Development of Cell-Based High-Throughput Chemical Screens for Protection Against Cisplatin-Induced Ototoxicity

Abstract

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.

CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss

“…We used an immortalized cell line (HEI-OC1) derived from mouse cochleae (postnatal day 7 [P7]; Kalinec et al, 2003 ) to conduct an unbiased screen for compounds protective against cisplatin ototoxicity ( Teitz et al, 2016 ). We screened a bioactive library of 4,385 unique compounds, including 845 FDA-approved drugs ( Morfouace et al, 2014 ) at a concentration of 8 µM, cotreating the cells with 50 µM cisplatin ( Fig.…”

“…Pifithrin-α was chosen as a reference compound for the screen, as it provided good protection against cisplatin ototoxicity by inhibiting caspase-3 cleavage with an IC 50 of 17 µM (Fig. S1 D; Zhang et al, 2003 ; Ding et al, 2012 ; Teitz et al, 2016 ). This screen has been described in detail elsewhere ( Teitz et al, 2016 ).…”

“…S1 D; Zhang et al, 2003 ; Ding et al, 2012 ; Teitz et al, 2016 ). This screen has been described in detail elsewhere ( Teitz et al, 2016 ). Assays were first optimized at the bench.…”

“…Pifithrin-α was added to each plate as a screening quality control. Among the top hits were several previously characterized otoprotective agents, such as zVAD-FMK (a known general, irreversible protein inhibitor of caspases [ Teitz et al, 2016 ]), ebselen (currently being tested against noise injury in a clinical trial; https://clinicaltrials.gov/show/NCT01444846, accessed February 21, 2018), olsalazine sodium (a derivative of salicylate; Fig. S1 F), and cyanocobalamin (vitamin B12; Fig.…”

A kinase inhibitor library screen identifies novel enzymes involved in ototoxic damage to the murine organ of Corti

“…These include, in particular, immortalized mammalian cell lines derived from inner ear cells (e.g. [ 14 ]) and the zebrafish lateral line (e.g. [ 15 ]).…”