Optimizing a Stapled-Peptide That Specifically Targets HSV-1 to Treat Herpes Ocular Keratitis

ABSTRACT Infection of the eye by Herpes Simplex Virus-1 (HSV-1) can result in Herpes Keratitis (HK), which is the leading cause of infectious corneal blindness worldwide. In the U.S., nearly 500,000 individuals experience ocular herpes infections that 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). However, emergence of ACV viral resistant mutants in 7-14% of ocular HK patients has created the urgent need to discover a second drug directed against a different HSV-1 target. A new class of antiviral targets are processivity factors (PFs) that are essential for tethering their cognate polymerases (Pols) to the template to enable continuous DNA synthesis. During HSV-1 replication, the DNA-anchored PF binds to the extreme C-terminus of Pol (C-Pol) to keep Pol from dissociating from the viral template. When co-crystallized with its PF, the C-terminus of the viral Pol (C-Pol) forms an α-helix, with one face making multiple bonds with several residues of PF while the other face is solvent exposed. Our goal is to develop a novel antiviral drug that specifically targets herpes PF for the purpose of treating ACV resistant HK. Stapled a-helical peptides have emerged for use in targeting protein-protein interactions that often display as long flat surfaces which are difficult for small molecules to bind efficiently. Peptide a-helices are ideal structural motifs for incorporating chemical staples to provide rigidity to make natural bonds with residues on the target protein. Moreover, staples can create a protease shield to prolong residence time. We have now engineered a stapled peptide of C-Pol (SPep7B) that blocks the mechanism of processive DNA synthesis in vitro and HSV-1 infection in human organotypic (3D) corneal epithelial cells; the unstapled peptide is non-inhibitory. SPep7B was shown to eliminate HSV-1 viral DNA in infected cells while failing to block a different virus. Importantly, SPep7B exhibits undetectable toxicity (CC50>100µM) by two different assays in human ocular epithelial cells. Our GOAL is to enter SPep7B into preclinical development as a Topical to treat HK. This will be accomplished by conducting a series of AIMS that include scaleup of SPep7B and evaluating two formulations for delivery of SPep7B into human 3D cultures and then ex-vivo excised rabbit cornea for permeability and toxicity. These studies are essential to confront the physiological and anatomical barriers to topical ocular delivery. In vivo studies will evaluate SPep7B for tolerability, genotoxicity, PK, and repeat dosing. An efficacy study will evaluate SPep7B for clinical cure and viral clearing. Backup analogs of SPep7B will be prepared and evaluated for risk mitigation. A model for new antiviral drugs: Closely aligned with this approach is the potential for using PFs and stapled peptides of other viruses for treating unmet medical needs caused by infections that lead to deleterious complications.