A new molecular therapy against ocular herpes

单纯疱疹病毒1型（HSV-1）是导致感染性失明的主要原因， 也是美国角膜移植的重要适应症。目前的治疗方案包括阿昔洛韦 及其衍生物更昔洛韦和膦甲酸。所有这些药物主要作用于病毒胸苷激酶， 病毒DNA复制，本质上具有类似的作用机制。虽然这些选择是有效的， 显示出减少眼部HSV-1感染（包括疱疹性基质角膜炎（HSK））的希望，药物的出现 近年来的耐药性在眼部HSV-1患者的临床护理中引起了严重的困扰。作为 因此，对于开发针对HSV-1的新治疗，尤其是针对HSV-1的新治疗， 依赖于新的行动模式。在过去的3年资助期内，我们已经确定了两名候选人的特征 针对HSV-1感染的两个不同阶段：病毒进入和病毒蛋白合成。针对病毒进入a 开发了靶向HSV-1糖蛋白D（gD）的高效抗HSV-1适体（DApt）。同样地， 我们首次证明了小分子PDK-1抑制剂BX 795可以阻断HSV-1蛋白， 合成.这两种候选人进行了测试，在体内局部治疗角膜HSV-1感染，我们报告 显著改善疾病预后。下一个资助期的主要目标是：（1）改善 DApt的体内疗效，（2）了解BX 795抗病毒作用的分子基础，以及（3）开发 BX 795用于眼疱疹的全身治疗。在第一个具体目标中，我们提出了几种改进方法， 功效我们将设计治疗性隐形眼镜，以持续输送DApt，改进DApt配方， 包括渗透剂如环糊精以增加适体被递送进入的深度 角膜组织，并开发更高的功效（协同或相加）的药物组合，使用DApt和 BX 795或核苷类似物。在第二个具体目标中，我们将解码分子机制 使用有偏和无偏方法，负责BX 795的抗病毒活性。把注意力集中在帽子上- 负责病毒蛋白质合成的依赖性翻译途径将构成偏倚方法;细胞- 游离和全细胞蛋白质组学定量质谱分析及翻译后研究 修改将构成无偏见的方法。在第三个具体目标中，我们将全面研究 静脉内施用的BX 795的药代动力学和毒理学特征。利用这些数据，我们最终 了解有效抑制原发性和继发性肿瘤所需的BX 795的安全有效剂量， 使用角膜感染的鼠模型重新激活HSV-1感染。综合我们的研究 将建立新的抗病毒机制，帮助设计新的预防和治疗方法， 眼睛的HSV-1感染。

Herpes simplex virus type-1 (HSV-1) with limited treatment options is a leading cause of infectious blindness and an important indication for corneal transplants in the US. The current treatment options include acyclovir and its derivatives, ganciclovir and foscarnet. All these drugs primarily act upon viral thymidine kinase to inhibit viral DNA replication and in essence have a similar mechanism of action. While these options are effective and show promise in reducing ocular HSV-1 infection including herpes stromal keratitis (HSK), emergence of drug resistance in the recent years has caused significant distress in the clinical care of ocular HSV-1 patients. As a result, there exists an unmet need for the development of new treatments against HSV-1 especially the ones that rely on novel modes of action. In our previous 3-year funding period we have characterized two candidates that target two different stages of HSV-1 infection; viral entry and viral protein synthesis. Against viral entry a highly effective anti-HSV-1 aptamer (DApt) was developed that targets HSV-1 glycoprotein D (gD). Similarly, we demonstrated for the first time that a small molecule PDK-1 inhibitor, BX795, blocks HSV-1 protein synthesis. Both candidates were tested in vivo for topical treatment of corneal HSV-1 infection and we reported significant improvements in disease prognosis. The major goals for the next funding period are to: (1) improve the in vivo efficacy of DApt, (2) understand the molecular basis of antiviral action by BX795, and (3) develop BX795 for systemic treatment of ocular herpes. In the first specific aim, we propose several ways to improve efficacy. We will engineer therapeutic contact lenses for sustained delivery of DApt, improve DApt formulation to include permeating agents such as cylodextrins to increase the depth to which the aptamer is delivered into the corneal tissue, and develop higher efficacy (synergistic or additive) drug combinations using DApt and BX795 or a nucleoside analog. In the second specific aim, we will decode the molecular mechanisms responsible for the antiviral activity of BX795 using both biased and unbiased approaches. Focus on the cap- dependent translation pathways responsible for viral protein synthesis will constitute the biased approach; cell- free and whole cell proteomic analyses using quantitative mass spectrometry and study of post translational modifications will constitute unbiased approaches. In the third specific aim, we will comprehensively study the pharmacokinetic and toxicological profiles of intravenously administered BX795. Using this data, we will finally understand the safe and effective dosage of BX795 required for the effective inhibition of primary and reactivated HSV-1 infection of the cornea using murine models of corneal infection. Taken together our studies will establish novel antiviral mechanisms and help design new prophylactic and therapeutic ways to control HSV-1 infection of the eye.