Core Binding Site of a Thioflavin-T-Derived Imaging Probe on Amyloid

Jan 30, 2018 - Structural information obtained from this study may aid in the molecular design of new probes for Aβ imaging. .... Figure 5. Orange-G-...
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Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Core Binding Site of a Thioflavin-T-Derived Imaging Probe on Amyloid β Fibrils Predicted by Computational Methods Ryoko Kawai,† Mitsugu Araki,‡,§ Masashi Yoshimura,† Narutoshi Kamiya,∥ Masahiro Ono,*,† Hideo Saji,† and Yasushi Okuno*,‡,§ †

Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Medicine, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan § RIKEN Advanced Institute for Computational Science, 7-1-26 Minatojima Minami-machi, Chuo-ku, Kobe, Hyogo 650-0047, JAPAN ∥ Graduate School of Simulation Studies, University of Hyogo, 7-1-28 Minatojima Minami-machi, Chuo-ku, Kobe, Hyogo 650-0047, Japan ‡

S Supporting Information *

ABSTRACT: Development of new diagnostic imaging probes for Alzheimer’s disease, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) probes, has been strongly desired. In this study, we investigated the most accessible amyloid β (Aβ) binding site of [123I]IMPY, a Thioflavin-T-derived SPECT probe, using experimental and computational methods. First, we performed a competitive inhibition assay with Orange-G, which recognizes the KLVFFA region in Aβ fibrils, suggesting that IMPY and Orange-G bind to different sites in Aβ fibrils. Next, we precisely predicted the IMPY binding site on a multipleprotofilament Aβ fibril model using computational approaches, consisting of molecular dynamics and docking simulations. We generated possible IMPY-binding structures using docking simulations to identify candidates for probe-binding sites. The binding free energy of IMPY with the Aβ fibril was calculated by a free energy simulation method, MP-CAFEE. These computational results suggest that IMPY preferentially binds to an interfacial pocket located between two protofilaments and is stabilized mainly through hydrophobic interactions. Finally, our computational approach was validated by comparing it with the experimental results. The present study demonstrates the possibility of computational approaches to screen new PET/SPECT probes for Aβ imaging. KEYWORDS: Alzheimer’s disease, amyloid-β, imaging probe, Thioflavin-T, computational approaches, binding site, fibril structure

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INTRODUCTION Alzheimer’s disease (AD), which is the leading cause of dementia in the elderly, is a chronic neurodegenerative disorder of the brain characterized by short-term memory loss, progressive cognitive impairment, and disorientation. The incidence of AD increases exponentially with age, and it is estimated that the number of AD patients will increase to 131.5 million by the year 2050 as the global population ages.1 In order to prevent a collapse of global health care systems, the early diagnosis of AD needs to be developed for early intervention and appropriate treatment.2 Post-mortem brains of AD patients present neuropathological features such as depositions of extracellular senile plaques and intraneuronal neurofibrillary tangles, which contain amyloid β (Aβ) aggregates and highly phosphorylated tau proteins, respectively.3 Although the molecular mechanisms that lead to AD onset are poorly understood, Aβ plaques are already detectable in patients a few decades before the clinical symptoms of AD appear. Thus, Aβ plaques have been proposed © XXXX American Chemical Society

to play an important role in AD onset according to the amyloid cascade hypothesis.4 To identify Aβ plaques using an imaging biomarker, the utilization of nuclear imaging techniques that enable Aβ plaques in brains to be imaged sensitively and precisely from outside the body has been explored.5 Several positron emission tomography (PET) and single photon emission computed tomography (SPECT) probes for Aβ imaging have been developed. Among them, 18F-labeled Thioflavin-T analogues, GE-067 (flutemetamol),6−8 BAY94-9172 (florbetaben),9−11 and AV45 (florbetapir),9,12,13 have been approved by the U.S. Food and Drug Administration (FDA) for clinical AD diagnosis (see Figure 1). Another Thioflavin-T analogue, [123I]IMPY (see Figure 1), is the first SPECT imaging probe to be tested in humans and is Received: October 9, 2017 Accepted: January 8, 2018

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DOI: 10.1021/acschemneuro.7b00389 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Figure 2. Comparative study of IMPY- and Orange-G-binding sites on the Aβ aggregates. Representative competition curves of IMPY (black) and Orange-G (red) on Aβ aggregates bound to [125I]IMPY. The IC50 (half-maximal inhibitory concentration) was estimated to be 6.62 nM for IMPY, but the value could not be determined for Orange-G.

Figure 1. Chemical structures of Thioflavin-T, [18F]GE-067, [18F]BAY94-9172, [18F]AV-45, IMPY, and Orange-G.

one of the standard radiotracers used for in vitro competitive studies to evaluate potential utility for imaging Aβ plaques in the AD brain.14,15 Structure−activity relationships based on the chemical structure of IMPY have been evaluated by a common method in medicinal chemistry, being used to discover derivatives with higher affinities toward Aβ plaques.16,17 There is, however, a difficulty regarding rational design for the development of promising new probes because little is known about the binding mode of currently used probes to Aβ fibrils. Several studies addressing the molecular mechanism of probe binding with Aβ fibrils have been reported. Although information on the experimental structure is limited, Landau et al. determined the crystal structure of an assembly of Aβderived peptides bound to Orange-G (see Figure 1), which is a synthetic azo dye used in histological staining.18 This cocrystallized structure indicated that Orange-G recognized the KLVFFA region in Aβ fibrils and that bound Orange-G is stabilized by hydrophobic interactions with phenylalanine side chains and electrostatic interactions with lysine side chains in the region. Also, computational studies based on a single protofilament model predicted that Thioflavin-T analogues preferentially bind to a similar protein region on the Aβ fibrils.19,20 However, it is doubtful that probes with different chemical scaffolds would bind to Aβ fibrils at the same site, and the presence of several binding sites has been suggested.21−23 Actually, our competitive inhibition assay revealed that a Thioflavin-T derivative, IMPY, and Orange-G bind to different sites on the Aβ fibrils (Figure 2). Here, we developed a protocol to precisely predict the probebinding site on Aβ fibrils using molecular dynamics (MD) and ligand-docking simulations, and we applied it to a multipleprotofilament model, which suggested that IMPY preferably binds to an interfacial pocket located between C-terminal βsheets consisting of the residues Gly33, Leu34, and Met35. First, we extensively explored feasible probe-binding sites on a twofold symmetry Aβ fibril after its MD simulation in isolation, and generated IMPY-binding structure candidates using liganddocking simulations. Next, the binding free energy (ΔG) of IMPY with the Aβ fibril was calculated for each binding site using one of the alchemical free energy perturbation methods, MP-CAFEE (Massively Parallel Computational of Absolute binding Free Energy with well-Equilibrated states),24 whose prediction accuracy has been assessed by us25,26 and in several medical fields.27−29 This accurate free energy computation

clearly discriminates two potent candidates for the probebinding sites on the Aβ fibril. The computational results closely supported the experimental results, and we confirmed the feasibility of the computational approach to elucidate the binding mechanism of imaging probes to the Aβ fibril. Structural information obtained from this study may aid in the molecular design of new probes for Aβ imaging.

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RESULTS AND DISCUSSION Comparative Studies of IMPY- and Orange-G- Binding Sites on Aβ Fibrils by a Competitive Inhibition Assay. The crystal structure of Orange-G bound to an assembly of Aβderived peptides suggests that Orange-G recognizes the KLVFFA region.18 On the other hand, Thioflavin-T derivatives were suggested to recognize several binding sites on Aβ fibrils.21 Thus, to ascertain whether both IMPY (a Thioflavin-T derivative) and Orange-G bind to the common site in Aβ fibrils, we performed a competitive inhibition assay of IMPY and Orange-G on Aβ aggregates bound to [125I]IMPY (Figure 2). Cold-IMPY displaced [125I]IMPY on the Aβ aggregates in a concentration-dependent manner, confirming that the inhibition assay works well. On the other hand, Orange-G could not displace [125I]IMPY at any concentration. When half-maximal inhibitory concentration (IC50) values were calculated from the inhibition curves, IMPY showed a value of 6.62 nM, but the IC50 of Orange-G could not be determined. This result indicates that IMPY and Orange-G bind to different sites in Aβ fibrils; that is, IMPY binds to another site that does not contain the KLVFFA region in Aβ fibrils. Hence, we have predicted an IMPY-binding site in Aβ fibrils using a computational approach consisting of molecular dynamics (MD) and docking simulations. Structural Properties of the Aβ Fibril Obtained from MD Simulation. In previous in vitro binding experiments, several chemical probes exhibited similar Ki/Kd values toward Aβ40 and Aβ42 aggregates formed under the same conditions,30 suggesting that similarly shaped probe-binding pockets are formed in both Aβ40 and Aβ42 fibrils. Therefore, the initial structure for the MD simulation was set to a twofold symmetry Aβ40 fibril structure (PDB ID: 2LMN), whose experimental conditions required for fibril formation were the most similar to those of the Aβ42 fibrils used in our assay, assuming that the same fibril formation conditions result in similar fibril structures B

DOI: 10.1021/acschemneuro.7b00389 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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The root-mean-square fluctuations (RMSF) of the backbone Cα atoms indicated that although the turn region consisting of Glu22−Ala30 in addition to the region in the N- and Cterminals was highly mobile, the fibril structure was stably maintained during the MD simulation (Figure S1A), concomitant with no observation of molecular dissociation and significant structural distortion. The overall Cα root-meansquare deviation (RMSD) with respect to the initial structure and principal component analysis of the MD structures showed conformational arrangement of the fibril during the first 20 ns and no significant changes thereafter (Figure S1B,C). Comparison between the initial structure (Figure 3A,B) and the MD structure after the 50 ns simulation (Figure 3C,D) indicated that the deviations in the RMSD observed in the first 20 ns are attributable to the formation of a left-handed twist in the Aβ fibril. A similar twist of Aβ fibrils has been observed with an electron microscope,31 suggesting that our simulation captures structural features of the Aβ fibril without breaking its initial structure. We thus extracted three energetically stable structures from the trajectory from 20 to 50 ns based on the potential energy profile of the intrafibril van der Waals plus Coulomb interactions (Figure S1D), considering that the fibril does not undergo significant conformational change during this time period. These three structures corresponding to the time points of 32.738 ns (−25995.64 kJ/mol), 34.142 ns (−25939.57 kJ/mol), and 49.776 ns (−25853.01 kJ/mol), named MD conformations I, II, and III, respectively, were used for the following ligand-docking simulations. Exploration of Probe-Binding Sites in the Aβ Fibril. Compound-binding sites were explored using the three energetically stable Aβ conformations that were extracted in the previous section. Protein pockets that exhibited positive PLB values were regarded as druggable sites capable of accommodating probe compounds (see Methods). According to the PLB index in MOE,32 6, 11, and 10 druggable sites were extracted from MD conformations I, II, and III, respectively (Figure S2), in which three sites were common among all MD conformations, named sites 1, 2, and 3 (Figure 4). Site 1 is formed by residues comprising the N-terminal β-sheet (Phe19, Phe20, and Ala21), those comprising the C-terminal β-sheet (Ile32 and Leu34), and those forming the turn region (Glu22, Asp23, Asn27, Lys28, and Ala30). This site includes the KLVFFA region, which has been suggested to be recognized by Orange-G.18 Site 2 is surrounded by C-terminal β-sheets and is formed by Gly33, Leu34, and Met35. Site 3 is formed by the N-

irrespective of a difference in the peptide length between Aβ40 and Aβ42. In the beginning, we conducted a 50 ns MD simulation using the NMR structure of the Aβ fibril (PDB code: 2LMN) to equilibrate the Aβ fibril in solution (see Methods). The fibril in the initial structure consists of 12 Aβ peptides (residues 9−40), and each peptide forms a β-hairpin in which the N-terminal β-strand (residues Leu17−Ala21) and C-terminal β-strand (residues Ile31−Met35) are linked by a turn spanning residues Glu22−Ala30. The C-terminal β-sheet in the U-shaped Aβ40 hexamer domain faces that in the other hexamer so that they are disposed in an antiparallel fashion (Figure 3A,B).

Figure 3. Initial structural model of the Aβ40 fibril (A, B) and the final structure after a 50 ns MD simulation (C, D). (A) Overall structures of the two hexamer domains of the Aβ fibril with twofold symmetry obtained from Protein Data Bank (PDB code: 2LMN). (B) Dimer structure unit of the two hexamer domains. Two β-hairpin structures form contacts at each C-terminal β-strand. Here, an N-terminal βstrand (Leu17−Ala21), a C-terminal β-strand (Ile31−Met35), and a turn (Glu22−Ala30) are colored in red, yellow, and blue, respectively, and amino acid side chains in the N- and C-terminal strands are depicted by stick models. (C, D) Structure of the Aβ fibril after a 50 ns MD simulation. A left-handed twist of the fibril was formed during the simulation.

Figure 4. Three candidates for IMPY-binding sites mapped on the representative structures of the Aβ fibril obtained from MD simulations. Three candidates for IMPY-binding sites (sites 1, 2, and 3) were extracted by the Site Finder module in MOE. Cavities in sites 1, 2, and 3, mapped on MD conformations I, II, and III, are indicated by magenta, cyan, and green spheres, respectively. Site 1 is located in the loops connecting the N- and Cterminal β-sheets, including the KLVFFA region. Site 2 is surrounded by the hydrophobic C-terminal β-sheets. Site 3 is composed of the N- and Cterminal residues. Colors of the backbone structure of the Aβ fibril are the same as those in Figure 3. C

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ACS Chemical Neuroscience Table 1. Docking Results of Orange-G toward Sites 1−3 on the Aβ Fibrila MD conformation I MD conformation II MD conformation III

site 1

site 2

site 3

−0.372 −0.397 −0.469 −0.412 ± 0.050

−0.354 −0.355 −0.385 −0.364 ± 0.018

−0.337 −0.297 −0.375 −0.336 ± 0.039

a

All values are in kcal/mol. The binding capability in each site was assessed with the LE score (see Methods). These ligand-docking simulations were performed for each of the energetically stable conformations (MD conformations I−III). Averages and standard deviations among MD conformations I−III were calculated.

Figure 5. Orange-G- and IMPY-binding modes on a twofold symmetry Aβ fibril. (A) Orange-G-binding mode in site 1. A binding structure with the best docking score toward MD conformation III is shown. Orange-G is shown by thick sticks (orange, carbon; red, oxygen; blue, nitrogen; yellow, sulfur). The side chains of residues closely contacting Orange-G (Ala21, Lys28, Gly29, Ala30, and Ile32) are represented by thin sticks. (B) IMPYbinding modes in site 1 (left) and site 2 (right). Two representative docking poses of IMPY toward MD conformation III are shown. In site 1, one is denoted “pose 1”, in which the iodine atom orients toward the N-terminal β-strand including Phe19, and the other is “pose 2”, in which the iodine atom orients toward the C-terminal β-strand. In site 2, one is “pose 1”, in which the iodine atom is exposed to the solvent, and the other is “pose 2”, in which the iodine atom is located in the fibril interior. IMPY is shown by thick sticks (green, carbon; blue, nitrogen; iodine, brown). The side chains of residues closely contacting IMPY (Phe19, Ile32, Leu34, and Met35) are represented by thin sticks. (C) Comparison of IMPY- and OrangeG-binding sites. Our computational prediction and crystal structural analysis18 suggest that Orange-G binds preferably to site 1, which includes the KLVFFA region. On the other hand, our computational prediction suggests that IMPY binds preferably to site 2, which is surrounded by C-terminal β-strands (yellow), through hydrophobic intermolecular interactions. Orange-G and IMPY are shown based on the Corey−Pauling−Koltun model (orange/green, carbon; white, hydrogen; red, oxygen; blue, nitrogen; yellow, sulfur; brown, iodine). Colors of the backbone and side-chain structures of the Aβ fibril are the same as those in Figure 3.

Table 2. Docking Results of IMPY and 55 IMPY-Competitive Compounds toward Sites 1−3 in the Aβ Fibrila site 1 MD conformation I MD conformation II MD conformation III

−0.446 −0.426 −0.506 −0.459

site 2

± 0.022 ± 0.030 ± 0.026 ± 0.042

−0.422 −0.463 −0.487 −0.458

± 0.057 ± 0.041 ± 0.040 ± 0.033

site 3 −0.387 −0.357 −0.357 −0.367

± 0.026 ± 0.020 ± 0.023 ± 0.017

a All values are in kcal/mol. The binding capability in each site was assessed with the LE score (see Methods), and averages and standard deviations among IMPY and 55 IMPY-competitive compounds are indicated. These ligand-docking simulations were performed for each of the energetically stable conformations (MD conformations I−III). Averages and standard deviations among MD conformations I−III were calculated.

Orange-G was docked at sites 1−3 on the Aβ fibril, which were predicted as candidate compound-binding sites. The binding capability of each site was assessed by its LE score (see Methods), showing that site 1 has the highest binding capability (Table 1). It is likely that this is attributable to a favorable Orange-G−Aβ fibril interaction property, in which negatively charged sulfonic acid groups in Orange-G form salt bridges with positively charged residues such as Lys28 (Figures 5A and S3A). Table S1 shows the LE scores for all of the 27 druggable protein sites (i.e., 6 sites in MD conformation I, 11 sites in MD conformation II, and 10 sites in MD conformation III), showing that site 1 has the highest binding capability. Also, Orange-G did not fit into site 2, which has a narrower cavity than site 1 (Figure S4). These prediction-based results were consistent with the Orange-G−fibril binding mode observed in

and C-terminal residues: Gly9, Try10, Glu11, Val12, and His13 (N-terminal) and Leu34, Met35, Val36, Gly37, Gly38, Val39, and Val40 (C-terminal). Since the results of RMSF analysis suggest that the N- and C-terminal regions show large fluctuations, it is inferred that especially site 3 would have a dynamic property. These three sites were identified as candidates for the probe-binding sites on the Aβ fibril, so we performed ligand-docking simulations. Validation of Our Probe-Binding Site Prediction Method. Prior to identifying an IMPY-binding site on the Aβ fibril, we predicted an Orange-G-binding site on the Aβ fibril to confirm the validity of our computational methodology, which had already been determined experimentally by crystal structure analysis.18 D

DOI: 10.1021/acschemneuro.7b00389 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Table 3. Binding Free Energy (ΔG) between the Aβ Fibril and IMPY and Electrostatic (Q)/van der Waals (vdW) Contributions to ΔGa site 1 MD conformation I

MD conformation II

MD conformation III

site 2

pose 1

pose 2

pose 1

pose 2

Q vdW ΔG Q vdW ΔG Q vdW ΔG

−0.57 ± 0.21 −17.66 ± 0.87 −18.24 ± 0.92 −0.82 ± 0.36 −15.90 ± 1.39 −16.72 ± 1.20 2.43 ± 0.18 −16.44 ± 0.84 −14.01 ± 0.94

1.11 ± 0.21 −18.23 ± 0.74 −17.12 ± 0.90 2.88 ± 0.05 −20.55 ± 0.76 −17.67 ± 0.74 1.85 ± 0.10 −17.08 ± 0.56 −15.23 ± 0.61

0.95 ± 0.06 −29.93 ± 0.86 −26.98 ± 0.86 1.95 ± 0.12 −25.04 ± 1.47 −23.09 ± 1.44 2.91 ± 0.13 −24.23 ± 1.21 −21.32 ± 1.25

1.95 ± 0.11 −20.16 ± 1.72 −18.21 ± 1.64 1.60 ± 0.03 −26.17 ± 2.15 −24.57 ± 2.15 2.42 ± 0.04 −28.09 ± 2.04 −25.67 ± 2.02

Q vdW ΔG

0.35 ± 1.81 −16.67 ± 0.90 −16.32 ± 2.14

1.95 ± 0.89 −18.62 ± 1.77 −16.67 ± 1.28

1.94 ± 0.98 −25.73 ± 1.94 −23.79 ± 2.90

1.99 ± 0.41 −24.81 ± 4.13 −22.82 ± 4.02

Q vdW ΔG

1.15 ± 1.55 −17.65 ± 1.65 −16.50 ± 1.59

1.96 ± 0.67 −25.27 ± 2.93 −23.31 ± 3.18

All values are in kcal/mol. ΔG for each of the three Aβ fibril conformations (MD conformations I−III) was calculated with the standard deviation of the six sets of independent simulations. Averages and standard deviations among three Aβ fibril conformations (× two docking poses) were calculated. a

is a plausible IMPY-binding site between sites 1 and 2, leaving the possibility of binding to both sites. A Plausible Recognition Site and Binding Mode of IMPY Predicted from the Binding Free Energy (ΔG) Calculation. To determine a plausible IMPY-binding site between sites 1 and 2 in the Aβ fibril, the binding free energy (ΔG) of IMPY with the Aβ fibril in each site was precisely calculated using MP-CAFEE. ΔG for two kinds of docking poses (pose 1 and pose 2, Figure 5B) in each site was evaluated. The averaged ΔG values for sites 1 and 2 were −16.50 ± 1.59 and −23.31 ± 3.18 kcal/mol (Table 3), respectively, indicating the higher binding affinity of IMPY for site 2 than that for site 1. ΔG for poses 1 and 2 at site 2 were −23.79 ± 2.90 and −22.82 ± 4.02 kcal/mol (Table 3), respectively, showing no significant difference between these two binding poses. These results suggest that IMPY has no preferable binding orientation, and multiple binding structures may exist in site 2. In site 2, the van der Waals contribution to the calculated ΔG was estimated to be −25.73 ± 1.94 kcal/mol for pose 1 and −24.81 ± 4.13 kcal/mol for pose 2, whereas the electrostatic contribution was 1.94 ± 0.98 kcal/mol for pose 1 and 1.99 ± 0.41 kcal/mol for pose 2 (Table 3), suggesting that intermolecular van der Waals interactions contribute dominantly to the binding affinity between IMPY and the Aβ fibril. This is because IMPY is mainly surrounded by hydrophobic residues (Gly33, Leu34, and Met35) in the C-terminal β-sheets in the Aβ fibril (Figures 5B and S3B). Since the binding orientation of ligands is generally stabilized by intermolecular electrostatic interactions (e.g., hydrogen bonds), comparable ΔG values between two opposite binding orientations and weak electrostatic interactions of IMPY with the Aβ fibril could account for the low specific orientation of IMPY bound to the Aβ fibril. IMPY is one of the radioisotope probes for Aβ imaging, developed from Thioflavin-T (a fluorescent probe) as a leading compound. Three PET probes approved by the FDA, [18F]GE-067 (flutemetamol), [18F]AV-45 (florbetapir), and [18F]BAY94-9172 (florbetaben), were designed on the basis of

the cocrystallized structure, validating our binding-site prediction method based on molecular dynamics and compound-docking simulations. Prediction of the IMPY-Binding Site on the Aβ Fibril. A total of 56 compounds, IMPY and other IMPY-competitive compounds (Table S2), were docked at sites 1−3 in the Aβ fibril. Since these compounds have a variety of molecular sizes, docking scores were normalized by the number of heavy atoms in the compounds. Averaged LE scores among the 56 ligands for sites 1−3 were −0.459 ± 0.042, −0.458 ± 0.033, and −0.367 ± 0.017 kcal/mol, respectively, showing that the binding capabilities for sites 1 and 2 were meaningfully higher than that for site 3 (Table 2). Table S3 shows the averaged LE scores for all of the 27 druggable protein sites (i.e., 6 sites in MD conformation I, 11 sites in MD conformation II, and 10 sites in MD conformation III), showing especially higher binding capabilities of sites 1 and 2. Thus, these two sites were extracted as more potent candidates of the Aβ-binding sites for IMPY and IMPY-competitive compounds. Figure 5B shows the docking poses of IMPY toward sites 1 and 2 in the Aβ fibril. In site 1, IMPY interacted with the side chains of Phe19, Ile32, and Leu34 in a few peptides. In site 2, IMPY interacted with the side chains of Met35 in six Aβ peptides. Within both sites, no hydrogen bonds or other electrostatic interactions were observed between IMPY and the Aβ fibril. The docking structures predicted in each site can be classified into two kinds of binding modes. In site 1, one is “pose 1”, in which the iodine atom orients toward the N-terminal β-strand including Phe19, and the other is “pose 2”, in which the iodine atom orients toward the C-terminal β-strand (Figure 5B). In site 2, one is “pose 1”, in which the iodine atom is exposed to the solvent, and the other is “pose 2”, in which the iodine atom is located in the fibril interior (Figure 5B). The docking scores (GBVI/WSA dG scores) for pose 1 (pose 2) were −9.25 ± 0.69 (−8.47 ± 0.91) kcal/mol for site 1 and −9.82 ± 0.48 (−9.69 ± 0.35) kcal/mol for site 2, and therefore it was difficult to judge which E

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based on the 2LMN structure suggested the highest binding affinity of IMPY toward this type of pocket, site 2. Also, previous computational studies employing other multipleprotofilament structures have reported that linear aromatic markers including Thioflavin-T analogues bind preferentially to the interfacial pockets.22,23 Therefore, this pocket region may function as the probe-binding site only when located at the junction between protofilaments. In addition, this interfacial site appears to have a limitation in accommodating chemical probes because Orange-G (Figure S4) and a linear aromatic marker23 were not fully inserted into the site because of their larger molecular sizes. On the other hand, chemical environments of another pocket region including the KLVFFA sequence are determined dependent primarily on the basis of intraprotofilament packing patterns. Whereas the regions observed in the 5KK3 and 2M4J structures are exposed to the solvent, those in the 2LMN, 2LMQ, and 2BEG structures are located between the N-terminal and C-terminal β sheets (Figure 6). Particulaly, pocket environments in the 2LMN and 2LMQ structures are quite similar, and our findings suggested that this pocket, site 1, more preferably accommodates OrangeG than IMPY. In contrast, the 2BEG structure has a unique pocket environment, and previous computational studies suggested that this site has the capability to bind several kinds of chemical probes including Thioflavin-T.19,20,22 These findings suggest that conformational differences in the Aβ fibril drastically change in the selectivity of chemical probes on the basis of the chemical environment of the probe-binding site.

the chemical structure of Thioflavin-T. Since IMPY and these three compounds have a common chemical scaffold, these compounds may commonly recognize this hydrophobic pocket. Selectivity of Chemical Probes on Twofold Symmetry Aβ Fibril and Comparison with That on Other Fibril Structures. In this study, the probe-binding site on the Aβ fibril was precisely predicted with a total simulation time of 7.9 μs, focusing on a twofold symmetry fibril (PDB ID: 2LMN) whose fibril formation conditions were the most similar to those for the fibrils used in our inhibition assay. As a result, we identified two distinct probe-binding sites, sites 1 and 2, which were suggested to preferentially recognize Orange-G and IMPY, respectively (Figure 5C). This prediction result was consistent with the experimental results of the competitive inhibition assay (Figure 2). Pocket regions corresponding to these two probe-binding sites appear to be universally present in other fibril structures deposited in Protein Data Bank (Figure 6). Protein segments constituting each pocket are shared

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CONCLUSIONS

In this study, we investigated the most accessible binding site of a Thioflavin-T-derived imaging probe, IMPY, on Aβ fibrils. The results of a competitive inhibition assay indicate that IMPY and Orange-G bind to different sites in Aβ fibrils. We next developed a protocol to predict probe-binding sites using molecular dynamics (MD) and ligand-docking simulations, and we then confirmed the validity of this protocol using Orange-G. The computational protocol was then applied to predict an IMPY binding site on a twofold symmetry Aβ fibril (PDB ID: 2LMN). First, using energetically stable Aβ conformations obtained from a 50 ns MD simulation (MD conformations I− III), three candidate sites were extracted (sites 1−3). Next, we performed ligand-docking simulations of IMPY and 55 IMPYcompetitive compounds, and two feasible candidate sites (sites 1 and 2) were identified by the binding capabilities of these compounds. Finally, the binding affinity of IMPY toward these two sites was precisely assessed by MP-CAFEE. Calculated ΔG values suggested that IMPY binds preferably to an interfacial pocket between C-terminal β-sheets (site 2) in the Aβ fibril. Site 2 is mainly composed of nonpolar residues (Gly33, Val34, and Met35), and thus van der Waals interactions between IMPY and the Aβ fibril may contribute dominantly to its binding affinity. These computational results clarified distinct recognition mechanisms of IMPY and Orange-G toward the Aβ fibrils, supporting the experimental results of the competitive inhibition assay. Structural features of interactions between the Aβ fibril and IMPY will lead to the possibility of computational screening and molecular design for the development of new probes in the future.

Figure 6. Representative Aβ fibril structures deposited in Protein Data Bank (PDB ID: 2BEG, 2LMN, 2LMQ, 2M4J, and 5KK3). Fibril structures are categorized by the length of Aβ peptides, Aβ1−40 and Aβ1−42. Compound-binding sites are explored on these fibril structures by using the Site Finder module in the MOE programs, and the detected pocket sites are depicted by magenta, cyan, and green spheres. Protein segments constituting the magenta- and cyan-colored pockets are shared among these Aβ structures (e.g., pocket sites indicated by magenta spheres commonly include the KLVFFA region, whereas those indicated by cyan spheres are surrounded by Met35). The side chains of Leu17, Val18, Phe19, Phe20, Ala21, and Met35 are represented by thin sticks. Colors of the backbone structure of the Aβ fibril are the same as those in Figure 3.

among these fibril structures (e.g., pockets indicated by magenta spheres include the KLVFFA region, and those indicated by cyan spheres are surrounded by Met35 in Figure 6). However, each pocket has different shapes and chemical properties because of distinct packing patterns among protofilaments in the fibril. In particular, the chemical environment of the pocket region surrounded by Met35 is drastically changed based on the presence/absence of packing between protofilaments. In a single protofilament model (PDB ID: 2BEG), this pocket is exposed to the solvent (Figure 6), and thereby Thioflavin-T and styrylbenzoxazole analogues were predicted to exhibit lower binding affinity toward this site.19,20 In multipleprotofilament models (PDB ID: 2LMN, 2LMQ, 2M4J, and 5KK3), this site is shielded from the solvent through packing between the C-terminal β sheets, forming interfacial pockets between protofilaments (Figure 6). Our computational study F

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individual Aβ fibril−IMPY complex system and solvated compound system, respectively. All MD runs were carried out with time steps of 2 fs, and snapshots were output every 2 ps to yield 500 snapshots per nanosecond of simulation. In MD simulations of the Aβ fibril, weak conformational constraints are often included to prevent the collapse of its initial structure.19 For example, a fibril structure consisting of five strands (PDB ID: 2BEG) may appear fragile because of its flat shape. In contrast, the initial structure used in our study (PDB ID: 2LMN) has a structural unit (protofilament) of a U-shaped β-sheet consisting of six Aβ molecules, and the C-terminal β-sheet in the hexamer domain faces a similar domain in the other hexamer, forming a cuboidlike fibril structure comprising 12 Aβ molecules (Figure 3). Therefore, conformational constraints were not applied in the production runs of the Aβ fibril, expecting the robustness of its fibril structure. Principal Component Analysis (PCA) of MD Structures of the Aβ Fibril. Using 15 000 Aβ structures extracted evenly from the 50 ns MD simulation, principal component analysis (PCA) was performed using Cartesian coordinates of heavy atoms in the whole region excluding the two N- and C-terminal residues in the fibril. After removing the overall translation and rotations, the covariance matrix was calculated using heavy-atom coordinates of the above-described regions and was diagonalized to obtain the principal component eigenvectors. Docking Simulations of Compounds toward the Aβ Fibril. First, the Site Finder module in the MOE programs was used to predict feasible compound-binding sites in the energetically stable Aβ conformations, which were extracted from a 50 ns MD simulation (see Results and Discussion). Compound-binding propensity at each site was assessed by the PLB index, which can be used to judge the probability of ligand binding on the basis of the idiosyncratic amino acid profile of each binding site.32 A site with a higher PLB index can be considered a more plausible compound-binding site. Next, we collected 55 IMPY-competitive inhibitors on Aβ fibrils. One of them is curcumin, shown by our competitive inhibition assay with IMPY (IC50 = 32.1 nM). The others are extracted from the previous literature.16,17,46−48 The structures of these compounds were constructed using the MOE Builder tool and were optimized using the MMFF94x force field.49 The 2D structures and experimental Ki values are given in Table S2. The total of 56 compounds were docked into each of the pocket sites that exhibit a higher PLB index, assuming that these compounds bind to the same pocket site in Aβ fibrils. Induced-fit docking simulations with the Amber10:EHT force field38 and reaction field solvation model were carried out with the MOE Dock program. The placement algorithm was Triangle Matcher in MOE. The binding capabilities of IMPY and 55 IMPY-competitive inhibitors in each site were assessed by the LE score, which is the Generalized Born Volume Integral/Weighted Surface Area (GBVI/WSA) dG score50 normalized by the number of heavy atoms. The LE scores were calculated using the ligand properties module in the MOE programs after energy minimization of bound ligands. Intermolecular interactions between the Aβ fibril and IMPY are drawn by the 2D Ligand Interaction module of MOE. Calculation of the Binding Free Energy between the Aβ Fibril and IMPY. The binding free energy between the Aβ fibril and IMPY was calculated by MP-CAFEE (Massively Parallel Computation of Absolute binding Free Energy with well-Equilibrated states). MPCAFEE is one of the alchemical free energy perturbation methods, especially the double annihilation method.51 For accurate prediction of the protein−ligand binding free energy, methods for selection of the initial structure for the free energy perturbation calculation and an optimal number of simulations for each λ state have been assessed.51,26 In this method, the protein−ligand binding free energy (ΔGbind) is calculated by the following protocol25 (Figure S5A). (1) For the protein−ligand complex system, 2 ns averages of protein−ligand interaction energies are calculated during preceding 50 ns × 5 simulations. If an energetically stable conformation is maintained for several nanoseconds, such a conformation is regarded as a sufficiently relaxed structural model and is used as the initial structure for the free energy simulation.

METHODS

Inhibition Assay. We determined the binding affinity of Orange-G and IMPY to Aβ aggregates according to the inhibition assay reported previously.16 In this assay, the final concentrations of Orange-G, nonradioactive IMPY, [125I]IMPY, and Aβ42 aggregates were adjusted to 0.415 pM to 20.25 μM, 0.512 pM to 5 μM, 0.025 nM, and 0.125 μg/mL, respectively. Computational System. The prediction accuracy of in silico drug discovery is often increased by incorporating protein flexibility, and the “ensemble docking” method is a common strategy that utilizes multiple crystallographically derived or MD-derived protein conformations for the docking simulation.33,34 In this study, we applied this method to identify the probe- binding site on the Aβ fibril that stably accommodates chemical probes in solution. The computational study consisted of the following protocols. First, for the structural sampling of a ligand-binding pocket in the Aβ fibril, we conducted an MD simulation of the isolated Aβ fibril. Next, structural models of the Aβ fibril in complex with IMPY were constructed by ligand docking simulations. Then, to evaluate the binding affinities of IMPY, MD simulations of the complex and binding free energy simulations were carried out. For selection of the initial Aβ fibril, we used a twofold symmetry Aβ1−40 fibril structure (PDB ID: 2LMN), whose experimental conditions for fibril formation (210 μM Aβ40 peptide in 10 mM sodium phosphate buffer containing 0.01% NaN3 at 22 ± 2 °C)35 were the most similar to our conditions for the fibril formation described above. The N-terminus residue Gly9 was capped with an acetyl group using the structure preparation module in Molecular Operating Environment (MOE, Chemical Group Montreal, Canada), version 2014.09. The dominant protonation state at pH 7.0 was assigned for titratable residues. The 3D structure of IMPY was optimized, and the electrostatic potential was calculated at the RHF/STO-3G level using the GAMESS program,36 after which the atomic partial charges were obtained by the RESP approach.37 Here, because IMPY contains an iodine atom, basis sets applicable up to period 5 elements (RHF/STO-3G, RHF/STO6G, RHF/3-21G, RHF/3-21G*) were employed. As a result, a significant difference in resulting partial charges was not observed among the four basis sets (Table S4). Therefore, the following MD simulations of the fibril−IMPY complex and solvated IMPY systems were performed with the atomic partial charges calculated at the RHF/ STO-3G level. The other parameters for the compound were determined by the general Amber force field (GAFF)38 using the antechamber module of AMBER Tools 12. The Amber99SB-ILDN force field was used for protein and ions39 unless stated otherwise, and TIP3P was used for water molecules.40 Water molecules were placed around the isolated Aβ fibril (Aβ fibril−IMPY complex) with an encompassing distance of 12 Å (8 Å), including roughly 41 000 (22 000) water molecules. Charge-neutralizing ions were added to neutralize the system. MD Simulation. All MD simulations were performed under periodic boundary conditions using the GROMACS 4 program41 on High Performance Computing Infrastructure (HPCI). Electrostatic interactions were calculated using the particle mesh Ewald (PME) method42 with a cutoff radius of 10 Å. van der Waals interactions were cut off at 10 Å. The P-LINCS algorithm was employed to constrain all bond lengths.43 After each of the fully solvated systems was energyminimized, the system was equilibrated for 100 ps under a constant volume (NVT) and run for 100 ps under a constant pressure and temperature (NPT), with positional restraints on protein heavy atoms and ligand atoms. Production runs were performed under NPT conditions without the positional restraints. In this procedure, the temperature was maintained at 298 K using velocity rescaling with a stochastic term44 and the pressure was maintained at 1 bar with Parrinello−Rahman pressure coupling,45 where the time constants for the temperature and pressure couplings to the bath were 0.1 and 2 ps, respectively. A 50 ns production run was performed for the isolated Aβ fibril system. Five sets of 50 ns production runs and three sets of 20 ns production runs were performed with different velocities for the G

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ACS Chemical Neuroscience ORCID

(2) The free energy difference for the protein−ligand complex (ΔGcomp) is calculated by considering 32 intermediate states between λ = 0 (the bound ligand interacts exactly with its surrounding molecules) and λ = 1 (the intermolecular interactions are completely uncoupled). (3) The free energy difference for the solvated ligand (ΔGlig) is calculated by considering 32 intermediate states between λ = 0 (the ligand interacts exactly with solvent molecules) and λ = 1 (the intermolecular interactions completely uncoupled). (4) The binding free energy (ΔGbind) is calculated by the equation ΔGbind = ΔGcomp − ΔGlig. To calculate the free energy differences for each protein−ligand complex (ΔGcomp) and solvated ligand (ΔGlig), we used a 32 λ parameter set for the Coulomb and van der Waals interactions,51 where the Coulomb interaction is first turned off by using 11 λ values (0.0, 0.1, 0.25, 0.4, 0.55, 0.65, 0.725, 0.8, 0.875, 0.95, and 1.0) and subsequently the van der Waals interaction is turned off by using 21 λ values (0.0, 0.1, 0.2, 0.3, 0.4, 0.475, 0.55, 0.6, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.95, and 1.0),51 and we performed six independent simulations with different initial velocities for each λ parameter.26 Thus, to calculate ΔGcomp or ΔGlig, we carried out a total of 192 (=6 × 32) simulations. During these simulations, the temperature was maintained at 298 K using the Nosé−Hoover thermostat52 and the pressure was maintained at 1 bar using the Berendsen barostat.53 Each simulation time was 2 and 1.2 ns for the individual protein−ligand complex and solvated ligand systems, respectively. The time constants for the temperature and pressure couplings to the bath were 0.3 and 1 ps, respectively. All simulations were performed on the K-computer (RIKEN, Japan) under the NPT conditions described above. After the free energy difference between neighboring λ points was calculated by the Bennett acceptance ratio method (BAR)54 from the work distribution in both directions, each free energy difference was accumulated as total free energy differences upon λ = 0 → 1. In the plot of free energy against simulation time, ΔGcomp and ΔGlig were calculated using the equilibrated time period (Figure S5B,C). The total simulation time was 7.9 μs ((2 ns ×192 + 50 ns ×5) × 12 complex states + (1.2 ns ×192 + 20 ns ×3) × 1 ligand states).

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Narutoshi Kamiya: 0000-0002-0527-6968 Masahiro Ono: 0000-0002-2497-039X Hideo Saji: 0000-0002-3077-9321 Author Contributions

R.K. and M.A. contributed equally to this work. M.O., H.S., and Y.O. designed the study. M.Y. carried out the competitive inhibition assay. R.K., M.A., and N.K. performed the molecular dynamics and docking simulations. R.K., M.A., M.O., and Y.O. wrote the paper. All authors discussed the results and reviewed the manuscript. Funding

This research was supported by MEXT, as “Priority Issue 1 on Post-K computer” (Building Innovative Drug Discovery Infrastructure Through Functional Control of Biomolecular Systems), Japan Research Foundation for Clinical Pharmacology, Takeda Science Foundation, and by a Grant-in-Aid for Scientific Research C (JP16K07331) from JSPS to N.K. This research used computational resources of the K computer provided by the RIKEN Advanced Institute for Computational Science through the HPCI System Research Project (Project ID: hp150272 and hp160213). Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information includes the following data: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.7b00389. Structural properties of the Aβ fibril during a 50 ns MD simulation; compound-binding sites mapped on the representative structures of the Aβ fibril obtained from MD simulations; interactions of the Aβ fibril with Orange-G and IMPY in the complex structures chosen from the docking results; 18 docking poses of Orange-G toward site 2 in the Aβ fibril; thermodynamic cycle in the MP-CAFEE method and free energy against simulation time in free-energy simulations for the fibril−IMPY complex and solvated IMPY systems; chemical structures of 55 IMPY-competitive inhibitors and their properties; docking results of Orange-G, IMPY, and 55 IMPYcompetitive compounds toward all predicted compoundbinding sites in the Aβ fibril; and dependence of the basis set on the partial atomic charges of IMPY (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(M.O.) E-mail: [email protected]. Phone: +81-75753-4608. Fax: +81-75-753-4568. *(Y.O.) E-mail: [email protected]. Phone: +8175-751-4881. Fax: +81-75-751-4881. H

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