Synthesis of berbamine analogs. (A) Design strategy, including the synthetic handle. (B) Specific analogs synthesized for otoprotection studies. BA-13 through BA-16 are monomers, while BA-17 is a fluorescent analog.

Berbamine analogs confer protection from neomycin. Analog BA-4 is shown here as an example. (A) Zebrafish labeled with the mitochondrial dye DASPEI to visualize neuromasts in the lateral line (white dots). Scale bar = 1 mm. (B) Zebrafish labeled with DASPEI after 200 μM neomycin resulting in reduced fluorescence. (C) Zebrafish labeled with DASPEI after pre-co treatment with analog BA-4 and 200 μM neomycin. (D,E) Representative IO3 neuromasts from Tg(Brn3c:GFP) zebrafish. (D) Zebrafish exposed to 200 μM neomycin exposure, resulting in hair cell loss. Scale bar = 10 μm. (E) Zebrafish exposed to a pre-co treatment of analog BA-4 and 200 μM neomycin.

Berbamine analogs confer protection from aminoglycosides. (A,B) Dose-response curves for berbamine analog polymers and berbamine (parent compound) against 200 μM neomycin and 100 μM gentamicin. (C,D) Dose-response curves for the remainder of the berbamine analog polymers, and the berbamine analog monomers (BA-13 to BA-16) against 200 μM neomycin and 100 μM gentamicin. Zebrafish were pretreated for 1 h with each berbamine analog at variable doses followed by co-treatment with analog and aminoglycoside. Data are normalized to controls. Twelve analogs protected hair cells from neomycin damage, while 11 protected from gentamicin. Ten of the berbamine analogs were protective against both neomycin and gentamicin in a dose-dependent manner. Hair cells were assessed via DASPEI. Data are presented normalized to vehicle controls. Data were analyzed by one-way ANOVA, N = 4–12, except for the gentamicin dose-response curves for BA-9, BA-10, BA-11, and BA-13, where N = 2 for the gentamicin-only groups. We are still confident in the data for these groups, based on the additional gentamicin data shown here and the data in Supplementary Figure S1. Bars are ± SEM. Refer to Table 1 for statistics.

Hair cells are viable after analog exposure. (A) Zebrafish were pretreated with the nuclear marker YO-PRO-1 for 1 h followed by treatment with the optimally protective concentration of each analog for 24 h. Zebrafish were then rinsed in embryo medium (EM) and treated with FM 1–43FX to assess mechanoelectrical transduction (MET) channel function after prolonged analog exposure and removal. Analog treatment did not alter FM 1–43FX or YO-PRO-1-labeling. Scale bar = 10 μm. (B) Examples of Tg(myo6b:EGFP) neuromasts treated with the optimally protective concentration of each analog for 24 h before hair cell assessment. In each case, neuromast morphology appeared normal. Scale bar = 10 μm. (C) Zebrafish were exposed to the optimal protective concentration (OPC) of berbamine analog for 24 h before hair cell assessment with DASPEI. None of the analogs impact hair cell survival. Data are normalized to controls and analyzed by one-way ANOVA, F_(4,48) = 0.6564, p = 0.6252. N = 11–12, bars are ± SEM.

Hair cell protection persists after 24 h. Zebrafish were pretreated with the OPC of analog for 1 h followed by 200 μM gentamicin treatment for 30 min. Fish were allowed to recover for 24 h before the assessment. All analogs tested significantly prevented hair cell death after 24 h suggesting true protection rather than a delay in cell death onset. Hair cells were assessed via DASPEI. Data are presented normalized to vehicle controls. Data were analyzed by one-way ANOVA, ****p < 0.0001, ***p < 0.001, F_(4,32) = 12.98, N = 5–10, bars are ± SEM.

Berbamine analogs reduce aminoglycoside uptake by hair cells. (A) Twelve of the analogs significantly reduced uptake of gentamicin conjugated with Texas Red (GTTR), as did berbamine. Zebrafish were pretreated with the optimally protective analog concentration (or with 25 μM berbamine, Kruger et al., 2016) for 1 h followed by a co-treatment with 50 μM GTTR for 18 min. Dimethylsulfoxide (DMSO) and high calcium are negative and positive controls, respectively. (B) Fluorescence intensity quantification using the OPC of analog. The intensity was measured in arbitrary units and the data were normalized by subtracting the background fluorescence. Data were analyzed via one-way ANOVA, F_(17,101) = 38.71, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, asterisks denote significantly different from vehicle controls, N = 4–8 for most compounds, except for N = 2 for compounds BA-1 and BA-5 and N = 15 for berbamine, bars are ± SEM. (C) Linear regression was conducted for analogs that protect hair cells from gentamicin to identify a relationship between analog potency and uptake (y = −0.0203x + 0.9162). The Pearson correlation coefficient of 0.2268 denotes no significant relationship. Berbamine data are replotted from Kruger et al. (2016).

Berbamine analogs differentially reduce mechanotransduction channel activity. Mechanosensitive-calcium signals from hair bundles of posterior lateral line neuromasts. (A) Side view of a neuromast expressing hair cell-specific membrane-localized GCaMP6s. Left: an imaging plane through the apical hair bundles can be used to measure mechanosensitive-calcium signals during fluid-jet stimulation. Right: a top-down view of the imaging plane showing the hair bundles expressing GCaMP6s. (B) Traces comparing the average GCaMP6s signals before and after application of 1 μM BA-16, N = 10 neuromasts. (C) Dot plots quantifying the peak GCaMP6s response per neuromast. BA-16 does not alter evoked calcium activity in hair cells. (D) Traces comparing the average GCaMP6s signals before and after application of 1 μM BA-9, N = 7 neuromasts. (E) Dot plots quantifying the peak GCaMP6s response per neuromast. BA-9 reduces MET-evoked calcium activity compared to baseline levels. Each dot in (C,E) averages the response of all hair bundles in each neuromast. A paired t-test was used in (C,E), ***p < 0.001. Error bars in (C,E) and dashed lines on either side of the solid line in (B,D) represent ± SEM.

Differential patterns of analog protection from gentamicin. Zebrafish were either pre-treated with the OPC of analog for 1 h, then co-treated with analog and gentamicin for 30 min (“pre-co” condition), co-treated with analog and gentamicin without a pretreatment period (“co” condition) or post-treated with analog for 5.5 h following gentamicin removal (“post” condition). All treatments used 200 μM gentamicin. Examples are shown for analogs that represent each pattern of protection. (A) Analog BA-1 was only protective when administered in the “pre-co” or “co” conditions suggesting that protection occurs via aminoglycoside uptake block. (B,C) Analogs BA-6 and BA-7 significantly protected hair cells in all three conditions, suggesting an intracellular mechanism of protection in addition to aminoglycoside uptake block. (D) Analog BA-16 is not protective of any treatment paradigm. Hair cells were assessed via DASPEI. Data are presented normalized to untreated controls, which are not shown but represent 100% hair cell survival. Data were analyzed via one-way ANOVA, ****p < 0.0001, **p < 0.01 (as compared to gentamicin-only controls), N = 7–12, bars are + SEM. Refer to Supplementary Table S1 for statistics.

Berbamine analogs enter hair cells. (A) The dose-response curve for the fluorescent analog (BA-17) demonstrates significant protection from 200 μM neomycin. Hair cells were assessed via DASPEI. Data are normalized to controls. One-way ANOVA, F_(4,49) = 50.22, p < 0.0001. N = 10–12, ****p < 0.0001, ***p < 0.001, bars are ± SEM. (B)Tg(α-tubulin: tdTomato) zebrafish were pretreated with BA-17 for 1 h before imaging on a confocal microscope. Fluorescent analog (green) is present in hair cells (red). Scale bar = 10 μm. (C) AB zebrafish were pretreated with BA-17 for 1 h followed by a 3-min exposure to 50 nM LysoTracker before confocal imaging. Fluorescent analog (green) does overlap with lysosomes (red) in some areas (white arrowheads). Scale bars in (B,C) = 10 μm.

Summary of berbamine analog protection and compound characteristics. Compound name, structure, molecular weight, protection class, likely mechanism of protection, ED₅₀, and OPCs against neomycin and gentamicin for all 16 berbamine analogs. The general protection classes were defined based on thresholds of protection levels by percent. Robust = 60% increase in hair cell protection compared to aminoglycoside group; Strong = over 40% increase in hair cell protection compared to aminoglycoside group; Modest = over 30% increase in hair cell protection compared to aminoglycoside group; No protection = less than 30% of hair cell protection compared to aminoglycoside group. The likely mechanism was determined by the GTTR experiments in Figure 6 and the time course experiments shown in Figure 8 and Supplementary Figure S1.