Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

An in vivo platform for identifying inhibitors of protein aggregation

Nature Chemical Biology volume 12, pages 94–101 (2016)Cite this article

Subjects

Abstract

Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, that is capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of islet amyloid polypeptide aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Split β-lactamase assay for protein aggregation identifies aggregation-prone sequences.
Figure 2: Antibiotic resistance of the tripartite fusion constructs correlates with peptide aggregation propensity in vitro.
Figure 3: βla-hIAPP aggregation in vitro and the effects of curcumin in vitro and in vivo.
Figure 4: In vivo screen identifies hIAPP aggregation inhibitors.
Figure 5: Identification of dopamine as an inhibitor of hIAPP aggregation.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Sipe, J.D. et al. Nomenclature 2014: amyloid fibril proteins and clinical classification of the amyloidosis. Amyloid 21, 221–224 (2014).

    Article  PubMed  Google Scholar 

  2. Knowles, T.P.J., Vendruscolo, M. & Dobson, C.M. The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 15, 384–396 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Tipping, K.W., van Oosten-Hawle, P., Hewitt, E.W. & Radford, S.E. Amyloid fibres: inert end-stage aggregates or key players in disease? Trends Biochem. Sci. 40, 719–727 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Pieri, L., Madiona, K., Bousset, L. & Melki, R. Fibrillar α-synuclein and huntingtin exon 1 assemblies are toxic to the cells. Biophys. J. 102, 2894–2905 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bulawa, C.E. et al. Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade. Proc. Natl. Acad. Sci. USA 109, 9629–9634 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Meng, F., Marek, P., Potter, K.J., Verchere, C.B. & Raleigh, D.P. Rifampicin does not prevent amyloid fibril formation by human islet amyloid polypeptide but does inhibit fibril thioflavin-T interactions: implications for mechanistic studies of β-cell death. Biochemistry 47, 6016–6024 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Butterfield, S., Hejjaoui, M., Fauvet, B., Awad, L. & Lashuel, H.A. Chemical strategies for controlling protein folding and elucidating the molecular mechanisms of amyloid formation and toxicity. J. Mol. Biol. 421, 204–236 (2012).

    Article  CAS  PubMed  Google Scholar 

  8. Foit, L. et al. Optimizing protein stability in vivo. Mol. Cell 36, 861–871 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kim, W. et al. A high-throughput screen for compounds that inhibit aggregation of the Alzheimer's peptide. ACS Chem. Biol. 1, 461–469 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Lee, L.L., Ha, H., Chang, Y.T. & DeLisa, M.P. Discovery of amyloid-beta aggregation inhibitors using an engineered assay for intracellular protein folding and solubility. Protein Sci. 18, 277–286 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Espargaró, A., Sabate, R. & Ventura, S. Thioflavin-S staining coupled to flow cytometry. A screening tool to detect in vivo protein aggregation. Mol. Biosyst. 8, 2839–2844 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Romero, D., Sanabria-Valentín, E., Vlamakis, H. & Kolter, R. Biofilm inhibitors that target amyloid proteins. Chem. Biol. 20, 102–110 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. McKoy, A.F., Chen, J., Schupbach, T. & Hecht, M.H. A novel inhibitor of amyloid β (Aβ) peptide aggregation: from high throughput screening to efficacy in an animal model of Alzheimer disease. J. Biol. Chem. 287, 38992–39000 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Morell, M., de Groot, N.S., Vendrell, J., Avilés, F.X. & Ventura, S. Linking amyloid protein aggregation and yeast survival. Mol. Biosyst. 7, 1121–1128 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hailu, T.T., Foit, L. & Bardwell, J.C.A. In vivo detection and quantification of chemicals that enhance protein stability. Anal. Biochem. 434, 181–186 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Quan, S. et al. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. Nat. Struct. Mol. Biol. 18, 262–269 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chen, Y.-R. & Glabe, C.G. Distinct early folding and aggregation properties of Alzheimer amyloid-β peptides Abeta40 and Abeta42: stable trimer or tetramer formation by Abeta42. J. Biol. Chem. 281, 24414–24422 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Bitan, G., Lomakin, A. & Teplow, D.B. Amyloid β-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins. J. Biol. Chem. 276, 35176–35184 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Young, L.M., Cao, P., Raleigh, D.P., Ashcroft, A.E. & Radford, S.E. Ion mobility spectrometry-mass spectrometry defines the oligomeric intermediates in amylin amyloid formation and the mode of action of inhibitors. J. Am. Chem. Soc. 136, 660–670 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Koo, B.W. & Miranker, A.D. Contribution of the intrinsic disulfide to the assembly mechanism of islet amyloid. Protein Sci. 14, 231–239 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Qiao, Q., Bowman, G.R. & Huang, X. Dynamics of an intrinsically disordered protein reveal metastable conformations that potentially seed aggregation. J. Am. Chem. Soc. 135, 16092–16101 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Sgourakis, N.G., Yan, Y., McCallum, S.A., Wang, C. & Garcia, A.E. The Alzheimer's peptides Abeta40 and 42 adopt distinct conformations in water: a combined MD/NMR study. J. Mol. Biol. 368, 1448–1457 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Young, L.M. et al. Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry. Nat. Chem. 7, 73–81 (2015).

    Article  CAS  PubMed  Google Scholar 

  25. Nesterov, E.E. et al. In vivo optical imaging of amyloid aggregates in brain: design of fluorescent markers. Angew. Chem. Int. Ed. Engl. 44, 5452–5456 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. O'Nuallain, B. & Wetzel, R. Conformational Abs recognizing a generic amyloid fibril epitope. Proc. Natl. Acad. Sci. USA 99, 1485–1490 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Eichner, T., Kalverda, A.P., Thompson, G.S., Homans, S.W. & Radford, S.E. Conformational conversion during amyloid formation at atomic resolution. Mol. Cell 41, 161–172 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Valleix, S. et al. Hereditary systemic amyloidosis due to Asp76Asn variant β2-microglobulin. N. Engl. J. Med. 366, 2276–2283 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Mangione, P.P. et al. Structure, folding dynamics, and amyloidogenesis of D76N β2-microglobulin: roles of shear flow, hydrophobic surfaces, and α-crystallin. J. Biol. Chem. 288, 30917–30930 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jespers, L., Schon, O., Famm, K. & Winter, G. Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat. Biotechnol. 22, 1161–1165 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Jespers, L., Schon, O., James, L.C., Veprintsev, D. & Winter, G. Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold. J. Mol. Biol. 337, 893–903 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Daval, M. et al. The effect of curcumin on human islet amyloid polypeptide misfolding and toxicity. Amyloid 17, 118–128 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sparks, S., Liu, G., Robbins, K.J. & Lazo, N.D. Curcumin modulates the self-assembly of the islet amyloid polypeptide by disassembling α-helix. Biochem. Biophys. Res. Commun. 422, 551–555 (2012).

    Article  CAS  PubMed  Google Scholar 

  34. Meng, F. et al. The sulfated triphenyl methane derivative acid fuchsin is a potent inhibitor of amyloid formation by human islet amyloid polypeptide and protects against the toxic effects of amyloid formation. J. Mol. Biol. 400, 555–566 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Meng, F. & Raleigh, D.P. Inhibition of glycosaminoglycan-mediated amyloid formation by islet amyloid polypeptide and proIAPP processing intermediates. J. Mol. Biol. 406, 491–502 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Palhano, F.L., Lee, J., Grimster, N.P. & Kelly, J.W. Toward the molecular mechanism(s) by which EGCG treatment remodels mature amyloid fibrils. J. Am. Chem. Soc. 135, 7503–7510 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Cheng, B. et al. Coffee components inhibit amyloid formation of human islet amyloid polypeptide in vitro: possible link between coffee consumption and diabetes mellitus. J. Agric. Food Chem. 59, 13147–13155 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Cheng, B. et al. Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide. Biochem. Biophys. Res. Commun. 419, 495–499 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Tu, L.-H. et al. Mutational analysis of the ability of resveratrol to inhibit amyloid formation by islet amyloid polypeptide: critical evaluation of the importance of aromatic-inhibitor and histidine-inhibitor interactions. Biochemistry 54, 666–676 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Aitken, J.F., Loomes, K.M., Konarkowska, B. & Cooper, G.J.S. Suppression by polycyclic compounds of the conversion of human amylin into insoluble amyloid. Biochem. J. 374, 779–784 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Aarabi, M.H. & Mirhashemi, S.M. The role of two natural flavonoids on human amylin aggregation. Afr. J. Pharm. Pharmacol. 6, 2374–2379 (2012).

    Article  CAS  Google Scholar 

  42. Zelus, C. et al. Myricetin inhibits islet amyloid polypeptide (IAPP) aggregation and rescues living mammalian cells from IAPP toxicity. Open Biochem. J. 6, 66–70 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Porat, Y., Mazor, Y., Efrat, S. & Gazit, E. Inhibition of islet amyloid polypeptide fibril formation: a potential role for heteroaromatic interactions. Biochemistry 43, 14454–14462 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Wu, C., Lei, H., Wang, Z., Zhang, W. & Duan, Y. Phenol red interacts with the protofibril-like oligomers of an amyloidogenic hexapeptide NFGAIL through both hydrophobic and aromatic contacts. Biophys. J. 91, 3664–3672 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Noor, H., Cao, P. & Raleigh, D.P. Morin hydrate inhibits amyloid formation by islet amyloid polypeptide and disaggregates amyloid fibers. Protein Sci. 21, 373–382 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Li, J., Zhu, M., Manning-Bog, A.B., Di Monte, D.A. & Fink, A.L. Dopamine and L-dopa disaggregate amyloid fibrils: implications for Parkinson's and Alzheimer's disease. FASEB J. 18, 962–964 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Lee, H.-J. et al. Dopamine promotes formation and secretion of non-fibrillar alpha-synuclein oligomers. Exp. Mol. Med. 43, 216–222 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Baell, J. & Walters, M.A. Chemistry: chemical con artists foil drug discovery. Nature 513, 481–483 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Baell, J.B. & Holloway, G.A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 53, 2719–2740 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Jelsch, C., Mourey, L., Masson, J.-M. & Samama, J.-P. Crystal structure of Escherichia coli TEM1 β-lactamase at 1.8 A resolution. Proteins 16, 364–383 (1993).

    Article  CAS  PubMed  Google Scholar 

  51. Williamson, J.A. & Miranker, A.D. Direct detection of transient α-helical states in islet amyloid polypeptide. Protein Sci. 16, 110–117 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Walsh, D.M. et al. A facile method for expression and purification of the Alzheimer's disease-associated amyloid β-peptide. FEBS J. 276, 1266–1281 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kad, N.M., Thomson, N.H., Smith, D.P., Smith, D.A. & Radford, S.E. β(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro. J. Mol. Biol. 313, 559–571 (2001).

    Article  CAS  PubMed  Google Scholar 

  54. Marek, P., Woys, A.M., Sutton, K., Zanni, M.T. & Raleigh, D.P. Efficient microwave-assisted synthesis of human islet amyloid polypeptide designed to facilitate the specific incorporation of labeled amino acids. Org. Lett. 12, 4848–4851 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Giles, K. et al. Applications of a travelling wave-based radio-frequency-only stacked ring ion guide. Rapid Commun. Mass Spectrom. 18, 2401–2414 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Schrödinger Release 9.3 (Schrödinger LLC, New York, 2014-2).

Download references

Acknowledgements

J.C.S. is co-funded by Innovate UK (131841) and the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/M01259X/1) and was previously funded by a BBSRC CASE studentship (grant number BB/H014713/1) sponsored by Avacta Analytical plc, Wetherby, UK. L.M.Y. is funded by a BBSRC CASE studentship (grant number BB/I015361/1) sponsored by Micromass UK Ltd./Waters Corporation, Manchester, UK. R.A.M. is funded by a BBSRC studentship (grant number BB/F01614X/1). The Synapt HDMS mass spectrometer was purchased with funds from the BBSRC (BB/E012558/1). M.P.J., D.J.B. and S.E.R. also acknowledge funding from the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement number 322408. R.J.F. and C.H.R. acknowledge the Biomedical Health Research Centre (University of Leeds) for funding. We thank J. Bardwell (University of Michigan) for his longstanding collaboration and advice at the beginning of the study. We thank S. Webster (formerly at Avacta Analytical plc) for his advice and helpful discussions. We are very grateful to D. Raleigh and L.-H. Tu (Stony Brook University) for kindly providing the synthetic hIAPP and rIAPP peptides. We thank R. Wetzel (University of Pittsburgh) for providing the WO1 antibody. We also acknowledge our collaborators and all members of our groups for helpful discussions.

Author information

Author notes

  1. Janet C Saunders and Lydia M Young: These authors contributed equally to this work.

Authors and Affiliations

  1. Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK

    Janet C Saunders, Lydia M Young, Rachel A Mahood, Matthew P Jackson, Charlotte H Revill, Richard J Foster, Alison E Ashcroft, David J Brockwell & Sheena E Radford

  2. School of Molecular and Cellular Biology, University of Leeds, Leeds, UK

    Janet C Saunders, Lydia M Young, Rachel A Mahood, Matthew P Jackson, Alison E Ashcroft, David J Brockwell & Sheena E Radford

  3. School of Chemistry, University of Leeds, Leeds, UK

    Charlotte H Revill & Richard J Foster

  4. Avacta Analytical plc, Wetherby, UK

    D Alastair Smith

Authors
  1. Janet C Saunders
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lydia M Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rachel A Mahood
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthew P Jackson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Charlotte H Revill
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard J Foster
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D Alastair Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alison E Ashcroft
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David J Brockwell
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sheena E Radford
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.C.S. and L.M.Y. contributed equally to this work. J.C.S. designed the study, purified the β-lactamase constructs, performed the in vivo experiments, performed the β-lactamase activity assays in vitro, performed TEM, analyzed results and wrote the manuscript. L.M.Y. conceived, designed and performed mass spectrometry experiments, performed TEM and thioflavin T fluorometry and analyzed results. R.A.M. purified Aβ40 and performed TEM and thioflavin T fluorometry. M.P.J. performed western blots, dot blots, and thioflavin T and NIAD-4 fluorometry. C.H.R. and R.J.F. designed and prepared the small-molecule screening library. R.J.F. also performed all PAINS analyses. D.A.S. contributed to experiment design. A.E.A. conceived and designed mass spectrometry experiments. D.J.B. and S.E.R. conceived and designed the experiments and wrote the manuscript. All authors contributed to manuscript preparation.

Corresponding authors

Correspondence to David J Brockwell or Sheena E Radford.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–15. (PDF 6345 kb)

Supplementary Data Set 1

Full list of all small molecules used in this study and their structures and chemical properties. (PDF 4471 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Saunders, J., Young, L., Mahood, R. et al. An in vivo platform for identifying inhibitors of protein aggregation. Nat Chem Biol 12, 94–101 (2016). https://doi.org/10.1038/nchembio.1988

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1988

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research