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

优化专门针对 HSV-1 的钉合肽以治疗疱疹性眼角膜炎

• 批准号: 9909297
• 负责人: ROBERT Paul RICCIARDI
• 金额: $ 30.91万
• 依托单位: FOX CHASE CHEMICAL DIVERSITY CENTER, INC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909297
• 关键词: Acyclovir; Affinity; Antiviral Agents; Binding; Biological Assay; Blindness; Cell Proliferation; Cell membrane; Cells; Chemistry; Clinical; Cornea; Crystallization; Cytolysis; DNA; DNA Viruses; DNA biosynthesis; Development; Epithelial Cells; Eye; Eye Infections; Face; Failure; Goals; Gold; Half-Life; Herpesviridae; Herpesviridae Infections; Herpesvirus 1; Herpetic Keratitis; Human; Hydrophobicity; In Vitro; Infection; Keratitis; Knowledge; Lead; Location; Measures; Molecular Conformation; Oral; Patients; Peptide Hydrolases; Peptides; Permeability; Pharmaceutical Preparations; Polymerase; Positioning Attribute; Poxviridae; Production; Property; Protein Conformation; Proteins; Proteolysis; Recurrence; Research; Resistance; Safety; Site; Solubility; Solvents; Specificity; Statistical Data Interpretation; Surface; Testing; Therapeutic; Thymidine Kinase; Tissue Viability; Toxic effect; Viral; Viral Load result; Virus; Virus Replication; alpha helix; cell injury; corneal epithelium; corneal scar; crosslink; drug discovery; drug resistant virus; genital herpes; improved; indexing; mutant; novel; novel therapeutics; peptide drug; prevent; protein folding; protein protein interaction; small molecule; small molecule libraries; standard care; standard of care; thymidine kinase 1; uptake

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.

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。 全球角膜失明的主要原因。眼疱疹感染经常复发， 进行性角膜瘢痕形成和视力丧失。金标准治疗是阿昔洛韦（ACV）， HSV-1胸苷激酶（TK）。虽然ACV对口腔疱疹非常有效， 失败后，在7-14%的眼部HK患者中出现对TK具有抗性的病毒突变体是引人注目的。一个新 需要针对不同HSV-1靶点的抗病毒药物来避免这种困境。一种新型的 抗病毒靶点是持续合成因子（PF），其对于将其聚合酶（Pol）连接到 模板，使连续的DNA合成。我们的目标是开发一种局部药物， 靶向HSV-1 PF作为预防HK的手段。最初，我们发现了一些小分子， 在体外进行DNA合成，但在我们试图提高效力和 毒性因此，我们进行了范式转变，专注于开发一种钉合肽， 防止HSV-1的PF（UL 42）与其同源Pol（UL 30）功能性相互作用。锁环肽 是一类新的治疗药物，适用于靶向蛋白质-蛋白质相互作用， 因为平坦表面难以使小分子有效结合。特别地，钉合的双螺旋 肽已经显示出有益的药物发现性质，包括稳定的构象， 在抵抗蛋白水解的同时有效地与它们的靶结合。当与UL 42 PF共结晶时， UL 30 Pol的末端显示形成α-螺旋，其中一个面与几个面形成多个键， UL 42的残留物，而另一面是溶剂暴露的。作为一个开始，我们现在已经合成了几个C-Pol 通过钉合位置以及通过缺失、添加或取代 具体的残留物。这些肽在体外显示出特异性地阻断HSV-1进行性DNA合成 并抑制HSV-1在人角膜上皮和BSC-1细胞中的感染。钉合肽不能 阻断体外进行性DNA合成或不同DNA病毒的细胞感染。虽然我们能够 要达到可接受的IC 50（1.1 µM），需要提高选择性指数（SI，14.2）。这个目标 该项目是开发一种钉合α-螺旋C-Pol肽，其IC 50 <1 µM; HC 50>200 µM且SI>100，并且 在人眼器官型角膜培养物中病毒负荷降低超过100倍。缝合的 还将测试肽的溶解性、聚集性、螺旋性、蛋白酶抗性和细胞进入性。最近 大量钉合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pI的范围，这是细胞进入的最重要参数， 对细胞膜的损伤。我们将把这些知识用于我们的长期目标， 一种钉合肽治疗剂，以满足对治疗疱疹性角膜炎的新药的强烈临床需求。