To screen for compounds that protect hair cells against ototoxic drugs, only minor
modifications are necessary to our toxicity screens. Our initial screens for hair cell
protectants were designed to identify compounds that conferred robust protection to
neomycin-treated hair cells [26]. We initially focused on small molecules within the
Chembridge Diverset E library. To efficiently screen a sample of 10,960 compounds from
the library, compounds were multiplexed five per well and then reassessed individually
when protection was observed. Two compounds, PROTO1 and PROTO2, exhibited robust
protection of lateral line hair cells over a broad range of neomycin concentrations. Further
testing of these compounds in mice revealed that they also protected hair cells in neomycintreated
utricular explants, validating the zebrafish lateral line as a model for discovery of
novel otoprotective drugs. Both PROTO1 and PROTO2 are related benzothiophene
carboximides, raising the possibility that they may confer their protective effects through
action on the same target.