SBIR-STTR Award

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 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.

Public Health Relevance Statement:
NARRATIVE Herpes Keratitis is the leading cause of corneal blindness in the USA and the world. The significant emergence of acyclovir-resistant herpes virus in eye infections necessitates the development of a new, safe and effective drug. The goal of this research is to prevent loss of vision by continuing the development of a novel, n0n-toxic drug that specifically targets herpes virus that infects the eye.

Project Terms:
Acyclovir; Affinity; alpha helix; Antiviral Agents; Binding; Biological Assay; Blindness; cell injury; Cell membrane; Cell Proliferation; Cells; Chemistry; Clinical; Cornea; corneal epithelium; corneal scar; crosslink; Crystallization; Cytolysis; Development; DNA; DNA biosynthesis; DNA Viruses; drug discovery; drug resistant virus; Epithelial Cells; Eye; Eye Infections; Face; Failure; genital herpes; Goals; Gold; Half-Life; Herpesviridae; Herpesviridae Infections; Herpesvirus 1; Herpetic Keratitis; Human; Hydrophobicity; improved; In Vitro; indexing; Infection; Keratitis; Knowledge; Lead; Location; Measures; Molecular Conformation; mutant; novel; novel therapeutics; Oral; Patients; peptide drug; Peptide Hydrolases; Peptides; Permeability; Pharmaceutical Preparations; Polymerase; Positioning Attribute; Poxviridae; prevent; Production; Property; Protein Conformation; protein folding; protein protein interaction; Proteins; Proteolysis; Recurrence; Research; Resistance; Safety; Site; small molecule; small molecule libraries; Solubility; Solvents; Specificity; standard care; standard of care; Statistical Data Interpretation; Surface; Testing; Therapeutic; Thymidine Kinase; thymidine kinase 1; Tissue Viability; Toxic effect; uptake; Viral; Viral Load result; Virus; Virus Replication

Infection of the eye by Herpes Simplex Virus-1 (HSV-1) can result in Herpes Keratitis (HK), which is theleading cause of infectious corneal blindness worldwide. In the U.S., nearly 500,000 individualsexperience ocular herpes infections that are often recurrent and culminate in progressive corneal scarringand 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 theurgent need to discover a second drug directed against a different HSV-1 target. A new class of antiviraltargets are processivity factors (PFs) that are essential for tethering their cognate polymerases (Pols) tothe template to enable continuous DNA synthesis. During HSV-1 replication, the DNA-anchored PF bindsto the extreme C-terminus of Pol (C-Pol) to keep Pol from dissociating from the viral template. Whenco-crystallized with its PF, the C-terminus of the viral Pol (C-Pol) forms an α-helix, with one face makingmultiple bonds with several residues of PF while the other face is solvent exposed. Our goal is to developa 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 oftendisplay as long flat surfaces which are difficult for small molecules to bind efficiently. Peptide a-helicesare ideal structural motifs for incorporating chemical staples to provide rigidity to make natural bondswith residues on the target protein. Moreover, staples can create a protease shield to prolong residencetime. We have now engineered a stapled peptide of C-Pol (SPep7B) that blocks the mechanism ofprocessive 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 infectedcells 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 SPep7Binto preclinical development as a Topical to treat HK. This will be accomplished by conducting aseries of AIMS that include scaleup of SPep7B and evaluating two formulations for delivery of SPep7B intohuman 3D cultures and then ex-vivo excised rabbit cornea for permeability and toxicity. These studies areessential to confront the physiological and anatomical barriers to topical ocular delivery. In vivo studieswill evaluate SPep7B for tolerability, genotoxicity, PK, and repeat dosing. An efficacy study will evaluateSPep7B for clinical cure and viral clearing. Backup analogs of SPep7B will be prepared and evaluatedfor risk mitigation. A model for new antiviral drugs: Closely aligned with this approach is thepotential for using PFs and stapled peptides of other viruses for treating unmet medical needs causedby infections that lead to deleterious complications.

Public Health Relevance Statement:
NARRATIVE Herpes Keratitis is the leading cause of corneal blindness in the USA and the world. The significant emergence of acyclovir-resistant herpes virus in eye infections necessitates the development of a new, safe and effective drug. The goal of this research is to prevent loss of vision by continuing the development of a novel, n0n-toxic drug that specifically targets herpes virus that infects the eye.

Project Terms:
<3-D><3D><3-Dimensional><α-helix>

<3D cell culture><3D culture>