Chemoprotection Against Aminoglycoside-Induced Ototoxicity

描述（由申请人提供）：氨基糖苷类抗生素（AGAs）是临床上治疗危及生命的革兰氏阴性细菌感染的主要药物。AGAs广泛可用且经济；然而，由于耳毒性和肾毒性，它们的使用受到限制。我们试图确定能够阻断氨基糖苷耳毒性而不阻断其抗菌活性的药物。利用斑马鱼（Danio rerio）体内神经肥大试验，我们从多种药物样化合物库中鉴定出两种结构相关的化合物（PROTO1和2），这些化合物可保护斑马鱼机械感觉神经肥大细胞免受新霉素诱导的细胞死亡。PROTO1/2的保护作用是剂量依赖性的，而不是由于抑制AGA进入斑马鱼神经鞘。此外，PROTO1和PROTO2都不能抑制AGA在大肠杆菌中的抗菌活性。PROTO1在动物体内是无毒的，并在外植的小鼠胞体和人肾细胞中产生AGA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，确定保护药物的分子靶点，并确定参与aga诱导耳毒性和PROTO1/2预防其的分子途径。具体而言，在Aim 1中，我们将合成和评估PROTO1/2类似物的毛细胞保护作用，目的有两个：1)提高保护活性的效力；2).建立结构活性关系（SAR），使我们能够修改PROTO1/2用于目标识别研究（目标3）。在Aim 2中，我们将使用人肾细胞、斑马鱼神经肥大毛细胞和外植的小鼠胞体进行aga诱导的毒性试验。在Aim 3中，我们将对proto1 /2衍生药物进行体内临床前评估。在Aim 4中，我们将使用SAR数据设计和合成基于PROTO1/2的亲和捕获试剂，并将进行靶标鉴定研究。这些融合和互补的研究将有助于基于靶标的毛细胞保护剂的开发，并扩大对aga诱导的细胞损伤的分子认识。许多药物的使用受到严重副作用的限制。我们已经发现了一种化合物，可以帮助减少一类重要抗生素引起的听力损失和肾脏损害。本提案的研究将开发出更有效的保护化合物，确定这些化合物如何起作用，并为其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.