Structural Mechanism of the Interaction of Alzheimer Disease Aβ Fibrils with the Non-steroidal Anti-inflammatory Drug (NSAID) Sulindac Sulfide

Background: The mechanism of interaction between small molecules and amyloid-β fibrils is unknown.

Results: Molecular modeling on the basis of solid-state NMR reveals that sulindac sulfide intercalates between β-strands of amyloid-β fibrils.

Conclusion: Sulindac sulfide interacts with amyloid-β fibrils in a specific manner and binds to hydrophobic cavities in the core of the fibrils.

Significance: Unraveling how small molecules interfere with amyloidogenic deposits will assist structure-based drug design for neurodegenerative disorders.

Abstract

Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aβ populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp²³-Lys²⁸ salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly³³, a residue involved in Met³⁵ oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.

Introduction

The self-assembly of amyloidogenic proteins into fibrils and oligomers plays a pivotal role in various diseases (1). The deposition of fibrils formed by the amyloid-β peptide (Aβ)³ into plaques in brain tissue is a major pathological hallmark in the progression of neurodegeneration in Alzheimer disease. Aβ peptides are generated through sequential proteolytic cleavages of the amyloid precursor protein (APP) by the β- and γ-secretases (2, 3). This results in the production of Aβ peptides of differing lengths (4), mainly Aβ_1–40 and Aβ_1–42 (5), and shorter variants, such as Aβ_1–39 (6). Soluble oligomers formed by Aβ_1–42 represent the toxic species responsible for the decline in cognitive function associated with neurodegeneration (7). Several studies have demonstrated that small molecules can interfere with the solubility of amyloid proteins and are, therefore, potential drug candidates (8, 9). Chronic inflammation significantly enhances Alzheimer disease pathogenesis (10). Fibrillar β-amyloid deposits co-localize with numerous chronic inflammatory mediators and activated microglia in the brain (11). The relation to inflammation suggests that non-steroidal anti-inflammatory drugs (NSAIDs) might be beneficial for the treatment of Alzheimer disease. In fact, epidemiological studies demonstrate a link between the use of anti-inflammatory drugs and the prevalence of Alzheimer disease (12). This work aims to investigate the interaction mechanism of the NSAID sulindac sulfide (Fig. 1a) with Alzheimer peptide Aβ fibrils. In addition to sulindac sulfide (13), NSAIDs, including ibuprofen, indomethacin (13), and flurbiprofen (14), have been identified as γ-secretase modulators. γ-Secretase modulators interfere with APP processing and modify the relative Aβ_1–42 population. In particular, sulindac sulfide decreases the relative amount of the amyloid-prone Aβ_1–42, whereas the production of shorter, less amyloidogenic Aβ peptides is increased (13–16).

FIGURE 1.

The influence of sulindac sulfide on Aβ. a, chemical structure of the NSAID sulindac sulfide. b, TEM images of 50 μm Aβ fibrils in the absence (bottom panel) and presence (top panel) of a 5-fold molar excess of sulindac sulfide. The fibrillar character is maintained, and deposits of sulindac sulfide can be observed. Scale bar = 200 nm. c, two-dimensional ¹³C-¹⁵N TEDOR spectra of Aβ amyloid fibrils incubated in the presence of a 5-fold molar excess of sulindac sulfide and 1% DMSO (red) and Aβ fibrils incubated with 1% DMSO (black) as a control. Corresponding ¹³C-¹³C correlation spectra are shown in supplemental Fig. S1. The obtained ¹³C line widths are in the order of 120–200 Hz (data not shown). Sequential assignments are obtained from three-dimensional NCACX and NCOCX experiments. Arrows indicate residues that experience large chemical shift changes. d, CSPs induced by sulindac sulfide on the NMR chemical shifts of Aβ fibrils. Differences in chemical shifts (Δδ (ppm)) were calculated for ¹³C and ¹⁵N resonances according to Δδ_C = [(δC_sul − δC_ref)²]^1/2 and Δδ_N = [(2 / 5 × (δN_sul − δN_ref))²]^1/2, respectively.

Solution-state NMR structures of the APP-TM (transmembrane) dimer have been solved for the wild-type (17) and a familial mutant (18). NMR and EPR experiments have revealed a potential cholesterol-binding site within the C-terminal Cys⁹⁹ sequence and highlight the significance of the GXXXG segments for binding (19). It has been suggested that NSAIDs can interact with lipids to form phospholipid complexes (20–22). This may provide a general mechanism for the interaction of APP with small molecules. Reports on the interaction between sulindac sulfide and the APP transmembrane sequence are, however, contradictory. Sulindac sulfide, among other γ-secretase modulators, binds to the APP transmembrane sequence of the Cys⁹⁹ motif (23) and to Cys¹⁰⁰ dimers in the presence of SDS micelles (24). Bacterial reporter assays show that γ-secretase modulators, including sulindac sulfide, bind to the GXXXG dimerization motif and, thereby, attenuate the dimerization of the APP transmembrane sequence (25), a process necessary for proteolytic cleavage (26). However, colloidal aggregation of sulindac sulfide in aqueous solutions can potentially induce nonspecific binding (27, 28). Contradicting data have been reported for the influence of sulindac sulfide on the Aβ peptide itself (29, 30).

So far, it is not understood how sulindac sulfide interacts with amyloids. NMR is a suitable technique to study Aβ-ligand interactions for various Aβ aggregation states (31). Solution-state NMR can be employed to study interactions of Aβ monomers and small molecule (32) or peptide inhibitors (33), nanoparticles (34), and various others. Besides monomers and fibrils, oligomeric intermediates formed by Aβ in solution constitute potential drug targets (35, 36). However, oligomeric intermediates and insoluble fibrils are not detectable by solution-state NMR because their lines are broadened beyond detection. Solid-state NMR spectroscopy is a powerful tool that allows the study of Aβ-small molecule interactions at atomic resolution. In the past, this technique has been applied successfully for the characterization of the interaction between Aβ and the polyphenol epigallocatechin gallate (37), curcumin (38, 39), and catechol-type flavonoids (40) and to study the interface of Congo red and amyloids formed by the prion domain of the HET-s protein (41). In this work, we investigate the interaction between sulindac sulfide and Aβ fibrils using solid-state NMR spectroscopy. On the basis of the gathered NMR data, we employ docking to derive a model for the intercalation of sulindac sulfide with Aβ fibrils.

Experimental Procedures

Aβ Expression and Purification and Sample Preparation

The uniformly ¹⁵N or ¹⁵N-¹³C-labeled Aβ_1–40 peptide was recombinantly expressed in Escherichia coli inclusion bodies and purified via reverse-phase chromatography as described previously (42). The construct contains an N-terminal methionine but shows the same biochemical properties as the wild-type peptide (43). To obtain monomeric Aβ in solution, the lyophilized peptide was initially dissolved in 10 mm NaOH, sonicated for 10 min in an ultrasonic bath, and centrifuged for 10 min at 14,800 rpm to remove potential nucleation seeds. The solution was then diluted in 2× buffer (100 mm sodium phosphate and 100 mm NaCl buffer (pH 7.3)) to yield the respective Aβ concentration.

Preparation of NSAIDs

Stock solutions of sulindac sulfide were prepared in dimethyl-sulfoxide (DMSO), and the respective amount of NSAID stock was added to Aβ. The final concentration of DMSO in aqueous solutions was 1%.

Solid-state NMR Sample Preparation

Aβ fibrils were obtained according to a protocol described previously (44). Briefly, monomeric Aβ at a concentration of 50 μm was seeded with sonicated fibrils (10% w/w) and incubated under agitation until the completion of fibrillation. This step was repeated for 11 generations. For solid-state NMR measurements, the last generation was allowed to fibrilize for 2 days before sulindac sulfide was added to the sample. Approximately 10 mg of Aβ fibrils was incubated with a 5-fold molar excess of sulindac sulfide (250 μm, 1% DMSO). After mixing, the sample was kept quiescent for 1 h at room temperature. As a reference, Aβ fibrils were incubated under the same conditions with 1% DMSO. The sulindac sulfide-incubated fibrils were sedimented into a 3.2-mm rotor. The reference fibrils were packed into a 4.0-mm rotor.

Solid-state NMR Measurements

¹³C-detected assignment experiments were carried out using a Bruker Avance wide-bore spectrometer operating at a ¹H Larmor frequency of 700 MHz (16.5 tesla). The spectrometer was equipped with a triple-resonance MAS probe (¹H, ¹³C, ¹⁵N). All measurements were recorded at a MAS rotation frequency of 17 kHz for 3.2-mm rotors or 13 kHz for 4.0-mm rotors and a temperature of 12 °C. In all experiments, ¹H-¹³C magnetization transfer was achieved through cross-polarization. Two-dimensional spectra were recorded using proton-driven spin diffusion (45, 46) for ¹³C-¹³C magnetization transfer with a mixing time of 200 ms or transferred echo double-resonance (TEDOR) (47, 48) for ¹³C-¹⁵N magnetization transfer. The three-dimensional NCACX and NCOCX (49) experiments were recorded employing TEDOR for ¹³C-¹⁵N magnetization transfer and dipolar assisted rotational resonance (50) for ¹³C-¹³C mixing.

To identify ¹³C atoms in Aβ that are located in the vicinity of the ¹⁹F atom of sulindac sulfide, we recorded ¹³C-¹⁹F rotational echo double resonance (REDOR) experiments (supplemental Fig. S8 a) (51). These were recorded on a Bruker Avance III wide-bore spectrometer operating at a ¹H Larmor frequency of 600 MHz (14 tesla). The spectrometer was equipped with a triple-resonance cross-polarization MAS probe (¹H, ¹³C, ¹⁹F). All measurements were recorded at a MAS rotation frequency of 14.6 kHz and a temperature of 12 °C. In the REDOR experiments, ¹³C-¹⁹F dipolar dephasing was preceded by a ¹³C-¹³C proton-driven spin diffusion mixing step. REDOR dephasing times were set to 1.1, 2.2, and 4.4 ms. REDOR spectra recorded with and without dephasing pulses were subtracted to identify fluorine-coupled carbons. In Fig. 2a, REDOR spectra are represented in one-dimensional mode. No ¹³C-¹³C cross peaks could be observed in the three-dimensional REDOR experiments because the sensitivity was too low. To detect the characteristic salt bridge between the carboxyl of Asp²³ and the amine of Lys²⁸ of mature Aβ fibrils, TEDOR experiments (supplemental Fig. S8 b) were carried out on the basis of the pulse sequence described by Jaroniec et al. (52). All spectra were recorded on a Bruker Avance III narrow-bore spectrometer operating at a ¹H Larmor frequency of 750 MHz (17.63 tesla) equipped with a triple-resonance MAS probe (¹H, ¹³C, ¹⁵N) at a MAS rotation frequency of 11 kHz and 4 °C. In the experiment, the TEDOR mixing time was set to 7.27 and 15.72 ms.

FIGURE 2.

¹³C-¹⁹F REDOR and ¹³C-¹⁵N TEDOR NMR experiments. a, ¹³C-¹⁹F REDOR NMR experiments recorded to observe dephasing of Aβ ¹³C nuclei in close proximity to the ¹⁹F atom of sulindac sulfide. Experiments were run with mixing times of 1.1 (data not shown), 2.2, and 4.4 ms. A list of all ¹³C-¹⁹F contacts observed can be found in supplemental Table S2. Recoupled and reference spectra were subtracted to identify affected ¹³C resonances (red). For reference, a one-dimensional ¹³C experiment without ¹⁹F recoupling, including all aliphatic resonances, is shown (gray). b, analysis of the salt bridge involving residues Asp²³ and Lys²⁸. ¹³C-¹⁵N TEDOR spectra show cross-peaks between Lys²⁸-Nζ and Asp²³-Cγ in the presence (red) and absence of sulindac sulfide (black). A TEDOR mixing time of 15.72 ms is employed. One-dimensional traces extracted at the Nζ chemical shifts are superimposed with the one-dimensional ¹³C reference spectra of the respective sample (gray).

Transmission Electron Microscopy (TEM)

Images were recorded on an EM 900 from Carl Zeiss SMT. Samples were stained with 4% uranyl acetate solution on formvar/carbon-coated grids.

Molecular Modeling

The 2-fold symmetric (PDB code 2LMN) (53) and the 3-fold symmetric (PDB code 2LMP) (54) Aβ_1–40 fibril NMR structural models were employed in molecular modeling studies. To identify internal cavities, each model of the Aβ fibril was analyzed separately. For the calculation of internal cavities, van der Waals volumes were derived using the Voronoi cell method (55, 56). Atomic volumes are calculated on the basis of the allocation of space among atoms using hyperbolic surfaces (applying a cubic lattice of 0.1-Å grid width) and applying the ProtOr (57) atom radius set for protein atoms that were determined analytically from reference structures. Internal cavities are determined analytically from each model structure by a Delaunay triangulation (58). An internal cavity is defined as the buried space within a structure that is big enough to accommodate at least a 1.4-Å radius water-sized probe. The size of a cavity is estimated from the average distance of the cavity center to the neighboring atoms and depicted by the radius of a sphere. The polarity of the cavities is assessed with the program DOWSER (59), which calculates potential positions for water not resolved by the original structure determination approach. This program assesses the hydrophilicity of protein cavities by determining the interaction energy between a water molecule and its surrounding atoms. Water molecules with interaction energies of less than −10 kcal/mol are considered “low-energy water molecules” and selected for output. Internal cavities harboring such an internal water molecule are denoted as polar (blue) and the remaining cavities as hydrophobic (gray) (Fig. 3 and supplemental Figs. S4 and S5).

FIGURE 3.

Packing analysis of Aβ structures. a and b, distribution of internal cavities in 3-fold symmetric Aβ_1–40 fibrils (PDB code 2LMP, model 1) (54) (a) and 2-fold symmetric Aβ_1–40 fibrils (PDB code 2LMN, model 1) (53) (b). Shown is a cross-section perpendicular to the fibril axis. Each cavity is depicted as a sphere. The radius of the sphere corresponds to the average distance from the cavity center to the Aβ van der Waals surface. Water-containing polar cavities are colored in blue and hydrophobic cavities in gray. The approximate position of cavity clusters #1 to #5 is indicated. The protein backbones are depicted as schematics, with side chains drawn as lines. Residues that are as close as ≤6 Å to the ¹⁹F atom of sulindac sulfide are highlighted using sticks. Residues Ile³² and Val³⁶, which define cavity clusters, are represented in purple. Ala³⁰, Leu³⁴, and Val³⁹ are shown in green. Phe¹⁹ is colored red. Termini are marked with N or C.

For induced fit docking (60, 61), the region between the two most hydrophobic cavity clusters (termed #1-#5) near Ile³² (#2) and Val³⁶ (#3) from the first NMR model was targeted. Docking was performed using the Schrödinger Maestro suite (62) following a standard protocol. Subsequently, a grid with auto-assigned box size was generated. The resulting box is centered near Leu³⁴ for the 3-fold symmetric structure and Ile³² and Val³⁶ for the 2-fold symmetric structure. Induced fit docking was done with the program Glide with single precision and with no constraints applied (63). To simulate receptor flexibility, side chain orientations were optimized to take into account ligand binding. At last, a Glide redocking was applied for the refined structure. Docking poses are ranked and filtered by means of the Glide score. This score assesses the binding probability and accounts for steric clashes, van der Waals and coulomb energy, lipophilicity, H-bonding, and bond rotation ability, among others. Only the energetically most favorable docking poses (with theoretical energies <30 kcal/mol higher than the best pose) are considered for further analysis.

Results

To probe the interaction between Aβ fibrils and sulindac sulfide (Fig. 1a), we titrated sulindac sulfide to preformed fibrils. Fibrillar Aβ is observed by TEM in the presence of the NSAID (Fig. 1b). The resulting ¹³C-¹⁵N (Fig. 1c) and ¹³C-¹³C (supplemental Fig. S1) correlation spectra show well dispersed peaks. The three-dimensional NCACX and NCOCX experiments allowed sequential assignment of resonances for residues Gln¹⁵-Val⁴⁰ for both samples (representative strip plots are shown in supplemental Fig. S2; all assigned resonances are listed in supplemental Table S1). We detected only one set of resonances for both samples, indicating that the fibrils exist in one conformation.

Sulindac sulfide has no significant effect on the fibrillar structure of Aβ because the spectra of both samples are relatively similar (Fig. 1c and supplemental Fig. S1). However, small but defined chemical shift perturbations (CSPs) are observed in the presence of sulindac sulfide, indicating specific interactions of the NSAID with the fibrils. This is remarkable because a non-quantitative and nonspecific binding of sulindac sulfide to Aβ fibrils would result in peak-splitting and line-broadening. Fig. 1d shows CSPs, Δδ (parts per million), upon addition of sulindac sulfide. We observe changes in chemical shift in particular for side chain resonances of Lys¹⁶, Val¹⁸-Cβ, Phe¹⁹-Cβ, Phe²⁰-Cβ, Asn²⁷-Cγ, and Met³⁵-Cβ as well as for the backbone resonances of Phe¹⁹, Phe²⁰, Ala²¹ and Gly³³. CSPs reflect ligand binding but could as well be a consequence of local or global structural rearrangements. To unambiguously probe ligand binding, we recorded ¹³C-¹⁹F REDOR experiments employing the NMR-active properties of the ¹⁹F atom of sulindac sulfide. In general, only aliphatic resonances of Aβ could be detected. For comparison, a one-dimensional ¹³C spectrum containing assignments for all aliphatic resonances is represented (Fig. 2a). The strongest dephasing effects are observed for the Cγ resonances of Val¹⁸ or Val³⁹. However, because of spectral overlap, these two peaks cannot be discriminated. Smaller signal attenuations are observed for Cγ of Val²⁴ and Val³⁶, Cβs of Ala²¹ or Ala³⁰ (overlap) as well as for Cγ2 and Cδ1 of Ile³² at longer mixing times. These resonances exhibit the most severe dephasing effects. The respective residues must therefore be located close to the fluorine atom of sulindac sulfide.

To gain further information on the potential effects of sulindac sulfide on the Aβ fibril structure, we investigated the effect on the salt bridge, which is typically formed between the side chains of residues Asp²³ and Lys²⁸ (53, 54). From sequential assignments, the chemical shift of the carboxylic group of Asp²³ in the presence of sulindac sulfide was assigned to 177.8 ppm and to 178.0 ppm for the reference fibrils. On the basis of one-dimensional ¹⁵N spectra, the chemical shift of Lys²⁸-Nζ was found to be 33.9 ppm and 34.3 ppm for the two preparations, respectively. In both samples, a cross-peak between Lys²⁸-Nζ and Asp²³-Cγ was detected, implying the presence of a salt bridge in both cases (Fig. 2b).

In the following, the CSPs in the two-dimensional ¹³C-¹³C proton-driven spin diffusion and ¹³C-¹⁵N TEDOR as well as the ¹³C-¹⁹F REDOR contacts were used as restraints to derive a model for sulindac sulfide in complex with Aβ_1–40 fibrils. We used both a 2-fold (53) and 3-fold symmetric (54) Aβ_1–40 fibril NMR structure as reference structures for modeling and docking experiments because they show the highest correlation with our chemical shifts (supplemental Fig. S3) compared with all structures and models analyzed (44, 53, 54, 64–68). The NMR structures contained 10 models each. The architecture of the Aβ_1–40 fibrils obviously differs comparing the 2-fold and 3-fold-symmetric structures. In each structure, Aβ adopts a β-strand—turn—β-strand fold. Fibrils are stabilized by hydrogen bonds connecting individual β-strands along the fibril axis. The 3-fold symmetric fibril structure contains three stacks of Aβ molecules in a triangular form. The 2-fold symmetric fibril structure is built from two antiparallel stacks of Aβ molecules. To detect potential binding sites for the hydrophobic sulindac sulfide, we performed a packing density analysis for each Aβ structure. In fact, all models revealed large packing defects. On average, we found eight cavities per stack of four Aβ molecules in both structures (supplemental Figs. S4 and S5), clustering in five different regions within one stack (clusters #1-#5) (Fig. 3). To dissect polar from unpolar cavities, we applied the tool DOWSER (59) to calculate positions of internal water molecules that are not resolved in the NMR structures. We found that the most hydrophobic cavities in each structure clustered in the rigid core region, with calculated water occupancies of <10% in cluster #2 and <30% in cluster #3 that are located on both sides of Phe-19 (supplemental Table S3). The 3-fold symmetric structure seemed to be more tightly packed around clusters #2 and #3, featuring fewer cavities in comparison with the 2-fold symmetric structure. At the same time, other clusters in the 3-fold symmetric structure seemed to contain a larger numbers of cavities. Clusters of polar cavities were found in the flexible turn region between residues Glu²² and Ile³¹ (cluster #1) and toward the termini of the peptide sequence (cluster #4). The two structures differed in their interface architecture between the termini to the adjacent loop region. Although, in the 3-fold symmetric structure, cavities of cluster #4 were stabilized through interactions with the loop, in the 2-fold symmetric structure, fewer polar cavities were found because the structure opened and cavities could become exposed. Cluster #5 (around M35) with polar cavities was located at the intersheet/contact region between termini and the loop region (3-fold symmetric structure) or at the interface of the antiparallel β-sheets (2-fold symmetric structure). In the intersheet region, where the contact interface between 3-fold and 2-fold symmetric structure was apparently different, the amount of cavities per stack (of four Aβ molecules) and their polarity did not differ. The amount of cavities and the respective water contents per cluster #1-#5 are shown in supplemental Table S3.

Clusters #2 and #3 contain large hydrophobic cavities, which could, in principle, bury a sulindac sulfide molecule. The exact size of the individual cavities depends on the rotameric state of Phe¹⁹. The 3-fold symmetric and the 2-fold symmetric structures differ slightly in the position of Phe¹⁹ with respect to the registry of the second β-sheet containing residues Ile³² to Val³⁶ (supplemental Fig. S6). In the 3-fold symmetric structure, Phe¹⁹ is oriented more toward Ile³², whereas, in the 2-fold symmetric structure, Phe¹⁹ is pointing toward Leu³⁴ and is, therefore, located more central in the core region. Surprisingly, the size, polarity, and distribution of the detected cavities is very similar in all analyzed structural models even though there are slight differences between the two models.

To test whether the hydrophobic cavities in clusters #2 and #3 are indeed suitable binding sites for sulindac sulfide, we applied an induced fit docking approach where the sulindac sulfide and side chains of Aβ were kept flexible. The size and accessibility of these cavities depends on the rotameric state of Phe¹⁹. In fact, docking into cluster #3 of the 3-fold symmetric structure allows identification of two scenarios that fit to the distance restraints obtained from the NMR analysis. In particular, we find that the distance of the sulindac sulfide ¹⁹F atom to the methyl groups of Ile³², Leu³⁴, or Val³⁶ is smaller than 6 Å. In pose 1, sulindac sulfide lies in the Gly³³ groove and is orientated along the fibril axes, being close to Ile³² and Leu³⁴. The aromatic side chains of Phe¹⁹ are directed toward Ala³⁰ (Fig. 4a, top panel). In pose 2, sulindac sulfide has rotated into a position with its conjugated ring system parallel to the β-sheets and orthogonal to Phe¹⁹ so that the ¹⁹F atom approaches Leu³⁴ and Val³⁶. (Fig. 4a, bottom panel). Both poses suggest an aromatic π stacking interaction between the conjugated ring system of sulindac sulfide and the Phe¹⁹ side chain. Similar interactions are obtained from docking to cluster #2 of the 2-fold symmetric structure (Fig. 4b). In pose 1, sulindac sulfide is orientated along the fibril axes, with its conjugated ring system parallel to Phe¹⁹, analogous to pose 1 in the 3-fold symmetric structure. Again, the ¹⁹F atom is positioned in the Gly³³ groove close to Ile³² and Leu³⁴. In contrast to the 3-fold symmetric structure, Phe¹⁹ is positioned here on the other side of sulindac sulfide, close to Leu³⁴, thereby shielding Val³⁶ so that sulindac sulfide cannot change into a position to contact Val³⁶ (Fig. 4b, top panel). An additional docking to cluster #3 yields poses 2 and 3, where the normal of the conjugated ring system of sulindac sulfide is orientated perpendicularly to the fibril axes. Thereby, sulindac sulfide extends into the terminal region (#4). The conjugated ring system is positioned parallel to the β-sheets and orthogonal to Phe¹⁹. Sulindac sulfide is either orientated in a way that the ¹⁹F atom faces Phe¹⁹, Leu³⁴, and Val³⁶ (Fig. 4b, center panel) or that the ¹⁹F atom is oriented toward the fibril exterior, contacting Val³⁶ and Val³⁹ (Fig. 4b, bottom panel).

FIGURE 4.

Induced fit docking of sulindac sulfide to Aβ. a and b, induced fit docking of sulindac sulfide to the hydrophobic cavity cluster #3 of the 3-fold symmetric Aβ_1–40 fibril structure (PDB code 2LMP, model 1, poses 1 and 2) (54) (a) and to the hydrophobic cavity clusters #2 and #3 of the 2-fold symmetric Aβ_1–40 structure (PDB code 2LMN, model 1; for cluster #2, pose 1 and cluster #3, poses 2 and 3) (53) (b). Sulindac sulfide is depicted as blue sticks and the protein backbones as a schematic. Residues within 6 Å of the ¹⁹F atom of sulindac sulfide are depicted as sticks in purple for the cavity cluster defining residues Ile³² and Val³⁶ and in green for Ala³⁰, Leu³⁴, and Val³⁹. Phe¹⁹ and Gly³³ are colored in red.

Discussion

Sulindac sulfide-incubated Aβ fibrils are highly similar to control fibrils, implying that sulindac sulfide has no significant effect on fibril structure. Both the NMR chemical shift patterns (Fig. 1c and supplemental Fig. S1) and the morphology in TEM images (Fig. 1b) are maintained. Analysis of ¹³C chemical shifts predicts β-strands as the main secondary structural element in both fibril preparations (supplemental Fig. S7). In addition, chemical shift analysis by torsion angle likeliness obtained from shift and sequence similarities+ (TALOS+) (69) yields secondary structural propensities that predict the presence of two β-strands typically observed in specific regions within Aβ_1–40 fibrils (44, 53, 54, 67, 70) and oligomers (71, 72). Furthermore, we find, by TEDOR experiments, that the Asp²³-Lys²⁸ salt bridge remains intact in the presence of sulindac sulfide (Fig. 2b). Even though the overall fibrillar character is maintained, we observe defined chemical shift changes, potentially indicating local conformational changes (Fig. 1d). Previous reports have stated that small molecules such as curcumin (39) are able to disrupt the characteristic salt bridge in Aβ_1–42 fibrils. Judging from EM data, curcumin has a more drastic effect on the general fibril architecture compared with sulindac sulfide because it destroys Aβ_1–42 fibrils (39). We note that only one set of resonances is observed for the sulindac sulfide-incubated Aβ fibrils. No resonance splitting is observed for cross-peaks. Rather, resonances move to new positions, indicating that each peptide in a fibril interacts specifically with one or more NSAID molecules.

Upon addition of sulindac sulfide, CSPs are observed in particular for hydrophobic residues such as Val¹⁸, Phe¹⁹ and Phe²⁰, Gly³³, and Met³⁵ but also for the polar side chain of Lys¹⁶. The most dramatic CSPs are detected for Lys¹⁶ and Gly³³ (Fig. 1d). ¹³C-¹⁹F REDOR experiments yield unambiguous distance restraints. The REDOR experiments show that sulindac sulfide binds in the vicinity of methyl groups of Val¹⁸ or Val³⁹ as well as Ile³¹ and Ile³² Cγ2.

To identify trends and avoid bias, we use both the 2-fold symmetric (PDB code 2LMN) (53) and the 3-fold symmetric (PDB code 2LMP) (54) Aβ_1–40 fibrils as reference structures for modeling and docking experiments. The polymorphism of these structures may differ from the fibrils investigated in this study. However, they show the highest similarities of all three wild-type Aβ_1–40 fibril structures currently available (53, 54, 64). Within the two Aβ fibril structural ensembles, several hydrophobic cavities are detected that are large enough to potentially host a sulindac sulfide molecule. We did not find significant deviations in the distribution, size, and polarity of the cavities between the analyzed structural ensembles and models. Therefore, changes in fibril polymorphism do not significantly affect the distribution of hydrophobic patches in amyloid fibrils. We conclude that the two employed structures provide a suitable basis for docking experiments, which is also supported by experimental NMR data, although high-resolution structures of a better defined polymorph will allow more accurate docking in the future.

An induced fit docking reveals that the NSAID can interact with fibrils in three different ways, and all models fulfill the NMR restraints. The apparently ambiguous REDOR restraints suggest that more than one sulindac sulfide molecule might be involved in binding. In the docking poses that are in best agreement with the NMR restraints, sulindac sulfide intercalates between the two β-strands of the Aβ fibril, with the long axis of the molecule either parallel (Fig. 4, a and b, pose 1) or perpendicular (Fig. 4, a, pose 2, and b, poses 2 and 3) to the fibril axis. Therefore, residue Phe¹⁹ seems to play a crucial role in sulindac sulfide binding because its rotameric state has an influence on the size and shape of cavities in clusters #2 and #3. Furthermore, the aromatic side chain is involved in π stacking with the conjugated ring system of the NSAID.

Pose 1 of the docking approach suggests that sulindac sulfide fits into the groove formed by Gly³³ (Fig. 4, a and b, top panels). Therefore, the large CSPs may be attributed to Gly³³ backbone atoms experiencing a change in chemical environment or even undergoing conformational changes to accommodate the NSAID. In theoretical studies, this glycine residue has been suggested to be involved in oxidation of Aβ_1–42 because of its close proximity to the side chain of Met³⁵ (73, 74). Furthermore, Gly³³ has been reported as the key amino acid for Aβ toxicity and to be responsible for driving Aβ into neurotoxic conformations (75). Both residues, Gly³³ and Met³⁵, have been hypothesized to stabilize reactive oxygen species (76, 77). Sulindac sulfide might, therefore, act by binding to the hydrophobic pocket in the vicinity of Gly³³, and preventing oxidation of the Aβ peptide. Oxidation of Aβ_1–42 reduces fibril assembly and aggregation because of the increased polarity introduced by the methionine sulfoxide (78). In accordance, we find that the Aβ-sulindac sulfide complex exists in a stable fibrillar state.

CSPs for Lys¹⁶ may be explained by a recent docking study involving sulindac sulfide and Aβ fibrils that suggested that sulindac sulfide may bind weakly to a shallow, solvent-exposed pocket in the vicinity of Lys¹⁶ and Val¹⁸ and does not interfere with fibrillation (30). This binding mode may, in addition, account for the large REDOR signal detected for the overlapping resonances Val¹⁸/Val³⁹ Cγ (Fig. 2a). We cannot exclude docking of sulindac sulfide to the fibril surface because our REDOR and CSP data are also in agreement with the blind docking model proposed by Yesuvadian et al. (30).

Sulindac sulfide has been reported to form colloidal aggregates above a critical micelle concentration in solution (28). This phenomenon is commonly observed for hydrophobic compounds (79, 80). The size of small-molecule colloidal particles is typically on the order of 50–600 nm (28). These colloids bind unfolded proteins in a promiscuous manner (79, 80) and have been shown to lead to precipitation and inhibition of protein function (81–83). However, the effect of sulindac sulfide on Aβ fibril chemical shifts reported here indicates not a promiscuous but a specific interaction. Aggregates of the compound are observed in TEM images (Fig. 1b), implying the presence of colloids in solution. This is expected because the concentration used (250 μm) lies above the critical micelle concentration of 50–100 μm (28). To account for the experimental single set of resonances, we must assume that individual NSAID molecules dissociate from the colloidal complexes and interact specifically with Aβ fibrils.

Binding of sulindac sulfide in different cavities for different Aβ peptides would result in splitting of the resonances and line-broadening. We observe, however, narrow lines, indicating that the small molecule must either bind simultaneously to different cavities or exchange between these cavities. Relaxation experiments will be carried out in the future to differentiate between these two scenarios.

In conclusion, we suggest that the NSAID sulindac sulfide is able to interact with Aβ fibrils in a rather specific manner. We find that several cavities can accommodate a sulindac sulfide molecule. This is supported by defined CSPs and ¹³C-¹⁹F REDOR contacts. Sulindac sulfide does not induce drastic architectural changes to the non-toxic fibrillar structure, as indicated by NMR and EM. In addition, the characteristic Asp²³-Lys²⁸ salt bridge and the length and positioning of the β-strands are not affected. Molecular modeling suggests that sulindac sulfide intercalates between the two β-strands at, presumably, more than one position. The presented data contribute to the elucidation of the mechanism by which small molecules bind insoluble amyloids. This understanding is crucial for the design of pharmacologically relevant molecules that interfere with Aβ species and that might, in the future, be employed for the treatment of Alzheimer disease.

Author Contributions

E. P. expressed and purified isotopically labelled peptide samples, designed and performed NMR experiments, and carried out the data analysis. R. S. implemented the solid-state NMR experiments. J. M. L. d. A. recorded the initial experiments and assisted with data analysis. G. A. O. carried out the fluorine REDOR experiments. G. M. performed biochemical experiments. H. J. B. and P. W. H. performed and analyzed the molecular modeling and docking. All authors discussed the results. B. R. and E. P. conceived the project and wrote the paper with input from all authors.