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

Abstract

Ototoxicity is a significant side effect of a number of drugs, including the aminoglycoside antibiotics and platinum-based chemotherapeutic agents that are used to treat life-threatening illnesses. Although much progress has been made, the mechanisms that lead to ototoxic loss of inner ear sensory hair cells (HCs) remains incompletely understood. Given the critical role of protein phosphorylation in intracellular processes, including both damage and survival signaling, we screened a library of kinase inhibitors targeting members of all the major families in the kinome. Micro-explants from the organ of Corti of mice in which only the sensory cells express GFP were exposed to 200 μM of the ototoxic aminoglycoside gentamicin with or without three dosages of each kinase inhibitor. The loss of sensory cells was compared to that seen with gentamicin alone, or without treatment. Of the 160 inhibitors, 15 exhibited a statistically significant protective effect, while 3 significantly enhanced HC loss. The results confirm some previous studies of kinase involvement in HC damage and survival, and also highlight several novel potential kinase pathway contributions to ototoxicity.

Screening Mammalian Cochlear Hair Cells to Identify Critical Processes in Aminoglycoside-Mediated Damage

“…It would be advantageous to be able to screen larger numbers of compounds in vitro using differentiated mammalian HCs. To meet this need, we developed an in vitro assay using micro-explants of neonatal mouse oC (see Noack et al, 2017 ; Ryals et al, 2017 ; for details of methods). In brief, oC was harvested from the cochlea of postnatal day 3–5 pou4f3 /eGFP (enhanced GFP) transgenic mouse pups, which selectively express eGFP in HCs (Masuda et al, 2011 ).…”

“…These limitations must be considered when interpreting the results from micro-explants assay studies. The oC micro-explant assay was used to screen a redox compound library (Noack et al, 2017 ) and a kinase inhibitor library (Ryals et al, 2017 ).…”

“…Many damage and survival pathways can be activated by exposure to ROS (Martindale and Holbrook, 2002 ). Therefore, we screened a library of 160 kinase inhibitors targeting specifically all the major families in the kinome, using the oC micro-explant assay (Ryals et al, 2017 ). Of those, 15 exhibited a significant protective effect: epidermal growth factor receptor (EGFR) Inhibitors I and PP3, PDGF Inhibitors I and IV, Compound C, p38 Inhibitors I and III, AKT inhibitors V and X, Cdk4 Inhibitor II, Fascaplysin, Flt-3 inhibitor, Compound C, Casein Kinase 2 Inhibitor III and protein kinase R (PKR) Inhibitor.…”

“… Schematic diagram illustrating the various intracellular damage signaling cascades (red arrows) implicated by the kinase inhibitor screen (Ryals et al, 2017 ), and the points at which the inhibitors identified in the screen (green text) are predicted to intercede. Of the 160 inhibitors, 15 exhibited a statistically significant protective effect (ANOVA).…”

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

“…Recently, Walters et al screened for p27 Kip1 transcriptional inhibitors to enhance regeneration of supporting cells in the ear by employing HeLa cells transfected with a luciferase reporter plasmid driven by the p27 Kip1 promoter [ 4 ] and Edge et al had used the embryonic kidney cell line (HEK) expressing a luciferase gene controlled by an Atoh1 enhancer and promoter sequences to screen for compounds activating Atoh1 gene expression in cells and thereby increasing regeneration [ 5 ]. More recently, murine cochlear micro-explants have been directly used for a medium-throughput screen for protective compounds against aminoglycoside ototoxicity [ 6 ]. Interestingly, screens for protective compounds against ototoxic insults have been successfully performed in zebrafi sh model systems for identifying compounds that can protect the lateral line neuromasts from aminoglycosides [ 7 -9 ] or cisplatin [ 9 -11 ].…”