Proceedings of The Physiological Society

Mitochondria: Form and function (London, UK) (2017) Proc Physiol Soc 38, PC05

Poster Communications

Identifying Novel Otoprotectants and Investigating their Mechanism of Action

M. O'Reilly1, N. Kirkwood1, M. Derudas2, E. Kenyon1, G. Richardson1, C. Kros1

1. Sussex Neuroscience, University of Sussex, Brighton, United Kingdom. 2. Sussex Drug Discovery Centre, University of Sussex, Brighton, East Sussex, United Kingdom.

Background - The aminoglycosides (AGs) are a class of broad-spectrum antibiotic widely prescribed to treat life-threatening bacterial infections such as septicaemia. Whilst the AGs are highly efficacious, they are ototoxic. The AGs enter sensory hair cells of the inner ear through their mechano-electrical transducer (MET) channels. Once inside the cell they are thought to trigger mitochondrial dysfunction, thereby initiating apoptosis. To identify potential otoprotectants that might prevent AG-induced ototoxicity I have conducted a screen of the Tocris ion channel library. Methods - Two assays were used, whereby gentamicin (a commonly-prescribed AG) was applied with or without the compounds for the duration of the experiment: 1) a 6-hour assay monitoring the death of zebrafish lateral line hair cells and 2) a 48-hour assay to assess the death of hair cells in mouse cochlear cultures. Electrophysiological recordings were subsequently carried out, examining ionic currents from mouse cochlear hair cells, to evaluate potential mechanisms of protection. Furthermore, an assay using isolated rat liver mitochondria was used to assess the effect that gentamicin may have on respiratory activities and whether any of the identified protectants work by combating this effect. Results - Of the 160 compounds tested, 13 consistently protected mouse cochlear hair cells from 5 µM gentamicin when tested at a concentration of 50 µM. These 13 compounds were re-screened at a higher concentration (100 µM) in the absence of gentamicin. Three of the 13 were without obvious toxic side effects and of these, all three protected at 10 µM, two at 500 nM, and only one (XE 991) at 10 nM. These three compounds were also protective at 100 µM against 10 µM gentamicin in the zebrafish assay. Electrophysiological recordings revealed that only one of the compounds blocks the MET channels whereas all three block the basolateral potassium channels (IK,neo) to varying degrees. For XE 991, which does not block the MET channel at 50 µM, the modest level of IK,neo block (~40% at 30 µM) argues against depolarisation as the mechanism of protection. Mitochondrial investigation shows that gentamicin stimulates state four (ADP-limited) and inhibits state three (ADP-dependent) respiration. Preliminary experiments show that the second most successful Tocris compound combats this effect, preventing the gentamicin-induced stimulation of state four respiration. Conclusions - Our screen of known ion channel blockers has identified compounds that protect hair cells from AG-induced death in vitro, with multiple mechanisms of action. One of these, XE 991, protects hair cells at nanomolar concentrations and may prove to be a suitable lead compound for the development of a clinically viable otoprotectant.

Where applicable, experiments conform with Society ethical requirements