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:

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry–mass spectrometry

Nature Chemistry volume 7, pages 73–81 (2015)Cite this article

Subjects

Abstract

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry–mass spectrometry (IMS–MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human islet amyloid polypeptide (hIAPP) aggregation or amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS–MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of hIAPP amyloid assembly.

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: Schematic diagram of the basis of the ESI–IMS–MS screen and a selection of the small molecules utilized for screen validation.
Figure 2: hIAPP forms an array of oligomeric species en route to long, straight amyloid fibrils.
Figure 3: Inhibition by FG of the hIAPP amyloid assembly.
Figure 4: Colloidal inhibition and nonspecific binding observed using ESI–IMS–MS.
Figure 5: ESI–IMS–MS demonstrates the mode of inhibition (specific/colloidal/nonspecific) or lack of inhibition of hIAPP amyloid formation by small molecules.
Figure 6: Aβ40 alone and with nonspecific, negative and specific binding small molecules.

Similar content being viewed by others

References

  1. Sipe, J. D. et al. Amyloid fibril protein nomenclature: 2012 recommendations from the Nomenclature Committee of the International Society of Amyloidosis. Amyloid 19, 167–170 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Grimster, N. P. et al. Aromatic sulfonyl fluorides covalently kinetically stabilize transthyretin to prevent amyloidogenesis while affording a fluorescent conjugate. J. Am. Chem. Soc. 135, 5656–5668 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Klabunde, T. et al. Rational design of potent human transthyretin amyloid disease inhibitors. Nature Struct. Mol. Biol. 7, 312–321 (2000).

    Article  CAS  Google Scholar 

  4. Connelly, S., Choi, S., Johnson, S. M., Kelly, J. W. & Wilson, I. A. Structure-based design of kinetic stabilizers that ameliorate the transthyretin amyloidoses. Curr. Opin. Struct. Biol. 20, 54–62 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hamrang, Z., Rattray, N. J. W. & Pluen, A. Proteins behaving badly: emerging technologies in profiling biopharmaceutical aggregation. Trends Biotech. 31, 448–458 (2013).

    Article  CAS  Google Scholar 

  6. Meier, J. J. et al. Inhibition of human IAPP fibril formation does not prevent β-cell death: evidence for distinct actions of oligomers and fibrils of human IAPP. Am. J. Physiol. Endocrinol. Metab. 291, E1317–E1324 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. 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 

  8. Harroun, T. A., Bradshaw, J. P. & Ashley, R. H. Inhibitors can arrest the membrane activity of human islet amyloid polypeptide independently of amyloid formation. FEBS Lett. 507, 200–204 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Woods, L. A. et al. Ligand binding to distinct states diverts aggregation of an amyloid-forming protein. Nature Chem. Biol. 7, 730–739 (2011).

    Article  CAS  Google Scholar 

  10. 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 

  11. Hyung, S. J. et al. Insights into antiamyloidogenic properties of the green tea extract (–)-epigallocatechin-3-gallate toward metal-associated amyloid-β species. Proc. Natl Acad. Sci. USA 110, 3743–3748 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Westermark, P. Amyloid in the islets of Langerhans: thoughts and some historical aspects. Ups. J. Med. Sci. 116, 81–89 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Selkoe, D. J. Alzheimer's disease: genes, proteins, and therapy. Physiol. Rev. 81, 741–766 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Feng, B. Y. et al. Small-molecule aggregates inhibit amyloid polymerization. Nature Chem. Biol. 4, 197–199 (2008).

    Article  CAS  Google Scholar 

  15. Bieschke, J. et al. EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity. Proc. Natl Acad. Sci. USA 107, 7710–7715 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Meng, F., Abedini, A., Plesner, A., Verchere, C. B. & Raleigh, D. P. The flavanol (–)-epigallocatechin 3-gallate inhibits amyloid formation by islet amyloid polypeptide, disaggregates amyloid fibrils, and protects cultured cells against IAPP-induced toxicity. Biochemistry 49, 8127–8133 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Howlett, D., Cutler, P., Heales, S. & Camilleri, P. Hemin and related porphyrins inhibit β-amyloid aggregation. FEBS Lett. 417, 249–251 (1997).

    Article  CAS  PubMed  Google Scholar 

  18. 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 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. 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 

  20. Ehrnhoefer, D. E. et al. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nature Struct. Mol. Biol. 15, 558–566 (2008).

    Article  CAS  Google Scholar 

  21. Daubenfeld, T., Bouin, A. P. & van der Rest, G. A deconvolution method for the separation of specific versus nonspecific interactions in noncovalent protein–ligand complexes analyzed by ESI-FT-ICR mass spectrometry. J. Am. Soc. Mass Spectrom. 17, 1239–1248 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Sun, N., Sun, J., Kitova, E. N. & Klassen, J. S. Identifying nonspecific ligand binding in electrospray ionization mass spectrometry using the reporter molecule method. J. Am. Soc. Mass Spectrom. 20, 1242–1250 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Sun, J., Kitova, E. N., Wang, W. & Klassen, J. S. Method for distinguishing specific from nonspecific protein–ligand complexes in nanoelectrospray ionization mass spectrometry. Anal. Chem. 78, 3010–3018 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Wang, W., Kitova, E. N. & Klassen, J. S. Influence of solution and gas phase processes on protein–carbohydrate binding affinities determined by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 75, 4945–4955 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. 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 

  26. Dupuis, N. F., Wu, C., Shea, J. E. & Bowers, M. T. Human islet amyloid polypeptide monomers form ordered beta-hairpins: a possible direct amyloidogenic precursor. J. Am. Chem. Soc. 131, 18283–18292 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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 

  28. Khurana, R., Uversky, V. N., Nielsen, L. & Fink, A. L. Is Congo red an amyloid-specific dye? J. Biol. Chem. 276, 22715–22721 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Kim, Y. S., Randolph, T. W., Manning, M. C., Stevens, F. J. & Carpenter, J. F. Congo red populates partially unfolded states of an amyloidogenic protein to enhance aggregation and amyloid fibril formation. J. Biol. Chem. 278, 10842–10850 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Porat, Y., Abramowitz, A. & Gazit, E. Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism. Chem. Biol. Drug. Des. 67, 27–37 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Thomas, T., Nadackal, G. T. & Thomas, K. Aspirin and diabetes: inhibition of amylin aggregation by nonsteroidal anti-inflammatory drugs. Exp. Clin. Endocrinol. Diabetes 111, 8–11 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Woods, L. A. et al. Ligand binding to distinct states diverts aggregation of an amyloid-forming protein. Nature Chem. Biol. 7, 730–739 (2011).

    Article  CAS  Google Scholar 

  33. Cao, P. & Raleigh, D. P. Analysis of the inhibition and remodeling of islet amyloid polypeptide amyloid fibers by flavanols. Biochemistry 51, 2670–2683 (2012).

    Article  CAS  PubMed  Google Scholar 

  34. 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 

  35. Tenidis, K. et al. Identification of a penta- and hexapeptide of islet amyloid polypeptide (IAPP) with amyloidogenic and cytotoxic properties. J. Mol. Biol. 295, 1055–1071 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Petkova, A. T. et al. A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR. Proc. Natl Acad. Sci. USA 99, 16742–16747 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Young, L. et al. Monitoring oligomer formation from self-aggregating amylin peptides using ESI-IMS-MS. Int. J. Ion Mobil. Spectrom. 16, 29–39 (2013).

    Article  CAS  Google Scholar 

  38. Aisen, P. et al. Alzhemed: a potential treatment for Alzheimer's disease. Curr. Alzheimer Res. 4, 473–478 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Gervais, F. et al. Targeting soluble Aβ peptide with tramiprosate for the treatment of brain amyloidosis. Neurobiol. Aging 28, 537–547 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Necula, M., Kayed, R., Milton, S. & Glabe, C. G. Small molecule inhibitors of aggregation indicate that amyloid β oligomerization and fibrillization pathways are independent and distinct. J. Biol. Chem. 282, 10311–10324 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Valler, M. J. & Green, D. Diversity screening versus focussed screening in drug discovery. Drug Discov. Today 5, 286–293 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. De Felice, F. G. et al. Targeting the neurotoxic species in Alzheimer's disease: inhibitors of Aβ oligomerization. FASEB J. 18, 1366–1372 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Ladiwala, A. R. A. et al. Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Aβ into off-pathway conformers. J. Biol. Chem. 285, 24228–24237 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yang, F. et al. Curcumin inhibits formation of amyloid β oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J. Biol. Chem. 280, 5892–5901 (2005).

    Article  CAS  PubMed  Google Scholar 

  45. Scherzer-Attali, R. et al. Complete phenotypic recovery of an Alzheimer's disease model by a quinone–tryptophan hybrid aggregation inhibitor. PLoS ONE 5, e11101 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Rapid Overlay of Chemical Structures (OpenEye, Scientific Software, Santa Fe, New Mexico, 2007).

  47. 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 

  48. 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 

  49. Platt, G. W., Routledge, K. E., Homans, S. W. & Radford, S. E. Fibril growth kinetics reveal a region of beta2-microglobulin important for nucleation and elongation of aggregation. J. Mol. Biol. 378, 251–263 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Maestro, version 9.3, Schrödinger Release 2014–2 (Schrödinger LLC, New York, 2014).

Download references

Acknowledgements

L.M.Y. is funded by a Biotechnology and Biological Sciences Research Council (BBSRC) CASE studentship (Grant Number BB/I015361/1) sponsored by Micromass UK Ltd/Waters Corporation. J.C.S. is funded by a BBSRC CASE studentship (Grant Number BB/H014713/1) sponsored by Avacta Analytical. 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 through its Research Equipment Initiative scheme (BB/E012558/1). S.E.R. acknowledges funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013; 322408). D.P.R. acknowledges support from the US National Institutes of Health (GM078114). We thank D. Walsh (Brigham & Women's Hospital) and S. Linse (Lund University) for provision of the Aβ40 peptide and vector and J. R. Ault (University of Leeds) for setting up the automated ESI-MS analyses. We also acknowledge all members of the Ashcroft, Radford and Raleigh groups for helpful discussions.

Author information

Author notes

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

Authors and Affiliations

  1. Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

    Lydia M. Young, Janet C. Saunders, Rachel A. Mahood, Charlotte H. Revill, Richard J. Foster, Sheena E. Radford & Alison E. Ashcroft

  2. School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK

    Lydia M. Young, Janet C. Saunders, Rachel A. Mahood, Sheena E. Radford & Alison E. Ashcroft

  3. School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK

    Charlotte H. Revill & Richard J. Foster

  4. Department of Chemistry, Stony Brook University, Stony Brook, New York, 11794-3400, USA

    Ling-Hsien Tu & Daniel P. Raleigh

Authors
  1. Lydia M. Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Janet C. Saunders
    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. Charlotte H. Revill
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Richard J. Foster
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ling-Hsien Tu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Daniel P. Raleigh
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sheena E. Radford
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alison E. Ashcroft
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.M.Y. and J.C.S. contributed equally to this work. L.M.Y., J.C.S., S.E.R. and A.E.A. conceived and designed the experiments. L.M.Y., J.C.S. and R.A.M. performed the experiments. C.H.R. and R.J.F. designed and prepared the screening library. L.H.T. and D.P.R. prepared the hIAPP. L.M.Y. analysed the data. All authors discussed the results, and wrote and commented on the manuscript.

Corresponding authors

Correspondence to Sheena E. Radford or Alison E. Ashcroft.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 8855 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Young, L., Saunders, J., Mahood, R. et al. Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry–mass spectrometry. Nature Chem 7, 73–81 (2015). https://doi.org/10.1038/nchem.2129

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.2129

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing