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.

Figure 1

Schematic diagram of the basis of the ESI-IMS-MS screen and a selection of the small molecules utilized for screen validation. (a-d) Schematic of expected ESI mass spectra resulting from different interactions between peptide/protein monomer (denoted m) and potential inhibitors (denoted L). Oligomers are denoted o; charge states are in superscript. (a) A specific ligand (termed positive) will result in a binomial distribution of bound peaks (pink); (b) the presence of a colloidal inhibitor will lead to spectra containing overlapping peaks resulting from the heterogeneous self-association of the small molecule (orange peaks); (c) a non-specific ligand will bind and result in a Poisson distribution of bound peaks (green); (d) the presence of a non-interacting small molecule (termed negative) will result in spectra similar to those of peptide alone; (e) list of ten small molecules analyzed initially for inhibition of hIAPP aggregation during ESI-MS screen validation. Colors correspond to binding-mode classification by mass spectra (a-d): specific = pink, colloidal = orange, non-specific = green, negative = gray.

Figure 2

hIAPP forms an array of oligomeric species en route to long-straight amyloid fibrils. (a) Primary sequence of hIAPP. The peptide has a disulfide bridge between Cys-2 and Cys-7 and an amidated C-terminus; (b) ESI-MS mass spectrum of hIAPP. Numbers above peaks denote oligomer order, with the positive charge state of ions in superscript; (c) ESI-IMS-MS Driftscope plot of the hIAPP monomer (1) through hexamer (6), present 2 min after diluting the monomer to a final peptide concentration of 32 μM in 200 mM ammonium acetate, pH 6.8. ESI-IMS-MS Driftscope plots show IMS drift time versus mass/charge (m/z) versus intensity (z = square root scale). Inset: negative stain TEM image of hIAPP fibrils after 5 days in 200 mM ammonium acetate pH 6.8 buffer (25 °C, quiescent) (scale bar = 100 nm).

Figure 3

Inhibition of hIAPP amyloid assembly by Fast Green FCF (FG). (a) ThT fluorescence intensity over time of hIAPP alone (black circles) (32 μM peptide, 200 mM ammonium acetate buffer, pH 6.8, 25 °C, quiescent) and with increasing FG:hIAPP molar ratios: 1:1 (orange) and 10:1 (green), showing dose dependent decrease in formation of ThT-positive hIAPP species upon addition of FG. (b) Negative stain TEM images of hIAPP incubated with (i) 1:1 or (ii) 10:1 molar ratios of FG:hIAPP for 5 days (25 °C, quiescent) (scale bar = 100 nm), showing lack of fibrillation (ii) and formation of small/amorphous aggregates (i) of hIAPP in the presence of FG. (c) Positive ion ESI mass spectra showing FG alone (i), or added at 32 μM (ii), or 320 μM (iii), to hIAPP (32 μM). FG binds to the 2+ and 3+ charge state ions of hIAPP monomer (bound peaks denoted with orange or green circles; number of circles represents number of ligands bound), and to the 4+ charge state of the hIAPP dimer (bound peak denoted with white circle). This binding mode is classified as specific.

Figure 4

Colloidal inhibition and non-specific binding observed using ESI-IMS-MS. (a) ThT fluorescence intensity of hIAPP (black) (32 μM peptide, 200 mM ammonium acetate buffer, pH 6.8, 25 °C, quiescent) with Congo red (CR):hIAPP molar ratios: 1:1 (orange) and 10:1 (red) and with 1H-benzimidazole-2-sulfonic acid (1H-B-SA):hIAPP molar ratio: 10:1 (blue)). Inhibition of the formation of ThT-positive species is observed only in the presence of excess CR. (b) Negative stain TEM images of hIAPP incubated with 1:1 (i) or 10:1 (ii) molar ratios of CR or a 10:1 molar ratio of 1H-B-SA (iii) (5 days, 25 °C, quiescent) (scale bar = 100 nm). Fibrils are observed in the presence of equimolar CR and excess 1H-B-SA but not in the presence of excess CR. (c) Positive ion ESI mass spectra showing CR added at 32 μM (i) or 320 μM (ii), or 1H-B-SA added at 320 μM (iii), to hIAPP (32 μM). CR is not observed to bind to hIAPP when added at 32 μM (i) or 320 μM (ii), however CR self-aggregates at 320 μM (ii) (denoted n^x+, where n is the number of CR molecules and x is the charge state of those ions (red peaks). This binding mode is classified as colloidal. Multiple copies of 1H-B-SA bind to the 2+ and 3+ hIAPP monomer ions (bound peaks denoted with blue circles, number of circles represents number of ligands bound), and to the hIAPP dimer (bound peaks denoted with white circles). This binding mode is classified as non-specific.

Figure 5

ESI-IMS-MS demonstrates the mode of inhibition (specific/colloidal/non-specific) or lack of inhibition of hIAPP amyloid formation by small molecules. ESI-IMS-MS Driftscope plots of hIAPP and (a) ibuprofen; (b) FG (bound peaks denoted with yellow (1³⁺ bound) or green (1²⁺ bound) circles, number of circles represents number of ligands bound); (c) 1H-B-SA (bound peaks denoted with blue circles; and (d) CR (colloidal aggregates are denoted n^x+, where n is the number of CR molecules and x is the charge state of the aggregate) (320 μM small molecule) to hIAPP (32 μM). An example of a negative (a), a positive (b), a non-specific (c) and a colloidal inhibitor (d) are illustrated. The numbers on the Driftscope plots indicate the oligomer order and the adjacent superscripted numbers show the charge state of those ions.

Figure 6

Aβ40 alone and with non-specific, negative and specific binding small molecules. (a) Primary sequence of recombinantly expressed Aβ40 (with an additional N-terminal methionine); (b) ESI mass spectrum of Aβ40. Numbers adjacent to peaks denote oligomer order, with the positive charge state of the ions in superscript; (c) ESI-IMS-MS Driftscope plot of Aβ40 alone (32 μM in 200 mM ammonium acetate, pH 6.8) showing IMS drift time versus m/z versus intensity (z = square root scale); (d) positive ion ESI mass spectra showing 320 μM tramiprosate (i), hemin (ii) or EGCG (iii) added to Aβ40 peptide (32 μM). Tramiprosate binds multiple copies to the 3+ and 4+ ions of Aβ40 monomer (bound peaks denoted with pink circles, number of circles represents number of ligands bound).This binding mode is classified as non-specific. Hemin (ii) does not bind and is classified as negative; EGCG (iii) binds to both the 3+ and 4+ ions of Aβ40 monomer (bound peaks are denoted with blue circles) and is classified as specific. (e) ThT fluorescence intensity of Aβ40 alone (black circles) in the presence of tramiprosate (pink circles), EGCG (blue circles) or hemin (orange circles) at small molecule:Aβ40 molar ratios of 10:1. Inhibition of the formation of ThT-positive species is observed in the presence of excess EGCG and interference with ThT fluorescence is observed in the presence of excess hemin. (f) Negative stain TEM images of Aβ40 alone (i) or incubated with 10:1 molar ratios of tramiprosate (ii), hemin (iii) or EGCG (iv) (5 days, 25 °C, quiescent); scale bar = 100 nm. Fibrils are observed by Aβ40 alone and in the presence of excess tramiprosate and hemin but not in the presence of excess EGCG.