Investigating the PROTO Targets in Regulating Drug-Induced Hair Cell Death

说明(申请人提供)：氨基糖苷类抗生素(AGA)被广泛用于治疗各种难治性细菌感染。AGAs最重要的副作用和治疗局限性是内耳损伤(耳毒性)和肾损伤(肾毒性)，前者可导致不可逆转的听力损失，后者可导致肾衰竭。预防AGA所致的耳毒性和肾毒性一直是生物医学研究的主要治疗目标。Julian Simon博士实验室的一个主要目标是确定耳保护性PROTO1类似物(PROTO1)的生物靶点，以确定它们在防止AGA诱导的内耳毛细胞死亡方面的作用机制。为了实现这一目标，实验室最近通过光亲和下拉结合质谱学确定了斑马鱼中的几个候选原生目标。在本奖学金提案中，三个特定目标旨在检验以下假设：即这些质谱学鉴定的Proto候选靶点之一在控制AGA诱导的毛细胞死亡和存活方面发挥关键作用：1)通过Western blotting和竞争下拉实验验证Proto与最有希望的质谱学确定的候选靶点之间的相互作用；2)使用表面等离子共振(SPR)生物传感器对Proto与其靶标之间的相互作用进行动力学分析；3)使用哺乳动物细胞系和条件基因敲除小鼠研究Proto靶点对AGA诱导的毛细胞毒性的影响。生物原靶的识别有望促进对调节毛细胞死亡和生存的细胞途径的理解，并促进新的治疗策略的发展，以预防听力和平衡障碍。该奖学金提案将在听力保护研究和临床前药物开发方面提供宝贵的实践经验和全面的知识，为作为听觉和生物医学研究领域的研究人员/科学家的独立研究生涯奠定坚实的基础。

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics (AGAs) are widely used in the treatment of a variety of refractory bacterial infections. The most important side effects and therapeutic limitations of AGAs are inner ear damage (ototoxicity) which can produce irreversible hearing loss and kidney damage (nephrotoxicity) which can lead to kidney failure. Prevention of AGA-induced ototoxicity and nephrotoxicity has been a major therapeutic objective in biomedical research. One major goal at the laboratory of Dr. Julian Simon is to identify the biological target of the otoprotective PROTO1 analogs (PROTO) to determine their mechanism of action in preventing AGA-induced inner ear hair cell death. To achieve this goal, the laboratory recently identified several candidate PROTO targets in zebrafish through photoaffinity pulldowns coupled with mass spectrometry. In this fellowship proposal, the three specific aims are designed to test the hypothesis that one of these mass spectrometry- identified PROTO candidate targets plays a critical role in controlling hair cell death and surviva in response to AGAs: 1) Validation of the interactions between PROTO and the most promising mass spectrometry-identified candidate targets using western blotting and competition pulldowns; 2) kinetic analysis of interactions between PROTO and its targets using a surface plasmon resonance (SPR) biosensor; 3) investigation of the effects of the PROTO targets on AGA-induced hair cell toxicity using mammalian cell lines and conditional knockout mice. Identification of the biological PROTO target is expected to advance the understanding of the cellular pathways that regulate hair cell death and survival and promote the development of new therapeutic strategies to prevent hearing and balance disorders. The fellowship proposal will provide an invaluable hands-on experience and comprehensive knowledge in hearing protection research and preclinical drug development paving a solid foundation towards an independent research career as an investigator/scientist in the field of auditory and biomedical research.