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

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.

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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.

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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.

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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.

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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

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