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 CLRN1N48K that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1N48K, 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 CLRN1N48K 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.

  • Compound

    4-chloro-1,3-dimethyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine

  • Compound

    2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethan-1-ol

  • Compound

    1-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-2-methylpropan-2-ol

  • Compound

    N-(3-(2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetamido)propyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

  • Compound

    4-chloro-1,3-dimethyl-5-(o-tolyl)-1H-pyrazolo[3,4-c]pyridazine

  • Compound

    4-chloro-5-cyclohexyl-1,3-dimethyl-1H-pyrazolo[3,4-c]pyridazine

  • Compound

    3-oxo-3-phenylpropanenitrile

  • Compound

    ethyl 2-(5-amino-3-phenyl-1H-pyrazol-1-yl)acetate

  • Compound

    3-phenyl-1H-pyrazol-5-amine

  • Compound

    ethyl 2-(5-acetamido-3-phenyl-1H-pyrazol-1-yl)acetate

  • Compound

    N-(3-phenyl-1H-pyrazol-5-yl)acetamide

  • Compound

    ethyl 2-(5-acetamido-4-iodo-3-phenyl-1H-pyrazol-1-yl)acetate

  • Compound

    N-(4-iodo-3-phenyl-1H-pyrazol-5-yl)acetamide

  • Compound

    ethyl 2-(5-acetamido-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate

  • Compound

    N-(3-phenyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide

  • Compound

    sodium 2-(5-amino-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate

  • Compound

    2-(5-amino-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)ethan-1-ol

  • Compound

    3-phenyl-4-(phenylethynyl)-1H-pyrazol-5-amine

  • Compound

    2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid

  • Compound

    4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine

  • Compound

    N-(1-(2-hydroxy-2-methylpropyl)-3-phenyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide

  • Compound

    1-(5-amino-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)-2-methylpropan-2-ol

  • Compound

    5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoic acid

  • Compound

    tert-butyl (3-aminopropyl)carbamate

  • Compound

    tert-butyl (3-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)propyl)carbamate

  • Compound

    N-(3-aminopropyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

  • Compound

    N-(1,3-dimethyl-1H-pyrazol-5-yl)acetamide

  • Compound

    N-(4-iodo-1,3-dimethyl-1H-pyrazol-5-yl)acetamide

  • Compound

    N-(1,3-dimethyl-4-(o-tolylethynyl)-1H-pyrazol-5-yl)acetamide

  • Compound

    1,3-dimethyl-4-(o-tolylethynyl)-1H-pyrazol-5-amine

  • Compound

    N-(4-(cyclohexylethynyl)-1,3-dimethyl-1H-pyrazol-5-yl)acetamide

  • Compound

    4-(cyclohexylethynyl)-1,3-dimethyl-1H-pyrazol-5-amine

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Acknowledgements

This work was supported by the Usher III Initiative (Y.I., K.N.A. and BioFocus group), the Maniglia Endowed Funds (K.N.A.), Hope for Vision (Y.I.), the Prince Family Foundation (Y.I.), start-up funding from Case Western Reserve University (CWRU) (Y.I.), the US National Institutes of Health (NIH) (grants R01-DC010816 (to K.N.A.), R01-EY020826 (to Y.I.) and R24-EY021126 (to K.P.)), and the Arnold and Mabel Beckman Foundation (K.P.). We thank the Elden family and the Prince Family Foundation for their generous support of our research via the Usher III Initiative, D. Saperstein for his leadership of the Initiative's research efforts, and A.R. Moise for useful suggestions and discussions before the inception of this project. We thank R. Papoian, S. Nelson and W. Seibel at the Drug Discovery Center (University of Cincinnati) for conducting the HTS. The monoclonal antibody E7 developed by M. Klymkowsky was obtained from the Developmental Studies Hybridoma Bank created by the National Institute of Child Health and Human Development (NICHD) of the NIH, and maintained at The University of Iowa, Iowa City, USA. Fluorescence microscopy was conducted to optimize the HTS assay at the light microscopy imaging core facility of CWRU. We thank Y. Chen at CWRU for providing the RHOP23H NIH/3T3 cell line, and L.T. Webster Jr. for insightful comments on this manuscript.

Author information

Author notes

    • Ruishuang Geng
    • , Nicola A Lindsay
    •  & Roland W Bürli

    Current addresses: Biological Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada (R.G.); AstraZeneca, Cambridge, UK (N.A.L.); and MedImmune, Cambridge, UK (R.W.B.).

Affiliations

  1. Otolaryngology Head and Neck Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio, USA.

    • Kumar N Alagramam
    • , Suhasini R Gopal
    • , Ruishuang Geng
    •  & Daniel H-C Chen
  2. Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio, USA.

    • Kumar N Alagramam
  3. Neurosciences, Case Western Reserve University, Cleveland, Ohio, USA.

    • Kumar N Alagramam
  4. Pharmacology and Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, Cleveland, Ohio, USA.

    • Ina Nemet
    • , Richard Lee
    • , Guilian Tian
    • , Krzysztof Palczewski
    •  & Yoshikazu Imanishi
  5. Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, USA.

    • Masaru Miyagi
  6. BioFocus, a Charles River company, Chesterford Research Park, Saffron Walden, UK.

    • Karine F Malagu
    • , Christopher J Lock
    • , William R K Esmieu
    • , Andrew P Owens
    • , Nicola A Lindsay
    • , Roland W Bürli
    •  & Angus M MacLeod
  7. Charles River Nederland BV, Leiden, the Netherlands.

    • Krista Ouwehand
    • , Faywell Albertus
    •  & David F Fischer
  8. Office of Translation and Innovation, Case Western Reserve University, Cleveland, Ohio, USA.

    • William E Harte

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Contributions

Y.I. and K.P. conceived the small-molecule screening experiments and designed the small-molecule HTS assay, and G.T. and R.L. contributed to the establishment of the assay; G.T., I.N. and R.L. conducted the experiments characterizing small molecules; I.N. and M.M. performed mass spectroscopy and associated experiments; K.N.A. conceived the transgenic mouse models TgAC1 and TgAC1;KI/KI; K.N.A., S.R.G., R.G. and D.H.-C.C. were involved in the generation of TgAC1;KI/KI mice; S.R.G. characterized the hearing loss profile in TgAC1;KI/KI mice; K.N.A., S.R.G. and D.H.-C.C. conceived the dosing scheme for regimen II; S.R.G. and D.H.-C.C. performed efficacy testing associated with regimens I and II; K.F.M., C.J.L., W.R.K.E. and A.P.O. performed the medicinal chemistry experiments; N.A.L. accommodated the small-molecule screening assay at BioFocus; K.O. and F.A. conducted the small-molecule screening assay at BioFocus; K.F.M., N.A.L., D.F.F., R.W.B., A.M.M. and W.E.H. provided project supervision at BioFocus; Y.I. and K.N.A. provided project supervision at CWRU; I.N. and K.F.M. wrote the results section associated with medicinal chemistry and target identification; and K.N.A., K.P. and Y.I. wrote the manuscript.

Competing interests

Usher III Initiative contracted K.F.M., C.J.L., W.R.K.E., A.P.O., N.A.L., K.O., F.A., D.F.F., R.W.B. and A.M.M. from BioFocus to carry out hit-to-lead optimization of small molecules described in this report. W.E.H. serves as the director of pharmaceutical development for the Usher III Initiative and as chief translational officer for the School of Medicine at CWRU. W.E.H., R.W.B., K.F.M., C.J.L., W.R.K.E. and A.P.O. are named on patents WO 2014066835, WO 2014066836 and WO 2012148994. The mouse Clrn1 cDNA used to generate transgenic mouse TgAC1 and the human ortholog are protected by provisional US patent 62/076,114 to K.N.A.

Corresponding authors

Correspondence to Kumar N Alagramam or Yoshikazu Imanishi.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Figures 1–13 and Supplementary Tables 1–4.

  2. 2.

    Supplementary Note

    Synthetic Procedures

  3. 3.

    Supplementary Data Set 1

    The 90 prioritized screening hits and 48 (highlighted structures) selected for further characterization.

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DOI

https://doi.org/10.1038/nchembio.2069

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