Department of Otolaryngology-HNS, University of Washington, Seattle, WA, USA; Virginia Merrill Bloedel Hearing Research Center, Seattle, WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Biological Structure, University of Washington, Seattle, WA, USA

■ OBJECTIVES: Identification of the molecular mediatorsinvolved in sensory hair cell death and survival, following ototoxic challenges, will yield a better understanding of the pathways and provide opportunities for therapeutic intervention. For this reason, we developed a whole organism drug toxicity-interaction screen to identify drug like small molecules that prevent aminoglycoside induced lateral line sensory hair cell death in zebrafish (Danio rerio).

■ METHODS:5-day post fertilization zebrafish larvae were exposed to 3μM FM 1-43 and 2μM Yo-Pro-1 to identify lateral line hair cells. Vital dye stained larvae were dispensed into 96 well glass bottom plates and pretreated with 10μM of compounds from a chemically diversesmall molecule library (10,960 compounds). Larvae were exposed to 200μM of neomycin and epifluorescent microscopy was used for visual assessment of hair cell integrity to identify compounds that protect against cell death. The dose-dependent toxicity and effectiveness, at increasing concentrations of neomycin, of the identified compounds was assessed. To evaluate their ability to provide protection in mammals, cultured utricles from three to six-week-old CBA/CaJ mice were pretreated with the exploratory compounds and substrate exposed controls were exposed to increasing doses of neomycin. We obtained post-treatment hair cell counts using immunohistochemistry with calbindin and calmodulin to identify surviving striolar and extrastriolar hair cells. Neomycin dose-dependent hair cell survival of compound pretreated and un-pretreated hair cells were compared.

■ RESULTS: We discovered two new benzothiophene carboxamides that significantly protect lateral line hair cells from neomycin-induced death. Both compounds protect hair cells over a wide range of aminoglycoside concentrations and over at least a 10- fold range of the benzothiophene carboxamide concentration. Neither compound altered the antibacterial properties of neomycin by minimum inhibitory and minimum bactericidal concentration assays. The benzothiophene carboxamides were then tested in a well documented in vitro assay using mature murine utricles and significant hair cell protection was observed.

■ CONCLUSIONS: Our findings represent 1.) a novel method by which high-throughput screening in one vertebrate organism can be translated to aid our understanding of the regulation of mammalian vestibular and auditory pathology and 2.) a viable method for identification of potential drug leads for therapeutic intervention.