A small molecule mitigates hearing loss in a mouse model of Usher syndrome III

Abstract

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

Figure 1. High-throughput screening identifies compounds that stabilize human CLRN1^N48K

(a) Inhibition of proteasomes by bortezomib increased CLRN1^N48K levels in a D6 cell line stably expressing CLRN1^N48K mRNA. CLRN1^N48K was tagged with an HA epitope that was detected by immunofluorescence microscopy. (b) Cell-containing areas were segmented to measure relative concentrations of CLRN1^N48K. Cells were outlined in the top image and colored in the bottom image. (c) Approximately 50,000 compounds were tested by high-throughput screening for stabilization of CLRN1^N48K, and the measured efficacies of these compounds were normalized to 25 nM bortezomib assayed on the same plate. The top 320 compounds (blue) were selected for further analysis. (d) The top 320 compounds were subjected to the same assay 6 times. Of these, 90 compounds with highest average percentage (%) activities are shown. Among them, 48 compounds (black) were selected for secondary screening but 42 compounds (grey) were eliminated due to unfavorable properties such as autofluorescence, the formation of dye-like structures, or chemical structures unsuitable for further pharmaceutical development^,. Data on the y-axis are presented as means ± SEMs (n = 6). Compounds O03, B03, M01, and K01 are labeled. Scale bars = 50 μm.

Figure 2. A dual-reporter assay eliminates pan-proteasome inhibitors

(a) Cells were engineered to co-express human CLRN1^N48K fused to Venus fluorescent protein (green) and DsRed-Express-DR (magenta). CLRN1^N48K-Venus and DsRed-Express-DR are both degraded by proteasome (top row). Thus, a proteasome inhibitor will cause both increased Venus fluorescence and DsRed fluorescence (middle row). A molecule specifically stabilizing CLRN1^N48K will cause increased Venus fluorescence but will minimally affect DsRed fluorescence (bottom row). (b) Dual-color assays conducted for 0 nM bortezomib (no treatment), 50 nM bortezomib, 16.8 μM K01, and 16.8 μM O03. O03 specifically increased CLRN1^N48K-Venus as compared to bortezomib and K01 which increased both Venus and DsRed. Scale bar = 50 μm. (c) Fluorescence intensities from Venus and DsRed normalized to intensity values obtained for 50 nM bortezomib. The specific and most significant increase in fluorescence with characteristics of Venus was observed with compounds B03, M01, and O03 (***P = 1.22 × 10⁻⁵, **P = 3.70 ×10⁻⁴, *P = 2.27 × 10⁻³, respectively, compared to no treatment; two-sided t-test). Results are expressed as means ± SDs (all images are taken in triplicate and 15 different areas from each replicate were picked for quantification). (d) Immunoblotting analysis of human CLRN1^N48K tagged with an HA epitope. Cells were treated with either bortezomib (50 nM), B03 (16.8 and 1.68 μM) or O03 (16.8 μM). CLRN1^N48K expression levels increased dramatically in cells treated with bortezomib and O03 but did not increase in cells treated with B03. Tubulin loading controls are indicated. See also Supplementary Figure 13.

Figure 3. The lead compound O03 stabilizes CLRN1^N48K by a post-translational mechanism

(a) Mouse CLRN1^N48K expressed in NIH/3T3 cells was stabilized by O03 treatment. (b) Quantitative RT-PCR of HEK293 cells stably expressing human CLRN1^N48K. CLRN1 mRNA levels were not affected by either bortezomib or O03 treatment (O03 vs. DMSO, P = 0.9995, two-sided t-test, n=4, means ± SDs). (c) Protein synthesis is not required for the effect of O03. Cells treated with proteasome inhibitors bortezomib and MG132 showed increased levels of CLRN1^N48K as did O03 treatment when compared to DMSO (lanes 1–4, T0). CLRN1^N48K levels remained similar after treatment for an additional 6 h in the presence of 100 μM cycloheximide (CX) (lanes 5–7, T1), indicating that O03 stabilized CLRN1^N48K in the absence of protein synthesis. To confirm that CLRN1^N48K can be effectively degraded in the absence of protein synthesis, cells were treated with bortezomib, MG132, or O03 for 6 h. Compounds then were washed out, and cells were further incubated for 6 h in the presence of CX (lanes 8 – 10, T1). CLRN1^N48K was undetectable in cells after removing the reversible proteasome inhibitor MG132 (compare lanes 3 and 9). Effects of O03 and bortezomib persisted 6 h after the washout. Note: Multiple immunopositive bands were detected by the HA-antibody which recognizes the Ct-tail of CLRN1^N48K (a and c). Based on the analysis of the primary structure, CLRN1 contains a few signal peptide cleavage sites; incomplete cleavage leads to a few differently sized bands. See also Supplementary Figure 13.

Figure 4. Design of BF844; identification of proteins bound to its pharmacophore

(a) Chemical structures of the lead compound (1, O03), the optimized compounds (2, BF981; 3, BF844) and biotinylated compound (4, BF071). BF844, BF981 and BF071 share the same core structure and BF071’s biotin moiety was used for avidin affinity purification. (b) CLRN1^N48K does not bind to the core structure of BF071. BF071 was used for affinity purification (AP) of binding proteins from cell homogenates which contained CLRN1^N48K. As a negative control (Con), target proteins in cell homogenates were blocked with BF981 prior to incubation and affinity purification with BF071. L, whole cell lysates prior to purification; E, the fraction eluted from avidin agarose. (c) BF071 binding proteins were analyzed by SDS-PAGE; protein bands (arrows) at the sizes of ~ 60 and 90 kDa were enriched by BF071 (AP) as compared to the negative control (Con) for which samples were blocked with BF981 prior to affinity purification. Based on quantitative mass spectroscopy, HSP60 was enriched 28 times in position 1 and HSP90 was enriched 10 times in position 2, compared to the control. Positions of the protein molecular mass markers (in kDa) are indicated (lane M). See also Supplementary Figure 13.

Figure 5. BF844 significantly preserves hearing in Tg;KI/KI mice

The y-axis indicates sound intensity in log scale (dB SPL). Each color symbol represents the average ABR threshold (“threshold”) from both ears of an individual mouse. Boxes represent the interquartile range. Horizontal lines within each box indicate the median values whereas the outermost bars present the extremes of the data. In each group, thresholds were recorded from the same mice at P46 and P55. Hearing at P46 (left column): The median threshold is 27.5 dB SPL for control mice and 60–62.5 dB SPL for Tg;KI/KI mice. For Regimen I, the median thresholds at 8, 16 and 32 kHz are 57.5, 45 and 50 dB SPL, respectively. For Regimen II, the median thresholds at 8, 16 and 32 kHz are 35, 37.5 and 45 dB SPL, respectively. ABR data at P55 (right column): The median threshold is 31.25 dB SPL for control mice and 92.5–95 dB SPL for Tg;KI/KI mice. For Regimen I, the median thresholds at 8, 16 and 32 kHz are 67.5, 57.5 and 60 dB SPL, respectively. For Regimen II, the median thresholds at 8, 16 and 32 kHz are 40, 50 and 55 dB SPL, respectively. At P55, Tg;KI/KI mice treated under Regimens I or II show a 30 to 40 dB SPL lower threshold compared to that of untreated Tg;KI/KI mice, indicating 1,000- to 10,000-fold more sensitive hearing across frequencies at P55 in BF844-treated mice. Statistics: ns (not significant) P≥0.05, * P≤0.05, **P≤0.01, and ***P≤0.001