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

摘要 由单纯疱疹病毒-1（HSV-1）引起的眼部感染可导致疱疹性角膜炎（HK），这是角膜炎的主要症状。 全球传染性角膜失明的主要原因。在美国，近50万人 经历眼部疱疹感染，经常反复发作并最终导致进行性角膜瘢痕形成 和视力丧失治疗的金标准是靶向HSV-1胸苷激酶的阿昔洛韦（ACV （TK）。然而，在7-14%的眼部HK患者中出现ACV病毒耐药突变体， 迫切需要发现针对不同HSV-1靶标的第二种药物。一类新的抗病毒药物 靶标是持续合成能力因子（PF），其对于将其同源聚合酶（Pol）连接到 使DNA的连续合成成为可能。在HSV-1复制过程中，DNA锚定的PF结合 至Pol的极端C末端（C-Pol），以防止Pol与病毒模板解离。当 病毒Pol（C-Pol）的C-末端与PF共结晶，形成α-螺旋，其中一个面使 多个键与PF的几个残基，而另一面是溶剂暴露。我们的目标是开发 一种特异性靶向疱疹病毒PF的新型抗病毒药物，用于治疗ACV耐药HK。 钉合α-螺旋肽已经出现用于靶向蛋白质-蛋白质相互作用， 显示为长的平坦表面，其难以使小分子有效地结合。肽a-螺旋 是理想的结构基序，用于结合化学斯台普斯，以提供刚性，从而形成天然键 与靶蛋白上的残基结合。此外，斯台普斯可以产生蛋白酶屏障， 时间我们现在已经设计了一种C-Pol的钉合肽（SPep 7 B），其阻断了 体外进行性DNA合成和人器官型（3D）角膜上皮细胞中的HSV-1感染; 未装订的肽是非抑制性的。SPep 7 B显示出在感染的细胞中消除HSV-1病毒DNA。 细胞，而无法阻止不同的病毒。重要的是，SPEp 7 B表现出不可检测的毒性， (CC50>100µM）。我们的目标是进入SPEP 7 B 作为治疗HK的外用药进行临床前开发。这将通过进行一项 一系列AIMS，包括SPep 7 B的规模扩大和评估两种用于将SPep 7 B递送到 人3D培养物，然后离体切除的兔角膜的渗透性和毒性。这些研究 这对于克服局部眼部递送的生理和解剖障碍是必不可少的。体内研究 将评价SPep 7 B的耐受性、遗传毒性、PK和重复给药。一项功效研究将评估 SPep 7 B用于临床治愈和病毒清除。将制备并评价SPEp 7 B的备用类似物 来降低风险。新型抗病毒药物的模型：与这种方法密切相关的是 使用PF和其他病毒的钉合肽治疗未满足的医疗需求的可能性 导致有害并发症的感染

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.