Identification of a series of hair-cell MET channel blockers that protect against aminoglycoside-induced ototoxicity

Abstract

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae selecting those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits share structural features and all block, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a known route of aminoglycoside entry into hair cells. Further characterisation of one of the strongest MET-channel blockers, UoS-7692, revealed it additionally protects against kanamycin and tobramycin, and does not abrogate the bactericidal activity of gentamicin. UoS-7692 behaves, like the aminoglycosides, as a permeant blocker of the MET channel, significantly reduces gentamicin-Texas Red loading into OHCs, and preserves lateral-line function in neomycin-treated zebrafish. Trans-tympanic injection of UoS-7692 protects mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserves hearing. The results confirm the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides, and provide a series of hit compounds that will inform the design of future otoprotectants.

Chronic neurotransmission increases the susceptibility of lateral-line hair cells to ototoxic insults

“…After initial entry through MET channels, aminoglycosides accumulate and remain confined inside hair cells where they can exert their pathological effects. Based on this premise, studies have established that blocking the MET channel is an effective method of protecting hair cells from aminoglycoside-induced cell death [28,42]. However, in our study we found that neurotransmission-deficient hair cells are protected despite intact MET channel function as well as normal levels of neomycin accumulation and rates of neomycin entry (Figure 1, 3).…”

In vivo real-time imaging reveals megalin as the aminoglycoside gentamicin transporter into cochlea whose inhibition is otoprotective

“…Our data provide conclusive evidence that megalin is a critical component of AG transport into endolymph and AGs are targeted to HCs through MET channel entry. We and others have shown that AG ototoxicity can be effectively reduced by preventing AG uptake into the HCs ( 34 – 36 , 63 ). Multiple sites of action of AGs can be targets for ototoxicity, and many of them overlap with antimicrobial targeting, making it difficult to eliminate ototoxicity without altering antimicrobial activity ( 17 ).…”

“…Possible therapeutic targets for preventing AG ototoxicity also have been explored by blocking one of the AG transport pathways or intracellular mechanisms ( 32 , 33 ). For example, MET channel blockers ( 34 – 36 ) as well as antioxidants and anti-apoptotic agents were proposed with some limited efficacy ( 37 – 39 ).…”

Regulation of membrane homeostasis by TMC1 mechanoelectrical transduction channels is essential for hearing

“…Auditory hair cells lacking TMC1 or carrying TMC1 deafness-causing mutations degenerate and die leading to hair cell loss, but the molecular mechanisms leading to hair cell degeneration and deafness are unknown. While wild type hair cells might use PS externalization and subsequent membrane blebbing and TMC1 removal to restore malfunctioning MET or prevent aminoglycoside overload and cell death (Kenyon et al, 2021), we propose that constitutive PS externalization in TMC1 M412K, D569N and D528N mutant hair cells may be the underlying mechanism leading to progressive hair cell loss, and ultimately, deafness in these mice and humans carrying the equivalent mutations (M418K and D572N). In fact, reduced TMC1 protein levels and immature-like bundles have been described in TMC1 D569N mice and hair cell loss can be observed in mice homozygous for any of these mutations as early as P15 (Beurg et al, 2019, Beurg et al, 2021, Marcotti et al, 2006).…”