DEVELOPMENT OF A NOVEL ANTIVIRAL TO TREAT AND PREVENT ACYCLOVIR RESISTANCE IN HUMAN OCULAR 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。在美国，近50万人经历了 经常复发并最终导致进行性角膜瘢痕形成和丧失的眼部疱疹感染 视觉。治疗HK的金标准是使用靶向HSV-1的阿昔洛韦（ACV）治疗 胸苷激酶（TK）。虽然ACV对口腔和生殖器疱疹都非常有效， 尽管药物失败可以忽略不计，但在7-14%的眼部HK患者中出现病毒耐药突变体， 引人注目。值得注意的是，耐药机制与突变改变直接相关 从对ACV无反应的HK患者中分离的HSV-1的TK基因。这种高强度的抵抗 如果ACV与针对不同靶点的第二种抗病毒药物联合使用， 逃避药物的突变体一类新的抗病毒靶点是持续合成因子（PF）， 对于保持其同源病毒聚合酶（Pos）与模板连接以进行连续的病毒复制是必不可少的。 DNA合成我们的目标是开发早期的主要抗病毒化合物， HSV-1合成能力因子（PF）。我们现在已经确定了一个有效的早期铅Z9445，它阻止 HSV-1感染，EC 50为280 nM。我们还确定了结构多样的后备线索。 随着动物模型验证的未来需求，我们处于一个独特的地位， 来自宾夕法尼亚大学兽医学院的感染同源猫病毒的猫患者（2期） 疱疹病毒-1（FHV-1）。这些猫科患者提供了人类疱疹的密切和自然的模型 角膜炎，可长期随访复发性感染。我们现在克隆了 FHV-1 PF/Pol基因与HSV-1具有相当大的同源性，并已证实， 我们的大多数阻断HSV-1的化合物也以相似的效力阻断FHV-1感染。 尽管猫科动物模型非常有吸引力，但我们的实验设计是集中的， 对HSV-1（但不是FHV-1）具有单一上级优势的药物将使用传统的 模型该提案的目的是紧密相连的，采用药物化学作为迭代 通过产生新的类似物来提高Z9445的治疗指数的方法， 没有检测到毒性的效力。基于Z9445与已知晶体对接的合理设计 PF靶蛋白的结构将是生产新Leads的一种手段。备份销售线索还将 如果需要增强溶解度或稳定性等特性，则应进行优化（第2阶段）。类似物 将测试体外阻断进行性DNA合成，与PF靶蛋白的物理结合， 毒性和细胞增殖，原代猫角膜上皮细胞和人体中的抗病毒活性 3D角膜组织。将进一步测试人3D角膜组织的渗透性和组织学。 .

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. 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 of care for HK is treatment with Acyclovir (ACV) that targets HSV-1 thymidine kinase (TK). Although ACV is extremely effective in both oral and genital herpes with negligible drug failure, the emergence of viral resistant mutants in 7-14% of ocular HK patients is compelling. Significantly, the mechanism of drug resistance is directly related to mutational alterations in the TK gene of HSV-1 isolated from HK patients unresponsive to ACV. This high level of resistance can be prevented if ACV is combined with a second antiviral directed against a different target to block mutants that escape either drug. One new class of antiviral targets is the processivity factors (PFs) that are essential to keep their cognate viral polymerases (Pols) tethered to the template for continuous viral DNA synthesis. Our objective is to develop early lead antiviral compounds that are directed against the HSV-1 processivity factor (PF). We have now identified a potent early Lead Z9445, which blocks infection of HSV-1 with an EC50 of 280 nM. We have also identified structurally diverse backup leads. With the future requirement of animal model validation, we are in a unique position to treat multiple feline patients (Phase 2) from the UPenn Vet School who are infected with the homologous Feline Herpes Virus-1 (FHV-1). These feline patients provide a close and natural model of human herpes keratitis that can be followed for extended periods for recurrent infections. We have now cloned the FHV-1 PF/Pol genes which share considerable homologies to those of HSV-1 and have validated that the majority of our compounds that block HSV-1, also block FHV-1 infection with similar potencies. Even though the feline model is highly attractive, our experimental design is focused, such that analogs that are singly superior for HSV-1 (but not FHV-1) will be developed further (Phase 2) using traditional models. The Aims of this proposal are tightly linked, employing medicinal chemistry as an iterative process to improve the therapeutic index of Z9445 by generating new analogs that have increased potency with no detectable toxicities. Rational design based on docking of Z9445 to the known crystal structure of the PF target protein will be one means on producing new Leads. Backup Leads will also be optimized should properties such as solubility or stability need to be enhanced (Phase 2). Analogs will be tested for blocking processive DNA synthesis in vitro, physical binding to the PF target protein, toxicity and cell proliferation, antiviral activities in primary feline corneal epithelial cells and in the human 3D corneal tissue. The human 3D corneal tissue will be tested further for permeability and histology. .