Insights on the Binding of Thioflavin Derivative Markers to Amyloid

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Insights on the Binding of Thioflavin Derivative Markers to Amyloid-Like Fibril Models From Quantum Chemical Calculations Jorge Ali-Torres, Albert Rimola, Cristina Rodríguez-Rodríguez, Luis Rodríguez-Santiago, and Mariona Sodupe J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp402807g • Publication Date (Web): 10 May 2013 Downloaded from http://pubs.acs.org on May 20, 2013

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The Journal of Physical Chemistry

Insights on the Binding of Thioflavin Derivative Markers to Amyloid-like Fibril Models from Quantum Chemical Calculations Jorge Alí-Torres,a Albert Rimola,a Cristina Rodriguez-Rodríguez,b Luis Rodríguez-Santiago,a Mariona Sodupea,* a

Departament de Química, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain

b

Medicinal Inorganic Chemistry Group, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada.

Corresponding author: [email protected]

ABSTRACT

Thioflavin-T (ThT) is one of the most widely used dyes for staining and identifying amyloid fibrils, which share a common parallel in register β-sheet structure. Unfortunately, ThT is a charged molecule, which limits its ability to cross the blood brain barrier and its use as an efficient dye for in vivo detection of amyloid fibrils. For this reason, several uncharged ThT derivatives have been designed and their binding properties to Aβ fibrils studied by fluorescence assays. However, there are still many unknowns on the binding mechanism and the role of non-covalent interactions on the affinity of these ligands towards β-sheet structures. The present contribution analyzes the binding of ThT (1) and neutral ThT derivatives (2-7) to a β-sheet model by means of quantum chemical B3LYP-D calculations and including solvent effects with the continuum CPCM method. Results show that, in all cases, ligand binding is mainly driven by dispersion interactions. In addition, ligands with –NH groups display

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hydrogen bond interactions with CO groups of the peptide strand, increasing the intrinsic affinity toward the β-sheet surface. Solvent effects notably reduce the affinity of charged ThT, as compared to neutral systems, due to its larger solvation energy. As a result, neutral derivatives display significantly higher affinities than ThT in solution, in agreement with experimental observations. Analysis of the hydrogen bonding network of the β-sheet structure indicates that stacking interactions upon ligand binding induce a shortening of interstrand hydrogen bonding, suggesting a strengthening of the β-sheet.

Keywords: Thioflavin markers, β-amyloid, DFT-D, ligand affinity, β-sheet, BTA


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INTRODUCTION Thioflavin-T (ThT) is a potent fluorescent marker, widely used for in vitro characterization of amyloid fibrils, the main component of senile plaques associated to Alzheimer’s disease (AD). Amyloid fibrils are ordered aggregates with a parallel in register β–sheet architecture,1 highly stabilized by H-bonding, with the β-sheet strands perpendicular to the long axis of the fibrils.2 Upon binding to amyloid deposits ThT fluorescence intensity increases by orders of magnitude.3 ThT refers to the cation of the 4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline chloride salt and consists of a N-methylated benzothiazole fragment linked to a dimethylaniline ring (Figure 1). Its photophysical properties are related to its behaviour as molecular rotor;4-5 that is, inhibition of internal rotation around the C-C bond enables radiative relaxation to the ground state from a locally excited state with a high oscillator strength, resulting in a high quantum yield of fluorescence.4 Amyloid binding is expected to induce this rotational lock, thereby leading to an enhancement of ThT fluorescence. The detailed knowledge of how ThT interacts with the fibers is, therefore, essential to understand this increase in fluorescence. For this reason, in the last years, many efforts have been devoted to unravel the binding modes of ThT to amyloid fibrils6-9 both experimentally8, 10-19

and by computational simulations.20-23 Several modes have been proposed; the most

accepted one being that first suggested by Krebs et al. in which ThT inserts into the βsheet cavities with its long axis parallel to that of the fibrils.10 ThT is a charged molecule due to the presence of the methyl group in the heterocyclic nitrogen of the benzothiazole ring, which limits its ability to cross the blood brain barrier (BBB) and its use as an efficient dye for in vivo detection of amyloid fibrils. For this reason, several uncharged ThT derivatives, with increased lipophilicity that


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facilitates the BBB crossing, have been designed and its binding properties to Aβ fibrils studied.24-29 In particular, compounds derived from the removal of the methyl group at the N position of the benzothiazole moiety such as the 2-arylbenzothiazole (BTA) compounds (2, 3 and 4, in Figure 1) or the Pittsburgh compound B (PIB), in which the methyl group at the sixth position of benzothiazole in 3 is replaced by a hydroxyl group, have been found to bind to Aβ(1-40) fibrils with a significantly higher affinity than ThT.26, 28, 30 Among the 6-CH3-BTA compounds (2, 3 and 4), the largest affinities were found for 3 and 4 to which additional methyl groups from the aminophenyl moiety had also been removed. From a very systematic study, in which the binding affinity of 18 ThT derivatives to Aβ fibrils was analyzed by fluorescent measurements, this greater affinity has been attributed to the formation of hydrogen bond interactions between the –N-H of the aryl group and the fibrils.28 Moreover, other factors such as dispersive stabilizing interactions have also been invoked28 to explain why compound 3 (RNH(CH3)) exhibits a larger affinity than 4 (R-NH2).26 Note that H-bond interactions alone would not explain the observed trend since the H-bond donor ability of N-H increases upon methyl removal. Fluorescence-based assays have provided important structural insights into the binding features of several ligands to Aβ fibrils.19,

26, 28, 30

There are still, however, many

unknowns about which are the most important stabilizing forces that determine the observed trends. In this context, and with the aim of providing structural information at the atomic level, in the present work we report quantum chemical calculations on the binding of 7 molecules to a model of a β-sheet structure. These molecules are shown in Figure 1 and include ThT (1), the three uncharged derivatives 2, 3 and 4 abovementioned and three previously designed multifunctional ligands (5, 6 and 7),31 that were inspired by ThT and clioquinol and contain both amyloid binding and metal


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chelating properties. Two major goals will be addressed in the present work: i) the binding modes and the non-covalent interactions that occur between the ligands and the β-sheet structure and ii) the changes induced on the β-sheet structure, particularly on the H-bond network, upon ligand binding. This latter point is also particularly important since recent experimental data have shown that ThT may promote the Aβ(1-40) peptide amyloid aggregation by changing solvent peptide interactions and stabilizing more ordered β-like conformations.32 Herein, we present quantum chemical calculations that provide useful information at the atomic level to help interpret experimental observations and also to assist in the rational design of more effective in vivo tracers, inhibitors, and therapeutics for AD.

COMPUTATIONAL DETAILS The β-sheet model considered consists of five strands of the triglycine peptide capped with acetyl and methylamine groups (Ac-GGG-NMe). This model was taken from our previous work,20 in which a docking exploration of the interaction of ThT with the NMR structure33 of Aβ1-42 (PDB code 2BEG) was carried out. Several binding sites were identified and, among them, the most stable favorable conformations were those in which the ligand was located in external binding sites (pose A and pose B1 in reference20). Our β-sheet model was obtained from pose A where ThT is accommodated into the crevices defined by residues 33-35 of Aβ1-42. Thus, we selected residues 32-36 of the five chains to build the model and substituted the terminal (-CO-) and (-NH-) groups and the side chains by H atoms. The simplification of the original β-sheet structure by the incorporation of hydrogen atoms instead of side chains (i.e. polyglycine


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systems) facilitates the understanding of the intrinsic effects of the ligand binding on the stability of the β-sheet, separating(avoiding) the influence of larger side chains. Calculations were performed at the B3LYP-D level; that is, using the hybrid non-local B3LYP density functional34-35 and adding the atom-atom additive damped

C6ij empirical correction of the form ED = − s6 ∑ f ( Rij ) 6 (S6=1.05) 36 to include longRij i< j range dispersion contributions to the computed ab initio B3LYP energy and gradients. This approach has been shown to provide good results in systems where dispersion forces play a fundamental role,36-37 as in the present case. Moreover, the inclusion of this dispersion correction has been shown to be essential to determine not only the adsorption energy but also the geometry of the adsorbed adduct in hydrophobic surfaces.38 Structure optimizations were carried out with the 6-31G(d) basis set, whereas binding energies were evaluated with the larger 6-311++G(d,p) basis, at the 6-31G(d) optimized geometries. This procedure was used in our previous work20 to study the binding of ThT to amyloid fibrils and it provides accurate enough results at a reasonable computational cost. Indeed, optimized geometries were found to be very similar with the two basis sets; i.e., the distances corresponding to stacking and CH-π interactions vary by less than 0.03 Å, and the B3LYP/6-311++G(d,p) binding energy was found to differ by around 2 kcal mol-1 regardless of the geometry used. It should be noted that in all geometry optimizations the terminal C atoms of each peptide chain were frozen at their positions in the NMR structure to avoid spurious deformation of the backbone. Additionally, binding energies for the most favorable configurations have been corrected for basis set superposition error using the counterpoise procedure.39 Finally, aqueous solvent effects were included by performing B3LYP/6-311++G(d,p) single


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point energy calculations with the CPCM continuum method,40-41 at the gas phase optimized geometries. The United Topological Model applied on atomic radii of the UFF force field (UA0), which is the default in Gaussian 03, was used to define the solvent

cavities.

Interaction

energies

are

computed

as:

∆E = E ( Ligand − βsheet) − E ( Ligand ) − E (βsheet) , with the energy of each species in ,CP gp solution being E Bsol3 LYP − D = E B 3 LYP− D + δ CP + ∆Gsolv ; i.e., including the gas phase energy at

the B3LYP-D level ( E Bgp3 LYP− D ), the counterpoise correction (δCP) and the solvation energy

( ∆Gsolv ). Note that these values do not include zero-point energy and thermal

corrections, which are expected to decrease the ligand affinity. All gas phase calculations were carried out with the Gaussian03 program.42

RESULTS AND DISCUSSION

Ligand-β-sheet binding. The interaction between the considered ligands (Figure 1) with the β-sheet structure has been examined by performing a series of geometry optimizations with different ligand orientations. In all cases the ligand has been placed in the groove of the β-sheet surface, parallel to the long axis of the fibril, in accordance with anisotropic fluorescence emission studies.10 Moreover, this kind of interaction maximizes stabilizing dispersion forces between the β-sheet surface and the ligand. Different orientations are those in which the sulfur atom of benzothiazole points toward/outward the surface or the methyl substituents of dimethylaniline lie above the smaller/larger side of the C12 pseudo-ring formed by H-bonding in the parallel β-sheet configuration (see Figure 2). The most stable pose for each ligand (including solvent effects) is shown in Figure 3. The main geometrical parameters for the optimized adducts are detailed in Table 1, while different energy-related values relative to the


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ligand binding are reported in Table 2. Data corresponding to all other considered structures are collected in the Supplementary Information (SI). It should be noted that except for two ligands (5 and 7), in all cases, the ground state structure in gas phase is the same as in solution. Analysis of all considered complexes indicate that, in general, ligands enclosing the benzothiazole moiety prefer to interact with the β-sheet structure with the S atom pointing toward to the bottom of the groove. Indeed, in all cases, except 4, the most stable structure shows this configuration (see Figure 3). For ligands such as 3 and 4 with the amino group partially or totally unsubstituted, however, the possibility of forming H-bonds with the β-sheet structure can modify this trend, if it hinders the formation of the H-bond. This is the case observed for 4, for which the formation of two H-bond interactions between the NH2 and the two O atoms of the C12 ring of the β-sheet structure leads to a complex in which the benzothiazole sulfur atom is pointing outward the groove. For ligands capable of forming hydrogen bonds, with the β-sheet, the intrinsic gas phase interaction energies for different ligand orientations can vary up to 78 kcal mol-1 depending on the strength of the H-bond, whereas for those ligands that do not establish H-bonds with the β-sheet surface, the energy changes associated with different orientations are less than 2 kcal mol-1 (see Supplementary Information). The ligands are stabilized over the β-sheet surface due mostly to the formation of stabilizing stacking interactions between the aromatic rings of the ligands and the peptide backbones, and by CH-π interactions between the aromatic rings and the CH2 groups of the peptide chain. The distances between the centroid of the phenyl/indene moieties and the average plane defined by the 12 backbone atoms of the three inner peptide strands immediately below the ligand are considered to describe the stacking


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interactions between the two aromatic rings and the β-sheet surface. Computed values range from 2.9 to 3.7 Å, similarly to those found for stacked DNA bases.43 CH-π interactions are observed between the CH2 groups of the peptide strands and the six member ring of the heterocyclic indene moiety. Computed distances between the C atom and the centroid of this ring range from 3.3 to 3.5 Å, similar to those found for the interaction of hydrocarbons with the π face of benzene,44-45 for all ligands except 3a and 4a, for which H-bond interactions leads to a ligand arrangement not suitable for such interaction. A closer look at the optimized structures, particularly at the geometrical parameters related to stacking, indicate that for compounds 5a, 6a and 7a, the distance between the heterocyclic indene moiety and the β-sheet surface (~2.9 Å) is significantly smaller than that of the phenyl one (3.6-3.7 Å), as expected due to the larger dispersion interactions with the bicyclic fragment. Moreover, for these ligands the phenyl ring holds an OH or NH2 substituent in the ortho position that leads to an increased repulsion with the βsheet. However, for 1a, 2a, and 3a, the reverse trend is observed; i.e. the phenyl moiety gets closer to the β-sheet surface than the heterocyclic indene fragment. This may result from different factors such as the presence of a CH3 group in the six member ring of benzothiazole, which leads to an increased repulsion with the surface, and the formation of H-bond interactions between the –NH(CH3) phenyl substituent and the β-sheet in 3a. Structure 4a is a particular case since both aromatic moieties exhibit similar stacking distances, resulting from a binding arrangement that maximizes the two hydrogen bond interactions between NH2 and the CO oxygen atoms of the C12 pseudo ring in the groove. Overall, the ligand conformation at the β-sheet surface results from a delicate balance between stabilizing interactions such as H-bonding, which is observed for 3a,


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4a and 7a with distances of about 2.0 -2.3 Å (see Table 1), dispersion forces and repulsion. Such a balance induces changes on the hydrogen bond network of the βsheet, defined by NH···O and the weak CαHα···O bifurcated hydrogen bonds,46 and also on the planarity of the ligand upon binding to maximize stabilizing forces. The binding of ThT (1a) to the β-sheet structure induces an increase of planarity responsible for its fluorescence enhancement.4, 8 For 2a, 3a, and 4a, which are planar when unbound, the interaction with the β-sheet structure induces a certain non-planarity, the Φ angle between the two aromatic moieties increasing up to 14 degrees for 2a. For structures 5a, 6a and 7a, changes on the torsional angle are small due to the presence of an intramolecular hydrogen bond. This interaction prevents the deformation of the molecules after attaching them to the β-sheet surface, as it is observed from the calculated distortion energies of the ligands (see below). Computed interaction energies, both in gas phase and in aqueous solution, are reported

has been decomposed into in Table 2. The total gas phase interaction energy ∆

two terms: the B3LYP (∆ ) and dispersion ( ∆ ) contributions to the energy. As

it is noted, the ligand binding is mainly driven by dispersion interactions. Note that electrostatic interaction is expected to be small considering relative dipole orientations of the β-sheet and the ligand. Indeed, pure B3LYP interaction energies are all positive, whereas the dispersion contribution to the binding is large and negative for all complexes, hence favoring the formation of the adsorbed complex. Comparing the different ligands, it is observed that the positively charged ThT (1a) is the one that exhibits a larger binding energy in gas phase, due to the presence of a positive charge, which leads to a larger electrostatic interaction. The neutral counterparts show interaction energies that are about 2-5 kcal mol-1 smaller, in absolute value, and their binding exhibit the following trend: 3a > 2a ~ 4a, i.e., with 3a being the ligand with the


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highest intrinsic affinity, in line with experimental observations.26, 28 In an attempt to evaluate the contribution of the hydrogen bonding interaction we have carried out calculations for ammonia interacting with the β-sheet model with the same hydrogen bond interactions as those found in 3a and 4a. Results indicate that the H-bond contribution to the binding energy is 3.8 kcal mol-1 for 3a and 4.7 kcal mol-1 for 4a. Thus, the larger binding energy of 3a as compared to 4a is due to the larger dispersion interactions of -NH(CH3) versus –NH2, as previously suggested.28 Ligand 3 also displays a larger dipole moment than 4, which contributes to a large electrostatic interaction. Complexes 5a, 6a and 7a, with a -OH or -NH2 substituent in the ortho position of the phenyl moiety, show smaller binding energies, due to the larger distortion energy of the β-sheet structure (see δEβsheet) and repulsion with the surface of the fibril model. Among them, 7 shows the greater affinity, which is attributed to its ability to form a H-bond between the NH group of the benzoimidazole moiety and a CO of the β−sheet. Let us now consider the influence of the solvent. It can be observed in Table 2 that the interaction energy decrease (in absolute value) upon including solvent effects. As expected, the major decrease corresponds to the positively charged ligand 1, since binding to the β-sheet partially screens the positive charge and thereby, solvation energies are more stabilizing in the unbound system. In complexes 3a, 4a and 7a the solvation energies are also somewhat destabilizing, leading to smaller binding energies. This is due to the presence of the already mentioned NH•••O=C H bonds; that is, in the complex the NH group is not exposed to the solvent as in the free ligands. For the remaining compounds, solvation energies have a small influence since the polar groups exposed to the solvent in the complex and in the separate fragments are quite similar.


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The computed higher affinity of neutral ThT derivatives 2, 3 and 4, as compared to ThT (1), toward the β-sheet surface is in perfect agreement with experiments conducted on native Aβ peptides,26, 28, 30 and is not due to a larger intrinsic affinity but to the fact that charged 1 is better solvated in aqueous solution. The trend observed for 2, 3 and 4, however, differs from experimental observations, which indicate that complex 3, with one methyl on the amine nitrogen, is the one that exhibits the highest affinity.26 While such a behavior is observed in gas phase (see Table 2), the inclusion of solvent effects changes the trend to: 2 > 3 > 4; that is, the larger the hydrophilicity the smaller the binding energy is. Differences on the binding energies are, however, small so that variations with respect to experimental observations may arise from the limitations of the computational approaches used, such as the use of an implicit solvent model or the non-consideration of side chains in the β-sheet. Indeed, the binding of BTA analogs could be influenced by the presence/absence of hydrophobic side chains present in native Aβ peptides but missing in our polyglycine model. Nevertheless, it is worth noting that our computed trend (2 > 3 > 4), that inversely correlates with the hydrophilic character of the ligand, is observed for other series of BTA ligands in which the R6=CH3 of benzothiazole is substituted, for instance, by OCH3, OH or H,30 indicating that the CH3 probably plays an important role in positioning the ligand on the β-sheet surface to minimize steric repulsion.

β-sheet hydrogen bond changes upon ligand binding. Ligand binding induces changes in the hydrogen bonding of the β-sheet. In order to evaluate these changes Table 1 reports the variations on the average interstrand hydrogen bond distances with respect to the computed value without the ligand. The values of all H-bond distances are reported in SI. One may observe that, in general,


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these values are negative, so that the H-bonds that held the different fibril strands become shorter (in average) in the complexes rather than in the free-ligand β-sheet. This variation is more noticeably if such variation is computed only considering the three innermost strands immediately below the ligand. Moreover, a detailed analysis of Hbond variations shows that, in general, H-bonds that experience a major decrease (~0.1 Å) are those immediately below the aromatic rings, particularly below the indene heterocyclic moiety. This fact seems to indicate that ligand binding, dominated by dispersion interactions, strengthens interstrand H-bonds. Changes on hydrogen bonding in DNA base pairs upon including stacking interactions have also been observed in previous studies,47-48 highlighting the mutual relationship between stacking and hydrogen bonding. Noticeably, the major changes on hydrogen bond distances are observed for complexes 5a, 6a and 7a. Accordingly, these complexes are the ones that exhibit the largest fibril deformation energies (see δEβ-sheet values of Table 2). This may be attributed to the closer proximity of the heterocyclic indene moiety, the computed stacking distance (~2.9 Å) being significantly smaller than for the remaining ligands (see above), which induces larger effects on the hydrogen bond network. In contrast, the interstrand hydrogen bond distances increase (on average) for 3a and 4a, which exhibit NHligand···O hydrogen bond interactions between the ligand and the carbonyl oxygens of the β-sheet. Such interactions compete with the β-sheet NH···O and CαHα···O hydrogen bonds, increasing their distances, particularly those involving the carbonyl oxygen interacting with the ligand. In order to analyze whether the geometrical changes on hydrogen bonding of the βsheet imply a strengthening of the interstrand hydrogen bonding, we have calculated the interaction energies of the three innermost strands (IS), ∆EHB(IS). We considered the innermost strands to avoid the end-effect, which we assume to be attenuated by the rest


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of the strands in the real system. These interaction energies have been calculated by performing single-point energy calculations on the system derived from removing the ligand and the two terminal strands and on every single strand, at the geometry of the ligand-βsheet complex. This energy represents the net interaction energy (i.e. without considering the distortion of the fibril after ligand attachment). Results given in Table 2 (∆∆EHB(IS)) are relative to the value obtained for the fully optimized fibril without the ligand. Negative values of ∆∆EHB(IS) imply larger interaction energies, i.e, a strengthening of the hydrogen bonding. As shown in Table 2, present calculations seem to indicate that, in most cases, the ligand binding increases the interaction between the strands (except for molecules 2a and 3a). A small strengthening is also observed for ThT, which may be related with the recent findings showing that ThT may promote the peptide amyloid aggregation.32 However, in order to be conclusive, calculations at higher levels of theory, which can only be carried out for much smaller systems, would be required. A larger interstrand strengthening is observed for ligands 5 and 6. This is probably due to the fact that these ligands do not establish H-bond interactions with the β−sheet, and show larger stacking interactions with the indene heterocyclic moiety (see above). For complexes that exhibit H-bond interactions with the β-sheet surface, the strengthening is small (4a and 7a) or it may even show a significant weakening such as in 3a. Overall, changes on the H-bonding network of the β-sheet structure upon ligand binding result from a balance between dispersion interactions, which appear to strengthen interstrand interactions, and the possibility of establishing H-bond interactions between the ligand and the β-sheet surface, which weakens them.


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CONCLUSIONS The binding of ThT (1) and neutral ThT derivatives (2-7) to a β-sheet structure has been analysed by means of quantum chemical B3LYP-D calculations and including solvent effects with the continuum solvent CPCM model. Considered ligands include ThT derivatives in which the methyl group of the heterocyclic nitrogen has been removed to eliminate the positive charge and phenyl moieties with –N(CH3)2, NH(CH3) and NH2 at 4’-position (2,3 and 4). In addition, we have considered three more ligands in which the methyl group of the heterocyclic indene moiety has been removed and the phenyl fragments hold an OH (5 and 6) or NH2 (7) substituent in the 2’-position. Two major goals have been addressed in the present work: i) the ligand- β-sheet binding and ii) the changes induced on the β-sheet structure, particularly on the H-bond network, upon ligand binding. The following conclusions have been obtained: i)

Stable structures with the ligand placed on the groove of the β-sheet surface, parallel to the long axis of the fibril are found for all cases, the ligand binding being mainly driven by dispersion interactions.

ii)

The removal of methyl groups on the amine nitrogen allows for the formation of H-bond interactions with the CO groups of the β-sheet surface, which for ligand 3 with –NH(CH3), leads to an increase of the intrinsic (gas phase) affinity toward the β-sheet.

iii)

The removal of the methyl group of the heterocyclic indene bicycle causes this moiety to be positioned closer to the β-sheet structure, causing larger changes on the β-sheet hydrogen bonding.


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iv)

Solvent effects notably reduce the affinity of charged ThT toward the surface, as compared to neutral systems, due to its larger solvation energy. As a result, neutral derivatives display significantly higher affinities in solution, in agreement with experimental observations. On the other hand, for 6-CH3-BTA series, solvent effects change the trend to: 2 > 3 > 4; that is, the larger the ligand hydrophilicity the smaller the binding energy is.

v)

Analysis of the hydrogen bonding network of the β-sheet seem to indicate that stacking interactions resulting from ligand binding induce a shortening of interstrand hydrogen bonding, suggesting a strengthening of the β-sheet. This is particularly noticeably for ligands 5 and 6 because these ligands do not establish H-bond interactions with the β−sheet and show larger stacking interactions with the indene heterocyclic moiety.

ACKNOWLEDGEMENTS Financial support from MICINN (CTQ2011-24847/BQU) and the Generalitat de Catalunya (SGR2009-638) and the use of the computational facilities of the Catalonia Supercomputer Center (CESCA) are gratefully acknowledged. MS also acknowledges support through 2011 ICREA Academia award. CRR wishes to thank Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) from Generalitat de Catalunya for the finantial support with the grant Beatriu de Pinós (BP-DGR-2009). AR is indebted to MICINN for a Juan de la Cierva contract.

Electronic Supplementary Information Available. Complete reference 42, interaction energies and optimized geometries for all poses, and β-sheet hydrogen bond distances. This information is available free of charge via the Internet at http://pubs.acs.org


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(15) Robbins, K. J.; Liu, G.; Selmani, V.; Lazo, N. D., Conformational Analysis of Thioflavin T Bound to the Surface of Amyloid Fibrils. Langmuir 2012, 28 (48), 16490-16495. (16) Sabate, R.; Lascu, I.; Saupe, S. J., On the Binding of Thioflavin-T to HET-s Amyloid Fibrils Assembled at pH 2. J. Struct. Biol. 2008, 162 (3), 387-396. (17) Sulatskaya, A. I.; Kuznetsova, I. M.; Turoverov, K. K., Interaction of Thioflavin T with Amyloid Fibrils: Stoichiometry and Affinity of Dye Binding, Absorption Spectra of Bound Dye. J. Phys. Chem. B 2011, 115 (39), 11519-11524. (18) Wolfe, L. S.; Calabrese, M. F.; Nath, A.; Blaho, D. V.; Miranker, A. D.; Xiong, Y., Protein-Induced Photophysical Changes to the Amyloid Indicator Dye Thioflavin T. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (39), 16863-16868. (19) Lockhart, A.; Ye, L.; Judd, D. B.; Merritt, A. T.; Lowe, P. N.; Morgenstern, J. L.; Hong, G. Z.; Gee, A. D.; Brown, J., Evidence for the Presence of Three Distinct Binding Sites for the Thioflavin T Class of Alzheimer's Disease PET Imaging Agents on Beta-amyloid Peptide Fibrils. J. Biol. Chem. 2005, 280 (9), 7677-7684. (20) Rodriguez-Rodriguez, C.; Rimola, A.; Rodriguez-Santiago, L.; Ugliengo, P.; Alvarez-Larena, A.; Gutierrez-de-Teran, H.; Sodupe, M.; Gonzalez-Duarte, P., Crystal Structure of Thioflavin-T and its Binding to Amyloid Fibrils: Insights at the Molecular Level. Chem. Commun. 2010, 46 (7), 1156-1158. (21) Wu, C.; Biancalana, M.; Koide, S.; Shea, J. E., Binding Modes of Thioflavin-T to the Single-Layer beta-Sheet of the Peptide Self-Assembly Mimics. J. Mol. Biol. 2009, 394 (4), 627-633. (22) Wu, C.; Bowers, M. T.; Shea, J. E., On the Origin of the Stronger Binding of PIB over Thioflavin T to Protofibrils of the Alzheimer Amyloid-beta Peptide: A Molecular Dynamics Study. Biophys. J. 2011, 100 (5), 1316-1324. (23) Wu, C.; Wang, Z. X.; Lei, H. X.; Duan, Y.; Bowers, M. T.; Shea, J. E., The Binding of Thioflavin T and Its Neutral Analog BTA-1 to Protofibrils of the Alzheimer's Disease A beta(16-22) Peptide Probed by Molecular Dynamics Simulations. J. Mol. Biol. 2008, 384 (3), 718-729. (24) Wang, Y. M.; Klunk, W. E.; Huang, G. F.; Debnath, M. L.; Holt, D. P.; Mathis, C. A., Synthesis and Evaluation of 2-(3 '-Iodo-4 '-Aminophenyl)-6-Hydroxybenzothiazole for in Vivo Quantitation of Amyloid Deposits in Alzheimer's Disease. J. Mol. Neurosci. 2002, 19 (12), 11-16. (25) Mathis, C. A.; Bacskai, B. J.; Kajdasz, S. T.; McLellan, M. E.; Frosch, M. P.; Hyman, B. T.; Holt, D. P.; Wang, Y. M.; Huang, G. F.; Debnath, M. L.; et. al., A Lipophilic Thioflavin-T Derivative for Positron Emission Tomography (PET) Imaging of Amyloid in Brain. Bioorg. Med. Chem. Lett. 2002, 12 (3), 295-298. (26) Klunk, W. E.; Wang, Y. M.; Huang, G. F.; Debnath, M. L.; Holt, D. P.; Mathis, C. A., Uncharged Thioflavin-T Derivatives Bind to Amyloid-beta Protein with High Affinity and Readily Enter the Brain. Life Sci. 2001, 69 (13), 1471-1484. (27) Wang, Y. M.; Mathis, C. A.; Huang, G. F.; Debnath, M. L.; Holt, D. P.; Shao, L.; Klunk, W. E., Effects of Lipophilicity on the Affinity and Nonspecific Binding of Iodinated Benzothiazole Derivatives. J. Mol. Neurosci. 2003, 20 (3), 255-260.


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(28) Yona, R. L.; Mazeres, S.; Faller, P.; Gras, E., Thioflavin Derivatives as Markers for Amyloid-beta Fibrils: Insights into Structural Features Important for High-Affinity Binding. ChemMedChem 2008, 3 (1), 63-66. (29) Cisek, K.; Kuret, J., QSAR Studies for Prediction of Cross-beta Sheet Aggregate Binding Affinity and Selectivity. Bioorg. Med. Chem. 2012, 20 (4), 1434-1441. (30) Mathis, C. A.; Wang, Y. M.; Holt, D. P.; Huang, G. F.; Debnath, M. L.; Klunk, W. E., Synthesis and Evaluation of C-11-Labeled 6-Substituted 2-Arylbenzothiazoles as Amyloid Imaging Agents. J. Med. Chem. 2003, 46 (13), 2740-2754. (31) Rodriguez-Rodriguez, C.; de Groot, N. S.; Rimola, A.; Alvarez-Larena, A.; Lloveras, V.; Vidal-Gancedo, J.; Ventura, S.; Vendrell, J.; Sodupe, M.; Gonzalez-Duarte, P., Design, Selection, and Characterization of Thioflavin-Based Intercalation Compounds with Metal Chelating Properties for Application in Alzheimer's Disease. J. Am. Chem. Soc. 2009, 131 (4), 1436-1451. (32) D'Amico, M.; Di Carlo, M. G.; Groenning, M.; Militello, V.; Vetri, V.; Leone, M., Thioflavin T Promotes A beta(1-40) Amyloid Fibrils Formation. J. Phys. Chem. Lett. 2012, 3 (12), 1596-1601. (33) Lührs, T.; Ritter, C.; Adrian, M.; Riek-Loher, D.; Bohrmann, B.; Döbeli, H.; Schubert, D.; Riek, R., 3D Structure of Alzheimer's Amyloid-β(1-42) Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (48), 17342-17347. (34) Becke, A. D., Density-functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98 (7), 5648-5652. (35) Lee, C.; Yang, W.; Parr, R. G., Development of the Colle-Salvetti CorrelationEnergy Formula into a Functional of the Electron Density. Phys. Rev. B 1988, 37 (2), 785-9. (36) Grimme, S., Semiempirical GGA-type Density Functional Constructed with a Long-Range Dispersion Correction. J. Comput. Chem. 2006, 27 (15), 1787-1799. (37) Jurecka, P.; Cerny, J.; Hobza, P.; Salahub, D. R., Density Functional Theory Augmented with an Empirical Dispersion Term. Interaction Energies and Geometries of 80 Noncovalent Complexes Compared with ab Initio Quantum Mechanics Calculations. J. Comput. Chem. 2007, 28 (2), 555-569. (38) Rimola, A.; Civalleri, B.; Ugliengo, P., Physisorption of Aromatic Organic Contaminants at the Surface of Hydrophobic/Hydrophilic Silica Geosorbents: a B3LYP-D Modeling Study. PhysChemChemPhys. 2010, 12 (24), 6357-6366. (39) Boys, S. F.; Bernardi, F., Calculation of Small Molecular Interactions by Differences of Separate Total Energies - Some Procedures with Reduced Errors. Mol Phys 1970, 19 (4), 553-566. (40) Barone, V.; Cossi, M., Quantum Calculation of Molecular Energies and Energy Gradients in Solution by a Conductor Solvent Model. J. Phys. Chem. A 1998, 102 (11), 19952001. (41) Cossi, M.; Rega, N.; Scalmani, G.; Barone, V., Energies, Structures, and Electronic Properties of Molecules in Solution with the C-PCM Solvation Model. J. Comput. Chem. 2003, 24 (6), 669-81.


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(42) Frisch, M. T., G.; Schlegel, H.; Scuseria, G.; Robb, M.; Cheeseman, J.; Montgomery, J.; Vreven, T.; Kudin, K.; Burant, J.; Millam, J.; et al. Gaussian 03, revision D.01; 2004. (43) Hobza, P.; Sponer, J., Structure, Energetics, and Dynamics of the Nucleic Acid Base Pairs: Nonempirical ab initio Calculations. Chem. Rev. 1999, 99 (11), 3247-3276. (44) Ringer, A. L.; Figgs, M. S.; Sinnokrot, M. O.; Sherrill, C. D., Aliphatic C-H/pi Interactions: Methane-Benzene, Methane-Phenol, and Methane-Indole Complexes. J. Phys. Chem. A 2006, 110 (37), 10822-10828. (45) Tsuzuki, S.; Honda, K.; Uchimaru, T.; Mikami, M.; Tanabe, K., The Magnitude of the CH/pi Interaction Between Benzene and Some Model Hydrocarbons. J. Am. Chem. Soc. 2000, 122 (15), 3746-3753. (46) Ho, B. K.; Curmi, P. M. G., Twist and Shear in beta-Sheets and beta-Ribbons. J. Mol. Biol. 2002, 317 (2), 291-308. (47) Mignon, P.; Loverix, S.; Steyaert, J.; Geerlings, P., Influence of the pi-pi Interaction on the Hydrogen Bonding Capacity of Stacked DNA/RNA Bases. Nucleic Acids Res 2005, 33 (6), 1779-1789. (48) Gil, A.; Branchadell, V.; Bertran, J.; Oliva, A., CH/pi Interactions in DNA and Proteins. A Theoretical Study. J. Phys. Chem. B 2007, 111 (31), 9372-9379.


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Table 1. Structural parameters for the different ligand-β-sheet complexes. φ refers to the angle between the two aromatic ring planes of the ligand. R(stacking)1 and R(stacking)2 refers to the distances between the centroid of the indene and phenyl moieties, respectively, to the plane defined by the 12 backbone atoms of the three inner peptide strands immediately below the ligand. R(CH-π) refers to the distance between the C atom of the closest CH2 group and the 6-membered ring centroid of benzothiazole. ∆HBAVG is the average hydrogen bond distance relative to the fully optimized β-sheet model without the ligand and ∆HBAVG(IS) is this average distance calculated using the interstrand H-bonds for the three innermost strands. Distances in Å and angles in degrees.

Structure

φa

R(stacking)1

R(stacking)2

Moiety 1

Moiety 2

H-bond distance

R(CH-π)

∆HBAVG

∆HBAVG(IS)

(N-H···O angle)

1a

21.0 (32.8)

3.516

3.227

3.479

-0.002

-0.010

2a

13.9 (0.0)

3.503

3.190

3.322

-0.005

-0.008

3a

10.8 (0.6)

3.318

3.109

3.967

2.020 (148.9)

0.008

0.005

4a

9.3 (0.2)

3.427

3.424

4.109

2.120 (143.4)

0.006

0.010

2.303 (131.0) 5a

4.3 (0.0)

2.864

3.664

3.384

-0.009

-0.015

6a

6.5 (0.0)

2.888

3.686

3.416

-0.005

-0.008

7a

7.6 (11.6)

2.862

3.575

3.381

-0.014

-0.020

a

2.055(158.6)

Values in parenthesis correspond to the non-interacting ligand.


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Table 2. Gas-phase interaction energies calculated at the pure B3LYP level (∆ );

dispersion contribution to the ligand-β-sheet interactions (∆ED ); total gas-phase

interaction energy accounting for dispersion (∆ ); counterpoise corrected

, ); counterpoise corrected interaction energy interaction energy in gas-phase (∆ , in solution ∆ ). Deformation energies of the β-sheet model (δEfib) and the

ligands (δElig) due to the ligand-β-sheet interaction. Changes upon ligand binding on the hydrogen bond energy of the β-sheet structure considering only the three innermost strands (∆∆EHB(IS)); negative values indicate a strengthening of the hydrogen bond interaction. All values in kcal mol-1.

Structure ∆ ∆

, , ∆ ∆ ∆ , δEβ-sheet

δElig

∆∆EHB(IS)

1a

6.2

-41.8

-35.6

-30.5

-10.9

4.5

2.7

-0.18

2a

15.8

-46.8

-31.0

-25.8

-23.6

1.3

1.6

0.38

3a

12.5

-45.8

-33.3

-28.5

-21.6

2.7

0.7

2.10

4a

14.2

-45.0

-30.8

-25.8

-17.3

2.6

0.7

-0.72

5a

20.2

-44.6

-24.4

-19.8

-19.8

3.7

0.4

-2.04

6a

19.3

-43.3

-24.0

-19.5

-19.1

3.5

0.3

-2.19

7a

17.6

-46.5

-28.9

-24.8

-20.4

4.8

1.2

-0.38


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Figure 1. Two dimensional representations of the ligands considered to interact with the fibril model.


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C12

C12

C12

Figure 2. Illustration of the 12 membered pseudo-ring formed for the H-bond systems in parallel β-sheets.


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Figure 3. B3LYP-D optimized geometries of the most stable structures for the different ligand-β-sheet complexes.


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Figure 3. continuation.


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Table of Contents of

Insights on the Binding of Thioflavin Derivative Markers to Amyloid-like Fibril Models from Quantum Chemical Calculations


27

Insights on the Binding of Thioflavin Derivative Markers to Amyloid

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