Development of Cell-Based High-Throughput Chemical Screens for Protection Against Cisplatin-Induced Ototoxicity.
Various compounds have been tested in recent years for protection against cisplatin-induced hearing loss, but no compound has yet been FDA approved for clinical use in patients. Towards this goal, we developed an unbiased, high-throughput, mammalian cochlear cell-based chemical screen that allowed quantification of the protection ability of bioactive compounds and ranked them for future testing ex vivo in cochlear explant cultures and in vivo in animal models. In our primary screens, protection in the HEI-OC1 organ of Corti immortalized cell line was measured by the ability of each compound to inhibit caspase-3/7 activity triggered by cisplatin treatment (50 μM cisplatin for 22 h). A total of 4385 unique bioactive compounds were tested in a single dose of 8 μM and promising compounds were validated by dose response curves covering ten, 1:3 serial diluted concentrations. Primary hits were defined as having more than 60 % inhibition of the caspase-3/7 activity. Toxicity of the top compounds was measured by a CellTiter-Glo (CTG) assay that measured the viability of the cells in the presence of compound alone in similar dose responsive analysis. A combination of the caspase-3/7 inhibition activity assay (as measured by IC50) and the CTG viability assay (as determined by LD50) identified the top protective compounds in the HEI-OC1 cells. In the future, the top hits in our screens will be tested for their protective ability ex vivo in mouse cochlear explants and in vivo in animal models. Our mammalian cochlear cell-based, high-throughput chemical screening assays described here can be further modified and represent an initial successful step towards therapeutic intervention of hearing disorders, an unmet medical need of our society.
Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA.
Tal
T
Teitz
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA.
Asli N
AN
Goktug
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA.
Taosheng
T
Chen
Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA. jian.zuo@stjude.org.
Jian
J
Zuo
D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D064420:Drug-Related Side Effects and Adverse Reactions / Q000378:metabolism / Q000517:prevention & control*; D005786:Gene Expression Regulation / Q000187:drug effects; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D009925:Organ of Corti / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*
D000970:Antineoplastic Agents; D054852:Small Molecule Libraries; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D002945:Cisplatin
Caspase 3/7-Glo assay; Cell Titer Glo viability assay; Cisplatin; HEI-OC1 cells; Hair cells; IC50, LD50