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A molecular imaging biosensor detects in vivo protein folding and misfolding

Journal of Molecular Medicine volume 94, pages 799–808 (2016)Cite this article

Abstract

Aberrant protein folding represents the molecular basis of many important human diseases. Although the discovery of new anti-misfolding drugs is a major priority in molecular therapeutics, there is currently no generalizable protein folding assay for use in cell-based high throughput screening (HTS) of chemical libraries, or for in vivo imaging. We molecularly engineered a bioluminescence-based biosensor composed of rationally split Firefly luciferase reporter fragments flanking a test protein, and used this in a protein-fragment complementation assay to quantitatively measure folding of the test protein. We comprehensively validated this biosensor in vitro, in cells, and by optically imaging protein folding and misfolding in living mice using several test proteins including enhanced green fluorescent protein, Renilla luciferase, Gaussia luciferase, and SIRT1. Applications of this novel biosensor are potentially far-reaching in both cell-based HTS approaches to discover new anti-misfolding drugs, and when using the same biosensor in validation studies of drug candidates in small animal models.

Key messages

  • Novel anti-misfolding drugs are needed as molecular therapeutics for many diseases.

  • We developed first in vivo imaging protein folding biosensor to aid drug discovery.

  • Biosensor created by flanking a test protein with rationally split Firefly luciferase.

  • Biosensor validated by detecting folding of test proteins EGFP, Rluc, Gluc, and SIRT1.

  • Generalizable molecular biosensor for translational applications in drug screening.

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Acknowledgments

We acknowledge use of the Stanford Center for Innovation in In-Vivo Imaging (SCi3) Core Facility and thank Dr. Sanjiv Sam Gambhir, Chairman, Department of Radiology, Stanford University, for his constant support. This work was supported by the National Institutes of Health (NIH grant R21CA185805 to T.F.M. and R.P.). T.F.M. was supported in part by the Ben and Catherine Ivy Foundation.

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Affiliations

  1. Laboratory of Experimental and Molecular Neuroimaging, Molecular Imaging Program at Stanford (MIPS), and Bio-X Program, Stanford University School of Medicine, Stanford, CA, 94305-5427, USA

    Anjali V. Sheahan, Thillai V. Sekar, Kai Chen, Ramasamy Paulmurugan & Tarik F. Massoud

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  1. Anjali V. Sheahan
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  2. Thillai V. Sekar
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  3. Kai Chen
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  4. Ramasamy Paulmurugan
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  5. Tarik F. Massoud
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Corresponding authors

Correspondence to Ramasamy Paulmurugan or Tarik F. Massoud.

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The authors declare that they have no competing interests.

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Sheahan, A.V., Sekar, T.V., Chen, K. et al. A molecular imaging biosensor detects in vivo protein folding and misfolding. J Mol Med 94, 799–808 (2016). https://doi.org/10.1007/s00109-016-1437-9

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  • DOI: https://doi.org/10.1007/s00109-016-1437-9

Keywords

  • Protein folding
  • Protein misfolding
  • Molecular imaging
  • Drug discovery
  • High throughput screening
  • Bioluminescence imaging