Chemoprotection Against Aminoglycoside-Induced Ototoxicity

描述（由申请方提供）：氨基糖苷类抗生素（AGA）是临床治疗危及生命的革兰氏阴性细菌感染的主要药物。AGA广泛可用且高度经济;然而，由于耳毒性和肾毒性，它们的使用受到限制。我们试图确定药物，将阻止氨基糖苷类耳毒性，而不阻止其抗菌活性。使用在体内斑马鱼（Danio rerio）神经肥大测定，我们确定了两个结构相关的化合物（PROTO 1和2）从不同的，药物样化合物的库，保护斑马鱼机械感觉神经肥大细胞从新霉素诱导的细胞死亡。PROTO 1/2的保护作用是剂量依赖性的，并且不是由于抑制阿加吸收到斑马鱼神经突。此外，PROTO 1和PROTO 2均不抑制阿加在大肠杆菌中的抗菌活性。杆菌PROTO 1在动物中是无毒的，并且在破裂的小鼠椭圆囊和人肾细胞中赋予阿加抗性。在本申请中，我们提出了四个具体目标，以进行先导化合物的临床前优化，以确定保护性药物的分子靶点，并确定参与AGA诱导的耳毒性及其通过PROTO 1/2预防的分子途径。具体来说，在目标1中，我们将合成和评估PROTO 1/2类似物的毛细胞保护作用，有两个目标：1）。以增加保护活性的效力，和; 2）.开发一个结构活性关系（SAR），使我们能够修改PROTO 1/2的目标识别研究（目标3）。在目标2中，我们将开发使用人肾细胞，斑马鱼神经肥大毛细胞和小鼠椭圆囊的AGA诱导的毒性试验。在目标3中，我们将对PROTO 1/2衍生药物进行体内临床前评价。在目标4中，我们将利用SAR数据设计和合成基于PROTO 1/2的亲和捕获试剂，并进行靶标识别研究。这些融合和互补的研究将有助于开发靶向毛细胞保护剂，并扩大对AGA诱导的细胞损伤的分子理解。许多药物的使用受到严重副作用的限制。我们已经发现了一些化合物，可以帮助减少由一类重要的抗生素引起的听力损失和肾脏损伤。该提案中的研究将开发出更有效的保护性化合物，确定这些化合物如何发挥作用，并为它们作为药物的开发提供初步数据。

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics (AGAs) are a mainstay in clinical management of life-threatening Gram-negative bacterial infections. AGAs are widely available and highly economical; however, their use is limited due to ototoxicity and nephrotoxicity. We sought to identify agents that would block aminoglycoside ototoxicity without blocking its antimicrobial activity. Using an in vivo zebrafish (Danio rerio) neuromast assay, we identified two structurally related compounds (PROTO1 and 2) from a library of diverse, drug-like compounds that protect zebrafish mechanosensory neuromast cells from neomycin-induced cell death. The protective effect of PROTO1/2 is dose-dependent and is not due to inhibition of AGA uptake into zebrafish neuromasts. Furthermore, neither PROTO1 nor PROTO2 inhibit AGA antibacterial activity in E. coli. PROTO1 is non-toxic in animals and confers AGA resistance in explanted mouse utricles and human kidney cells. In this application we propose four Specific Aims to carry out pre-clinical optimization of the lead compounds, to identify the molecular targets of the protective drugs and determine the molecular pathways that are involved in both AGA-induced ototoxicity and its prevention by PROTO1/2. Specifically, in Aim 1, we will synthesize and evaluate the hair cell protective effects of PROTO1/2 analogues with two goals: 1). to increase the potency of the protective activity, and; 2). to develop a structure activity relationship (SAR) that would allow us to modify PROTO1/2 for target identification studies (Aim 3). In Aim 2, we will develop AGA-induced toxicity assays using human kidney cells, zebrafish neuromast hair cells and explanted mouse utricles. In Aim 3, we will carry out in vivo pre-clinical evaluation of PROTO1/2-derived agents. In Aim 4, we will use SAR data to design and synthesize an affinity capture reagent based on PROTO1/2 and will carry out target identification studies. These convergent and complementary studies will aid in the development of target-based hair cell protective agents and expand the molecular understanding of AGA-induced cell damage. The use of many drugs is limited by serious side effects. We have discovered compounds that may help reduce hearing loss and damage to kidneys caused by an important class of antibiotics. Studies in this proposal will develop more potent protective compounds, identify how these compounds work and provide preliminary data for their development as drugs.