Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin

Abstract

Loss of mechanosensory hair cells in the inner ear accounts for many hearing loss and balance disorders. Several beneficial pharmaceutical drugs cause hair cell death as a side effect. These include aminoglycoside antibiotics, such as neomycin, kanamycin and gentamicin, and several cancer chemotherapy drugs, such as cisplatin. Discovering new compounds that protect mammalian hair cells from toxic insults is experimentally difficult because of the inaccessibility of the inner ear. We used the zebrafish lateral line sensory system as an in vivo screening platform to survey a library of FDA-approved pharmaceuticals for compounds that protect hair cells from neomycin, gentamicin, kanamycin and cisplatin. Ten compounds were identified that provide protection from at least two of the four toxins. The resulting compounds fall into several drug classes, including serotonin and dopamine-modulating drugs, adrenergic receptor ligands, and estrogen receptor modulators. The protective compounds show different effects against the different toxins, supporting the idea that each toxin causes hair cell death by distinct, but partially overlapping, mechanisms. Furthermore, some compounds from the same drug classes had different protective properties, suggesting that they might not prevent hair cell death by their known target mechanisms. Some protective compounds blocked gentamicin uptake into hair cells, suggesting that they may block mechanotransduction or other routes of entry. The protective compounds identified in our screen will provide a starting point for studies in mammals as well as further research discovering the cellular signaling pathways that trigger hair cell death.

Figure 1

Dose response testing reveals broad ranges of protective effects against neomycin, gentamicin, kanamycin and cisplatin. Graphs show mean % hair cell survival ± 1 SEM of zebrafish treated with varying concentrations of protective compound (“By Compound”) or by varying concentration of toxin (“By Toxin”) for four protective compounds: fluoxetine (A), paroxetine (B), loperamide (C) and benzamil (D). Zebrafish were assayed following 1 hr pretreatment in putative protective compound followed by co-exposure to a toxin: neomycin (neo) for 1 hr, gentamicin (gent) for 6 hr, kanamycin (kan) for 24 hr or cisplatin (cis) for 24 hr with protective compound. Larvae were stained in vivo with DASPEI to evaluate neuromasts. Each group consists of >9 fish. In the left column, treating with protective compound had a significant effect (1-way ANOVA, p<0.05) for all groups. In the right column, adding protective compound to a wide range of toxin concentrations had a significant effect (2-way ANOVA, p < 0.05 for treatment, concentration and interaction) for all groups.

Figure 2

Cisplatin-induced cancer cell death is not inhibited by addition of benzamil or paroxetine. Graphs show the mean percentage ± 1 SEM of A549 cells surviving 72 hr treatment with cisplatin and paroxetine (A) or benzamil (B). Mean % cancer cell survival was determined using an ATP luminescence assay. Each point shows the mean of three replicate experiments. Paroxetine did not significantly alter toxic effects of cisplatin (2-way ANOVA, interaction: p = 0.91, paroxetine concentration: p = 0.60, cisplatin concentration: p < 0.0001). Benzamil significantly enhanced cisplatin toxicity (2-way ANOVA, interaction: p = 0.21, benzamil concentration: p < 0.001, cisplatin concentration: p < 0.0001).

Figure 3

Pretreatment is necessary to confer protection against neomycin with methiothepin and phenoxybenzamine. Zebrafish hair cells exposed to methiothepin (A) or phenoxybenzamine (B) along with 1 hr exposure to neomycin showed significantly decreased protection as “time in pretreatment” decreased (1-way ANOVA, p < 0.05 for both compounds; by Bonferroni’s Multiple Comparison test: * = p < 0.05; *** = p < 0.001.). Treatment groups were tested along with control group treated with 1 hr exposure to neomycin only (“neo only”). Y-axes show mean % hair cell survival ± 1 SEM normalized to untreated controls. X-axis categories show minutes of time in pretreatment in protective compound before toxin exposure.

Figure 4

Gentamicin-Texas Red (GTTR) entry into hair cells is altered by exposure to protective compounds. A) Images of a zebrafish neuromast pre-labeled with YOPRO-1 after a 3 min exposure to GTTR. YO-PRO1 labels hair cell nuclei (left, green channel). GTTR localized to puncta the apical region of hair cells, down and to the left in this image and a lower levels throughout the cytoplasm (middle, red channel). An image merging the red and green channels is shown to the right. The scale bar is 10 μM. B) Mean fluorescence intensity ± 1 s.d. of neuromasts exposed to protective compound and GTTR, embryo media only (EM only), or GTTR only (GTTR) for 3 min. Intensity is normalized and shown as a mean percentage of the GTTR only case. GTTR fluorescence intensity is significantly altered by addition of the protective compounds raloxifene (p<.001 ), ractopamine (p< .01), phenoxybenzamine (p<.05) and benzamil (p<.05). Shaded bars represent compounds that did not protect hair cells from gentamicin toxicity after 6 hr exposure. C) Plot of GTTR intensity following 3 min exposure to 50 μM GTTR (% intensity normalized to GTTR only) vs. hair cell survival following 6 hr exposure to 50 μM gentamicin (% hair cell staining) for each protective compound.

Figure 5

Five selective estrogen receptor modulators (SERMs) protect hair cells from neomycin, while only raloxifene and MPP protect hair cells from long-term gentamicin exposure. Dose response curves show mean % hair cell survival ± 1 SEM following exposure to 200 μM neomycin or 50 μM gentamicin and tamoxifen (A), toremifene (B), afimoxifene (C), raloxifene (D), and MPP (E; 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride). Larvae were pretreated with protective compound for 1 hr before co-exposure of protective compound and neomycin for 1 hr or co-exposure with gentamicin for 6 hr, and then stained with DASPEI to count hair cells. Each group is n >9 fish. Treating with protective compound had a significant effect (1-way ANOVA, p < 0.05) on neomycin toxicity for all groups and a significant effect (1-way ANOVA, p < 0.05) on gentamicin toxicity for raloxifene and MPP but no significant effect for tamoxifen, toremifene and afimoxifene.

Figure 6

Chemical structures of estrogen receptor ligands tested for protective effects in this study. Those that protected against aminoglycosides had similar structural elements to either tamoxifen (toremifene, afimoxifene; A) or raloxifene (MPP; B) while the other compounds (PPT, DPN, R,R THC, 17-beta estradiol and fulvestrant; C) had noticeably different structures.