cognate Pol (UL30). Stapled peptides are a new class of therapeutics that are applicable for targeting protein-protein interactions that often display as flat surfaces which are difficult for small molecules to bind efficiently. In particular, stapled ?-helical peptides have demonstrated beneficial properties for drug discovery including stabilized conformations to effectively engage their targets while resisting proteolysis. When co-crystallized with UL42 PF, the extreme C- terminus of UL30 Pol was shown to form an ?-helix, where one face makes multiple bonds with several residues of UL42 while the other face is solvent exposed. As a start, we now have synthesized several C-Pol ?-helical peptides that differ by the position of the staple as well as by deletion, addition or substitution of specific residues. These peptides were shown to specifically block HSV-1 processive DNA synthesis in vitro and inhibit HSV-1 infection in human corneal epithelial and BSC-1 cells. The stapled peptides were unable to block in vitro processive DNA synthesis or cell infection by a different DNA virus. While we are able to achieve an acceptable IC50 (1.1 µM), the selectivity index (SI, 14.2) needs to be improved. The goal of this project is to develop a stapled ?-helical C-Pol peptide with an IC50 <1 µM; HC50>200 µM and SI>100 and a greater than 100-fold reduction in viral burden in human ocular organotypic corneal cultures. The stapled peptides will also be tested for solubility, aggregation, helicity, protease resistance and cell entry. Recent detailed knowledge and statistical analysis of large numbers of stapled peptides provides the optimal percent ranges for hydrophobicity, helicity and pI, which are the most important parameters for cell entry with minimal damage to the cell membrane. We will incorporate this knowledge towards our long-range goal of producing a stapled peptide therapeutic to meet the strong clinical need for a new drug to treat Herpes Keratitis.
