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

摘要 眼部感染单纯疱疹病毒-1(HSV-1)可导致疱疹角膜炎(香港)，这是 世界范围内导致角膜失明的主要原因。眼部疱疹感染通常反复发作，最终导致 进行性角膜疤痕和视力丧失。黄金标准疗法是以阿昔洛韦(ACV)为靶点的 单纯疱疹病毒1型胸苷激酶(TK)。虽然阿昔洛韦对口腔疱疹非常有效，药物可以忽略不计 失败，在7-14%的眼科HK患者中出现对TK耐药的病毒突变是令人信服的。一种新的 需要针对不同的HSV-1靶点的抗病毒药物来绕过这一困境。一类新奇的 抗病毒靶标是将它们的聚合酶(POL)拴在 模板，以实现持续的DNA合成。我们的目标是开发一种特效的外用药物 以HSV-1 PF为目标，作为预防香港的一种手段。最初，我们确定了阻止 在体外进行DNA合成，但在我们试图提高效力和 毒性。因此，我们进行了范式转换，将重点放在开发一种可以机械地 阻止HSV-1的PF(UL42)与其同源POL(UL30)在功能上相互作用。装订多肽 是一类新的疗法，适用于靶向蛋白质-蛋白质相互作用，经常表现为 作为小分子难以有效结合的平坦表面。特别是，装订的-螺旋 多肽已显示出有益于药物发现的特性，包括稳定的构象以 有效地吸引他们的目标，同时抵抗蛋白质分解。当与UL42PF共结晶时，极C- UL30Pol的末端形成一个-螺旋，其中一个面与多个面形成多个键 当另一面暴露在溶剂中时，会留下UL42的残留物。作为开始，我们现在已经合成了几个C-Pol -不同基序位置以及缺失、添加或取代的螺旋多肽 特定残留物。这些多肽被证明在体外特异性地阻断HSV-1过程中的DNA合成 抑制HSV-1在人角膜上皮细胞和BSC-1细胞中的感染。装订的多肽不能 阻断体外进行的DNA合成或细胞感染不同的DNA病毒。当我们能够 要达到可接受的IC50(1.1微米)，则需要提高选择性指数(SI，14.2)。这样做的目的是 该项目是开发一种具有IC501微米和200微米的α-螺旋C-POL多肽和SI100和 在人眼器官型角膜培养中，病毒负荷减少了100倍以上。订书机 还将测试多肽的溶解性、聚集性、螺旋性、蛋白酶抗性和细胞进入。近期 对大量装订多肽的详细知识和统计分析提供了最佳百分比 疏水性、螺旋度和等电点的范围，这是最重要的单元格输入参数 对细胞膜的破坏。我们将把这些知识融入到我们生产的长期目标中 满足临床对治疗疱疹角膜炎新药的强烈需求的装订多肽疗法。

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 α-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.