Binding of Polythiophenes to Amyloids: Structural ... - ACS Publications

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Letter

Binding of polythiophenes to amyloids: structural mapping of the pharmacophore Anne K. Schuetz, Simone Hornemann, Marielle Aulikki Wälti, Ladina Greuter, Cinzia Tiberi, Riccardo Cadalbert, Matthias Ganter, Roland Riek, Per Hammarström, K. Peter R. Nilsson, Anja Böckmann, Adriano A Aguzzi, and Beat H. Meier ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00397 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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ACS Chemical Neuroscience

Binding of polythiophenes to amyloids: structural mapping of the pharmacophore Anne K. Schütz1‡, Simone Hornemann2‡, Marielle A. Wälti1, Ladina Greuter2, Cinzia Tiberi2, Riccardo Cadalbert1, Matthias Gantner1, Roland Riek1, Per Hammarström3, K. Peter R. Nilsson3, Anja Böckmann4*, Adriano Aguzzi2*, and Beat H. Meier1* ‡

These authors have contributed equally to this work.

1

Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, 8093 Zurich, Switzerland

2

Institute of Neuropathology, University Hospital of Zurich, University of Zürich,

Schmelzbergstrasse 12, 8091 Zürich, Switzerland 3

Department of Physics, Chemistry and Biology (IFM), Linköping University, 58183

Linköping, Sweden. 4

Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS/Université de Lyon 1,

7 passage du Vercors, 69367 Lyon, France

Corresponding authors: [email protected], [email protected], [email protected]

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Abstract Luminescent conjugated polythiophenes bind to amyloid proteins with high affinity. Their fluorescence properties, which are modulated by the detailed conformation in the bound state, are highly sensitive to structural features of the amyloid. Polythiophenes therefore represent diagnostic markers for the detection and differentiation of pathological amyloid aggregates. We clarify the binding site and mode of two different polythiophenes to fibrils of the prion domain of the HET-s protein by solid-state NMR and correlate these findings with their fluorescence properties. We demonstrate how amyloid dyes recognize distinct binding sites with specific topological features. Regularly spaced surface charge patterns and wellaccessible grooves on the fibril surface define the pharmacophore of the amyloid, which in turn determines the binding mode and fluorescence wavelength of the polythiophene.

Keywords Amyloid, Pharmacophore, Luminescent conjugated polythiophenes (LCP), Diagnostic marker, Fluorescence, Solid-state NMR

The presence of amyloid deposits composed of protein with β-sheet-rich fibrillar morphology is the hallmark of a series of degenerative human diseases.(1, 2) Currently there is an unmet medical need for accurate amyloid diagnosis with the help of selective, specific and sensitive small-molecule indicators and tracers for detection in in vitro tissue cross sections(3) and in vivo imaging techniques like MRI and PET. (4-6) Of particular interest are specific dyes designed to distinguish different polymorphic forms, which are linked to the appearance of strains in prion diseases(7) and possibly also in other amyloid-related diseases.(8-11) Since the fluorescence properties of luminescent conjugated polythiophenes (LCPs) are sensitively modulated by their conformation in the bound state, they are able to report on structural features of the amyloid. The binding affinity of marker molecules depends critically on the pharmacophore presented by the amyloid, in particular the surface geometry and the distribution of 2 ACS Paragon Plus Environment

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charges. While the rigid β-sheet part of the amyloid structure remains largely unaffected by the interaction,(12) the conformation of the marker changes, leading to a readout in its optical properties like fluorescence wavelength or birefringence.(13) Whereas classical dyes like Congo red (CR) and Thioflavin T display little variation in spectral properties, e.g. with respect to fluorescence wavelength and intensity, indicating only the presence of amyloid plaques, LCPs have proven themselves as sensitive reporters on the details of the protein pharmacophores.(7, 14-16) They are made up of a flexible polymer backbone of conjugated aromatic thiophene rings of variable length with defined functional groups (for two examples, see Figure 1 a,b). Intensity and shape of the LCP fluorescence emission spectrum – the florescent fingerprint - is highly sensitive to the precise conformation of the LCPs when bound to the amyloid.(15, 17) Hence they represent optical probes capable of distinguishing between protein aggregates with a sensitivity that can address subtle conformational variations, e.g. different prion strains(7) or amyloids polymorphs in vivo such as Aβ peptide, tau or α-synuclein.(18-20) Early structural intermediates in the formation of amyloid fibrils such as oligomers and prefibrillar aggregates(21) are suspected to play an important role in amyloid toxicity(22) and can be detected by LCPs much earlier than by Thioflavin T(23). LCPs are also promising drug leads against diseases associated with amyloid deposition and were shown to prolong the survival of prion-infected mice by two fold.(24) The HET-s(218-289) amyloid fibril was used as a model because an atomic-resolution structure is available.(25,

26)

In the HET-s(218-289)E265K variant, residue E265 is

substituted by lysine creating a parallel in-register β-sheet structure with a ladder formed by positively charged side-chains with a 4.8 Å spacing (or multiples of it) - a common structural motif in amyloids(9, 27-30), which is essential for binding of some but not all LCP(24, 31) and for CR-binding.(12) As we have previously demonstrated for CR and a different LCP (LIN5001), solidstate NMR spectroscopy can detect amyloid-dye interactions by two complementary approaches, namely chemical-shift perturbations (CSP) and direct polarisation transfer (PT).(12,

24)

CSPs(32) are caused by small variations of the amyloid

conformation or its local electron density upon dye binding, whereas PT directly



probes the distance between protons of the dye and

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13

C nuclei on the protein by

heteronuclear polarization transfer mediated via dipolar couplings. Two representative LCPs were studied here: LIN1001 (PTAA, poly-thiophene-3acetic acid(14)) and LIN7002 (h-HTAA, heptameric hydrogen thiophene acetic acid(15)) (Figure 1a,b). The former has been shown to discriminate different strains of the prion protein PrPSc by its fluorescence fingerprint(7) and the latter is able to detect Aβ species that escape the conventional amyloid dye Thioflavin T(15). The two dyes differ in the number and position of negatively charged carboxylate sidechains (every ring system for LIN1001, every second for LIN7002) and in the number of rings (polydisperse with 11-20 thiophene rings for LIN1001, monodisperse with 7 rings for LIN7002). In order to determine their binding mode to the HET-s(218-289) amyloid, we compared two-dimensional (2D)

13

C-13C correlation spectra(33) of LCP-stained and

unstained [13C,15N]-labeled fibrils to map specific 13C CSPs upon binding (Figure 1ce and Figure S1a-d). Residues whose resonances shift (marked in Figure 1c-e) indicate either direct interaction sites or allosteric changes. In the case of LIN7002, clear CSPs were observed for the E265 and K229 Cα-Cβ correlations (see Figure 1c) as well as for nine other resonances (Table S1 and Figure S1a). For LIN1001, in contrast, no significant CSPs were observed (see Figure S1b). The E265K mutation does not globally change the fibril structure, inducing only small changes at the mutation site (Figure S1c) yet it dramatically changes LIN1001 binding. Strong CSPs (Table S1) similar to those for CR and LIN7002 with wildtype (wt) HET-s(218-289) are observed (Figure 1d, S1d). LIN1001 and LIN7002 binding to HET-s(218289)E265K result in very similar CSPs (Figure S1e,f) . Strong correlations were observed in PT spectra of LIN7002 and indicate direct contact between dye and protein, which cannot be allosteric (Figure 1f, S2a). Five distance restraints were derived (Table S1). Only weak PTs and to a set of different residues could be detected in the presence of LIN1001, namely for residues R236, R238, E272 and R274 in wt HET-s(218-289) (Figure 1h, S2b) while strong PT signals were observed for mutant HET-s(218-289)E265K again for residues 227-229 and 265(Figure 1g, S2c).


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Figure 1i-k summarizes the binding-sensitive atoms indicating only CSP in green, only PT in orange or both in red. The CSP’s are localized near the binding site and no further perturbations, e.g. from ring currents, were detected. For wt HET-s(218-289) bound to LIN7002 and HET-s(218-289)E265K bound to LIN1001, resonances are similar to the ones found for Congo Red

the disturbed

(12)

, and a direct comparison is

provided in Figure S3. In contrast, for wt HET-s(218-289) binding to LIN1001, the position of the residues involved in binding is entirely different. Distance restraints derived from CSP and PT experiments (Table S1) were used for in silico docking of LIN7002 and LIN1001 to wt HET-s(218-289) and of LIN1001 to HET-s(218-289)E265K

using

the

program

HADDOCK.(34)

Figure

2

shows

representations of the docked complexes and Table S2 compares the docking parameters. The most significant contribution to the interaction is the electrostatic energy, which increases even stronger than linearly with the number of negatively charged groups on the ligand (2 for CR, 4 for LIN7002, 8 for LIN1001). Analogously to CR,(12) the LIN7002 molecule is embedded in a groove running parallel to the fibril long axis lined by K229/E265 on one side and by S227/S263 on the other (Figure 2 a,b). The polythiophene backbone assumes an extended conformation in which the distance between sequentially adjacent carboxylate moieties matches the position of the K229 ε-amino groups, creating four electrostatic anchors with a spacing of 9.5 Å (Figure S4). The negative charges on the E265 residues are avoided by the carboxylates. In contrast to CR, where only every second K229 is contacted by negatively charged sulfate groups on the dye, all K229 are contacted for LIN7002 and thus have a very similar environment. Hence, only in the case of CR two nonequivalent populations – in direct contact with a charged group of the LCP, or not - of K229 and E265 residues arise. This manifests as a splitting of the E265 Cα-Cβ correlation in the NMR spectra with CR but not with LIN7002 (Figure S3b versus S3g). The pronounced CSP effects for E265 could be due to the glutamate sidechain moving away from the negatively charged LCP - a similar phenomenon has been observed for negatively charged sugars binding to proteins(35). In the HADDOCK-derived model, octameric LIN1001 occupies the same binding site on the E265K mutant fibril as CR, now contacting four K229 and four mutant K265 residues (Figure 2 c,d). All lysines, spaced by about 4.8 Å, are thus involved in



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binding. Such a binding mode is impossible for the wt fibril where residue 265 has a negatively charged glutamate side chain. The binding of LIN1001 to the wild-type protein (Figure 2 e,f) uses a completely different site at the “backside” of HET-s(218-289) and is mediated by three surfaceexposed positively charged arginine residues (R236, R238, R274). We next investigated how specific modifications to the amyloid surface, and specifically, the changes observed for mutant HET-s(218-289)E265K, would affect the optical spectral properties of the LCPs. We investigated the relationship between residue types displayed on the surface and the observed fluorescence of a series of single and double point-mutated HET-s(218-289) fibrils in the presence of LIN1001 and LIN7002, which were selected based on their impact on charge distribution and polarity on the amyloid surface and are listed in Figure 3a. All mutant fibrils were previously confirmed to have closely similar structures to the wt fibrils.(36) Fluorescence-emission spectra of LIN1001 mixed with fibrils were recorded between 480 and 750 nm after excitation at 430 nm (Figure S5) and can be conveniently visualized by a correlation diagram plotting the wavelength at maximum emission, λ, versus the emission at 532 nm normalized by the maximum emission (Figure 3a for measurements in PBS and Figure S5 in Tris/HCl). A maximum emission wavelength comparable to the unbound control is obtained for the mutant R238E, which destroys the pattern of positive charges at the secondary “backside” binding site by introducing a negative charge (Figure 4b) and for the double mutant R238A-R274A, which replaces two of the relevant positive charges on the secondary binding site by alanine residues. These two mutants bind LIN1001 very weakly or not at all. The E265K mutant, in contrast, establishes a row of positive charges on the primary binding site and leads to a strong redshift indicative of strong binding and a planarized polythiophene conformation.(7) Fluorescence microscopy images visually confirmed the yellow to greenish appearance of wild-type and the orange appearance of HETs(218-289)E265K aggregates stained with LIN1001 (Figure 3b and 3c). The fluorescence intensity acquired from these images greatly increases for E265K compared to wt fibrils (Figure S6). For the HET-s(218-289)–CR interaction, a false negative in the traditional birefringence test was obtained after K229A mutation that abrogates the electrostatic driving force of the binding.(12) In analogy, LIN7002 fluorescence is strongly reduced 6 ACS Paragon Plus Environment

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for the K229A mutant as evidenced by hyperspectral microscope emission spectra and fluorescence images (Figure 3d,e and Figure S7).

The binding between LCPs and amyloids requires an electrostatic match of the regioregular pattern of charges presented by the amyloid and the charged functionalities of the LCP. The relevant pharmacophores for HET-s(218-298) and point mutants thereof are shown in Figure 4a and b. CR, LIN7002 and LIN1001 in their quasiplanar conformation carry negative charges with a spacing of 19 Å (4x4.75 Å), 9.5 Å (2x4.75 Å) and 4.75 Å, respectively, (Figure 4c) to match amyloids with a positive charge at least every four β-strands, every two β-strands or every β-strand, respectively. At binding site I (Figure 4a) LIN7002 fits into a groove running along the fibril long axis lined by a ladder of alternating positive (K229) and negative (E265) charges. The carboxylates of LIN7002 contact the K229 side chain of each protein monomer with a distance of roughly 9.5 Å (Figure 2a). Another set of positive charges presented at the intermediate β-strands is not essential for binding. As a result, wt and E265K fibrils bind LIN7002 equally well (Figures S1a versus S1e) and cannot be discriminated using this dye as a marker. In contrast, LIN1001 cannot be accommodated into the same groove because half of the LCP carboxylates would not be able to establish electrostatically favorable contacts. Binding site I can be reestablished for LIN1001 by mutation of residue E265 to a positively charged lysine. This allows for simultaneous binding of all carboxyl moieties of LIN1001 (Figure 2c). The conformation of both LIN7002 and LIN1001 when embedded in the groove is ordered and extended, with all carboxylate side-chain residues aligned in the same direction and with regular spacings (Figure S4). At the alternative binding site II (Figure 4b) LIN1001 occupies a less pronounced grove on the “backside” of HET-s lined with positive charges (R236,238,274). The binding is weaker as judged by the adsorption maximum, which is close to the wt, the absence of CSPs and weak PT effects. The pharmacophore of this site is less favorable and the dye binds in an irregular conformation (see the HADDOCK model of Figure 2e and Figure S4) as the carboxylates contact multiple arginines in different



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layers of the β-sheet. Binding at this site has only been observed in the case where binding to site I is not detected (wildtype protein and LIN1001). Our results indicate that the electrostatic match between pharmacophore of the amyloid and functional groups on the LCP is a necessary but not sufficient condition for binding. Two distinct binding sites were identified here but only one of them could be detected in CSP experiments and induced a pronounced redshift in the fluorescence spectrum of the LCP. In addition, we note that a further potential binding site, the (E234-K270) ladder, shows no binding by NMR, although it presents the same ladder as the best binding site (K229-E265). Other factors thus also contribute to LCP affinity, including the charges and polarity of neighboring residues, as well as the existence of accessible surface-exposed grooves in the β-sheet. Small polar residues like serine and threonine on the amyloid surface have been identified as favorable for LCP affinity by molecular modeling.(24) The fluorescence data for LIN1001 in Figure 3 correlate with the NMR findings and pick up very subtle changes to the pharmacophore. Binding at site II (if site I is not available for LIN1001, e.g. for the wt) leads to only a moderate redshift of fluorescence compared to the control (free LIN1001), still indicating an overall more planar dye conformation than for the reference. By destroying stabilizing features of binding site II in R274A, R238A, R238A/R274A and R238E, a decreasing redshift is obtained up to a degree that there is no difference to free LIN1001 indicating very weak or no binding. As LIN1001 in complex with K229A fibrils also displays a slight blue shift, compared to wt, there must be some weak LIN1001 interaction involving residue K229 in the wt fibril, which was not detected in the NMR. Binding site I can be greatly improved for LIN1001 binding in the E265K mutant, compared to the almost non-existing binding in the wt, and a large redshift is observed. Interestingly, while removal of positive charges can abolish binding altogether, removal of negative charges, at the non-interacting layers, such as in E272A mutation in site I and E265A mutation in site II, does lead to only small changes in the fluorescence pattern. Such detailed observations can help to define and predict both the potential and the limitations of LCPs with differently charged side chain functionalities in discriminating amyloids with similar surface geometry yet different charge distribution.


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The geometrical arrangement of the LCP’s charged functional groups determines the specificity of the binding to the pharmacophore of an amyloid, whereas the resulting backbone conformation when bound (characterized by the torsion angles around the single carbon-carbon bonds linking two thiophene units) is reflected in the optical properties, which provide a sensitive proxy for the arrangement of surface charges of its binding partner. In order to optimally bind the amyloid, the LCP must (i) meet a specific regio-regular arrangement of positively charged amino acids on the amyloid surface that match its negative charges; (ii) encounter a well-accessible groove as primary binding site; and be able to favorably interact with the specific charges and polarity of the neighboring residues. These atomistic parameters of the protein determine the spectroscopic properties of the LCPs and their reporting function on the conformation of the pharmacophore. An optimized binding site induces an approximately planar conformation for the LCPs and leads to a pronounced red-shifted wavelength and intense fluorescence. Future studies could focus on how oligomers and prefibrillar aggregates, which are also amenable to nuclear magnetic resonance techniques

(37, 38)

,

are recognized by LCPs. Fluorescence fingerprinting has the potential to provide the crucial link between the pharmacophore of pathological aggregates identified in vivo and in vitro fibril preparations. While in vivo atomic-resolution structure determination is a distant dream, this link can connect the in vitro structure determination of pure polymorphic forms of fibrils(39) and the in vivo deposits. This information will not only be crucial for the rational design of diagnostic and therapeutic molecules for monitoring disease progression and accurate typing of different polymorphs and strains, but also help to create novel therapeutic molecules to cure amyloid diseases.(24)



Figures

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CSP

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36 R238CB-CA

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j

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β2a

R238 R236 A237 A228

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S273

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R274

S263 K265

Figure 1 CSPs and PTs induced by LIN7002 and LIN1001 for wt HET-s(218-289) and HET-s(218-289)E265K fibrils. (a, b) Chemical structures of LIN7002 (a) and LIN1001 (b). (c, d, e) Regions of interest in the 50 ms PDSD spectra of free HETs(218-289) fibrils (black contours), fibrils in complex with LIN7002 (left column, blue) and with LIN1001 (right column, purple). The middle column contains data for HET-s(218-289)E265K in free form (black) and in complex with LIN1001 (magenta). 10 ACS Paragon Plus Environment

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Correlations that display significant changes upon binding are labeled. (f, g, h) Regions of interest from the PT spectrum. The spectra of untreated HET-s(218-289) and HET-s(218-289)E265K fibrils are shown in grey. The blue contours represent a DREAM PT spectrum of fully deuterated fibrils with an 8 ms polarization transfer from LIN7002, the purple contours from LIN1001 to HET-s(218-289) and the magenta contours from LIN1001 to HET-s(218-289)E265K fibrils. (i, j, k) Observed CSP and PT effects on the hydrophobic core of the HET-s(218-289) amyloid are summarized. Red spheres denote nuclei with significant CSP effects combined with PT from the ligand, orange spheres nuclei with PT only, green spheres nuclei with CSPs only. Full spectra and experimental details are given in Supporting Figures 1-3 and in Supporting methods.



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Figure 2 Molecular HADDOCK models of LIN7002 and LIN1001 docked to HETs(218-289) and HET-s(218-289)E265K fibrils. (a, c, e) Side views of the complexes. Key residues involved in binding are indicated in dark blue (K229) and cyan (K265) for binding site I and in blue (R236), cyan (R238) and pale blue (R274) for binding site II. (b, d, f) Top view of the complexes for better visualization of the LCP binding grooves. The two dyes occupy different binding sites on the wild-type fibrils (a and c versus e). A point mutation, E265K, creates a ladder of positive charges every 4.8 Å at binding site I, making it attractive for LIN1001 (e). Figures were produced with Swiss-PDB viewer.(40)


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Figure 3 Fluorescence data for LIN1001 and LIN7002 in complex with wt HETs(218-289) and different HET-s(218-289) mutants. All scale bars: 50 µm. (a) Correlation diagram of the wavelength at maximum emission from LIN1001 bound to HET-s(218-289) mutant fibrils in PBS. Data in Tris/HCl are given in Figure S5. Error bars represent the standard deviation of quadruplicate experiments. LIN1001 denotes the spectrum of free probe (b, c) Hyperspectral fluorescence images of fibrillar aggregates of wt HET-s(218-289) (b) and HET-s(218-289)E265K (c) stained with LIN1001 in PBS. (d, e) Representative fluorescence images of fibrillar aggregates



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stained with LIN7002 of wt HET-s(218-289) (d) compared to HET-s(218-289)K229A (e) in PBS.

Figure 4 Pharmacophore presented by the HET-s(218-289) amyloid. (a,b) Side-view of the fibril showing binding site I (a) and II (b) as in Figure 2. Surface-exposed residues are highlighted in red and blue for negatively and positively charged sidechains, respectively. The point mutants are sorted from right to left in order or increasing redshift induced in fluorescence of LIN1001 in PBS (see Figure 3a). (c) The regio-regular spacing of negative charges presented by CR, LIN7002 and LIN1001, drawn to scale with the amyloid in (a).

Acknowledgements This work was supported by the Swiss National Science Foundation (Grant 200020_159707 and 200020_146757), the French ANR (ANR-14-CE09-0024B).


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Methods Detailed experimental methods are provided in the supporting information for cloning, protein expression, sample preparation, NMR spectroscopy, fluorescence spectroscopy and microscopy and molecular modeling. Supporting information available: Experimental methods, supplementary figures S1-S7 and supplementary tables S1-3.

Author contributions A.K.S. collected and analyzed NMR data and performed molecular docking; S.H., L.G. and C.T. collected and analyzed fluorescence data; M.A.W. , R.C. and M.G. produced protein samples, P.H. and K.P.R.N. provided LCPs; R.R. , A.B., A.A. , and B.H.M. designed and supervised the study. All authors were involved in the manuscript writing and editing process.

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Nilsson, K. P. R., Åslund, A., Berg, I., Nyström, S., Konradsson, P., Herland, A., Inganäs, O., Stabo-Eeg, F., Lindgren, M., Westermark, G. T., Lannfelt, L., Nilsson, L. N. G., and Hammarström, P. (2007) Imaging distinct conformational states of amyloid-beta fibrils in Alzheimer's disease using novel luminescent probes., ACS Chem Biol 2, 553-560. Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate., J Biol Chem 272, 22364-22372. Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo., Nature 416, 535-539. Åslund, A., Sigurdson, C. J., Klingstedt, T., Grathwohl, S., Bolmont, T., Dickstein, D. L., Glimsdal, E., Prokop, S., Lindgren, M., Konradsson, P., Holtzman, D. M., Hof, P. R., Heppner, F. L., Gandy, S., Jucker, M., Aguzzi, A., Hammarström, P., and Nilsson, K. P. R. (2009) Novel Pentameric Thiophene Derivatives for in Vitroand in VivoOptical Imaging of a Plethora of Protein Aggregates in Cerebral Amyloidoses, ACS Chem Biol 4, 673-684. Herrmann, U. S., Schütz, A. K., Shirani, H., Huang, D., Saban, D., Nuvolone, M., Li, B., Ballmer, B., Åslund, A. K. O., Mason, J. J., Rushing, E., Budka, H., Nyström, S., Hammarström, P., Böckmann, A., Caflisch, A., Meier, B. H., Nilsson, K. P. R., Hornemann, S., and Aguzzi, A. (2015) Structure-based drug design identifies polythiophenes as antiprion compounds., Sci Transl Med 7, 299ra123. Wasmer, C., Lange, A., Van Melckebeke, H., Siemer, A. B., Riek, R., and Meier, B. H. (2008) Amyloid fibrils of the HET-s(218-289) prion form a beta solenoid with a triangular hydrophobic core., Science 319, 15231526. Van Melckebeke, H., Wasmer, C., Lange, A., Eiso, A. B., Loquet, A., Böckmann, A., and Meier, B. H. (2010) Atomic-Resolution ThreeDimensional Structure of HET-s(218-289) Amyloid Fibrils by Solid-State NMR Spectroscopy, J Am Chem Soc 132, 13765-13775. Schütz, A. K., Vagt, T., Huber, M., Ovchinnikova, O. Y., Cadalbert, R., Wall, J., Güntert, P., Böckmann, A., Glockshuber, R., and Meier, B. H. (2015) Atomic-resolution three-dimensional structure of amyloid β fibrils bearing the Osaka mutation., Angew Chem Int Ed Engl 54, 331-335. Tuttle, M. D., Comellas, G., Nieuwkoop, A. J., Covell, D. J., Berthold, D. A., Kloepper, K. D., Courtney, J. M., Kim, J. K., Barclay, A. M., Kendall, A., Wan, W., Stubbs, G., Schwieters, C. D., Lee, V. M. Y., George, J. M., and Rienstra, C. M. (2016) Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein., Nat Struct Mol Biol 23, 409-415. Colvin, M. T., Silvers, R., Ni, Q. Z., Can, T. V., Sergeyev, I., Rosay, M., Donovan, K. J., Michael, B., Wall, J., Linse, S., and Griffin, R. G. (2016) Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils., J Am Chem Soc 138, 9663-9674. Wälti, M. A., Ravotti, F., Arai, H., Glabe, C. G., Wall, J. S., Böckmann, A., Güntert, P., Meier, B. H., and Riek, R. (2016) Atomic-resolution structure



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Page 19 of 24

Table of Contents graphics

590

580

λmax

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


570

560

550


Neuroscience LIN7002 & wildtype ACS Chemical LIN1001 & E265K

a

O

O

C

C

O

S

S

O

O

C

C

S

O

b

O O

C

O

O C

O

O

S

S

S

d

K229CA-CB

O

O C

O

S

S

O

C

O

O C

O

K229CA-CB

30

32

28

S

S

O

C

30

S

S

O

O C

O

O

C

O C

O

O O

C

O C

O

S

S

S

S

S

e

K265CA-CB K229CA-CD

E265CA-CB 32

C

S

S

K265CA-CG 28

32

g

h

30 28 δ2 -13C (ppm)

R236CG-CA R274CB-CA

K229CD-CE 44

42

E272CB-CA

K229CD-CE K265CD-CE

40

38

j

44

42

40

R236CB-CA 38

β2a

R236 A228

A228

S227 β1a

β1b

K229

β4a

β3b

k

55

S263 β3a E265

R238 A237

S227

K229 E272

V264

53

R274 S273

V264

ACS Paragon Plus Environment S263

K265

R238CB-CA 55

53

34

PT

δ1 -13C (ppm)

δ1 -13C (ppm)

O

CSP

1 2 3 4c 58 5 6 7 60 8 9 10 11 12f 13 28 14 15 16 30 17 18 19 20i 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

O

Page 20 of 24 & wildtype

36

Page 21 of 24

LIN7002 & wildtype LIN1001 & E265K Binding site I a c

LIN1001 & wildtype Binding site II

e

sideview

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


d

f

topview

b

ACS Paragon Plus Environment


Figure 3 160x233mm (300 x 300 DPI)


Page 22 of 24

Pharmacophore ACS Chemical Neuroscience

Page 23 of 24

a12

Binding site I

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Binding site II

b

Amyloid dyes

increasing LIN1001 redshift

c

19 Å CR 9.5 Å

E265K strong binding large redshift

E265A wildtype

K229A LIN7002 4.8 Å ACS Paragon Plus Environment

wildtype E272A

R274A R238A

R238E R238R274A no binding, no redshift

LIN1001



Page 24 of 24

Binding of Polythiophenes to Amyloids: Structural ... - ACS Publications

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