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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的通过单纯疱疹病毒1（HSV-1）感染眼睛的眼睛会导致疱疹角膜炎（HK），这是全球角膜失明的主要原因。眼疱疹感染通常是复发的，并在进行性角膜疤痕和视力丧失。黄金标准治疗是针对的acyclovir（ACV） HSV-1胸苷激酶（TK）。尽管ACV对用可忽略不计的药物非常有效失败，在7-14％的眼HK患者中，抗TK具有抗TK具有抗性的病毒突变体是令人信服的。一个新需要针对不同的HSV-1靶标的抗病毒来避免这种困境。一类抗病毒靶标是加工性因子（PFS），这对于将其聚合酶（POL）绑定到模板启用连续DNA合成。我们的目标是开发一种专门的局部药物以HSV-1 PF为目标，以防止HK。最初，我们确定了阻塞的小分子在体外进行过程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，专注于开发一个阶段的肽，该肽将机械化防止HSV-1的PF（UL42）与其同源POL（UL30）在功能上相互作用。钉肽是一种新的理论，适用于靶向蛋白质 - 蛋白质相互作用，经常显示作为小分子很难有效结合的平坦表面。特别是上演螺旋肽已经证明了对药物发现的有益特性，包括稳定的构象在抵抗蛋白水解的同时有效地参与目标。与UL42 PF共结晶时，极端的C- 显示ul30 pol的末端形成一个螺旋，其中一张脸与多个粘结形成多个键 ul42的残留物，而另一个面则暴露了。首先，我们现在合成了几个C-POL 螺旋肽因主食的位置以及删除，添加或取代而有所不同特定残差。这些肽在体外表现出特异性阻断HSV-1处理器DNA合成并抑制人角膜上皮和BSC-1细胞中的HSV-1感染。上演的胡椒无法通过不同的DNA病毒阻断体外过程DNA合成或细胞感染。虽然我们能够达到可接受的IC50（1.1 µM），需要改进选择性指数（SI，14.2）。目标的目标项目是开发带有IC50 <1 µm的α-螺旋C-Pol胡椒粉； HC50> 200 µm和Si> 100和A 人眼有机角膜培养物中病毒伯嫩的降低超过100倍。钉了还将测试肽的溶解度，聚集，螺旋，抗蛋白酶耐药性和细胞进入。最近的大量分阶段的胡椒粉的详细知识和统计分析提供了最佳百分比疏水性，螺旋性和PI的范围，这是细胞进入的最重要参数细胞膜的损害。我们将向生产的远程目标纳入这些知识雄鹿肽疗法满足了对治疗疱疹性角膜炎的新药的强烈临床需求。