Abstract
Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious bacterial infections but have use-limiting side effects including irreversible hearing loss. Here, we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo zebrafish assays and investigated its mechanism of protection which, we found, may be mediated by a block of the hair cell's mechanoelectrical transducer (MET) channel, the major entry route for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues were prepared and evaluated for their effect against AG-induced damage as well as their affinity for the MET channel. One derivative was found to confer greater protection than carvedilol itself in cochlear cultures and also to bind more tightly to the MET channel. At higher concentrations, both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting a good therapeutic window under in vivo conditions.
Highlights
The AGs can enter the sensory hair cells of the inner ear through both endocytic processes5 and specialized cation channels, the mechanoelectrical transducer (MET) channels that are located at the tips of the stereocilia and are responsible for the detection of sounds and body movements.6−9 The mechanism of ototoxicity is not fully understood and it differs amongst the various AGs, with neomycin and gentamicin, for instance, being shown to activate different cell-death pathways once inside zebrafish lateral line hair cells.10−12 Once inside the cell, they are thought to interact with various targets such as ribosomes, the endoplasmic reticulum, and mitochondria,13,14 leading to the production of cytotoxic levels of reactive oxygen species (ROS) which, in turn, cause apoptosis
Mouse cochlear cultures were used to assess whether carvedilol can protect mammalian hair cells from the death induced by exposure to 5 μM gentamicin for 48 h
A concentration of 5 μM gentamicin is optimal, as it is close to the estimated concentration of 1 μM gentamicin reached in the endolymph in vivo at the onset of ototoxic symptoms,25 and it kills >90% of the basal cells while sparing the apical cells, consistent with the predominantly highfrequency hearing loss observed in patients treated with AGs
Summary
Aminoglycosides (AGs) are broad-spectrum antibiotics widely prescribed to treat severe bacterial infections.− Despite being highly efficacious, they cause unfortunate side effects such as reversible nephrotoxicity and irreversible hearing loss, with the latter occurring in up to 25% of treated patients. The AGs can enter the sensory hair cells of the inner ear through both endocytic processes and specialized cation channels, the mechanoelectrical transducer (MET) channels that are located at the tips of the stereocilia and are responsible for the detection of sounds and body movements.− The mechanism of ototoxicity is not fully understood and it differs amongst the various AGs, with neomycin and gentamicin, for instance, being shown to activate different cell-death pathways once inside zebrafish lateral line hair cells.− Once inside the cell, they are thought to interact with various targets such as ribosomes, the endoplasmic reticulum, and mitochondria, leading to the production of cytotoxic levels of reactive oxygen species (ROS) which, in turn, cause apoptosis. It is this AGinduced hair cell death that underlies the hearing loss associated with clinical drug treatments. Compound 13 (dark green) was the most effective MET channel blocker of this series, providing almost 100% block of the MET current at all membrane potentials This result is in accordance with its protective effect, as 13 showed protection of the OHCs from gentamicin damage at Figure 9. One further property of the MET channel interaction that was investigated for both carvedilol and 13 was the kinetics of the block, to determine whether or not these compounds are open-channel blockers, similar to berbamine and D-tubocurarine, or can reside in the closed channel, similar to the permeant MET channel blocker FM1-43.6 The time course of the block is revealed by applying large force steps to the hair bundles both before and during exposure to the compound and recording the resulting currents. Neither of the compounds resulted in an observable reduction in gentamicin antimicrobial activity (Figure S2A−C)
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