Chirality-selected chemical modulation of amyloid aggregation

2 hours ago - Intriguingly, Phe-modified POMs showed stronger inhibition effect than other amino acid-modified POMs, as evidenced by multiple biophysi...
1 downloads 0 Views 733KB Size
Subscriber access provided by UNIV OF LOUISIANA

Article

Chirality-selected chemical modulation of amyloid aggregation Nan Gao, Zhi Du, Yijia Guan, Kai Dong, Jinsong Ren, and Xiaogang Qu J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 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

Journal of the American Chemical Society

Chirality-selected chemical modulation of amyloid aggregation Nan Gao,† Zhi Du,†,§ Yijia Guan,†,§ Kai Dong,† Jinsong Ren,† and Xiaogang Qu*,†

Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China.



University of Chinese Academy of Sciences, Beijing 100039, China.

§

KEYWORDS: Chirality, chemical modulation, β-amyloid, polyoxometalates, Alzheimer’s disease

ABSTRACT: Due to the composed α-helical/β-strand structures, β-amyloid peptide (Aβ) is sensitive to chiral environments. The orientation and chirality of the Aβ strand strongly influence its aggregation. Aβ formed fibrils have a cascade of chirality. Therefore, for selectively targeting amyloid aggregates, chirality preference can be one key issue. Inspired by the natural stereoselectivity and the β-sheet structure, herein, we synthesized a series of D- and L-amino acid-modified polyoxometalate (POM) derivatives, including positively charged amino acids (D-His and L-His), negatively charged (D-Glu and L-Glu) and hydrophobic amino acids (D-Leu, L-Leu, D-Phe and L-Phe), to modulate Aβ aggregation. Intriguingly, Phe-modified POMs showed stronger inhibition effect than other amino acid-modified POMs, as evidenced by multiple biophysical and spectral assays, including fluorescence, circular dichroism, NMR, molecular dynamic simulations and isothermal titration calorimetry. More importantly, D-Phe-modified POM had an 8-fold stronger inhibition effect than L-Phe-modified POM, indicating high enantioselectivity. Furthermore, in vivo studies demonstrated that the chiral POM derivatives crossed the blood–brain barrier, extended the life span of AD transgenic Caenorhabditis elegans CL2006 strain and had low cytotoxicity, even at a high dosage. INTRODUCTION Chirality, as widespread in nature, shows typically distinct preferences and strict selectivity for human beings. For instance, only D-sugars are included in the formation of DNA, and L-amino acids are found in proteins; thus, these chiral molecular units could decide both molecular conformations and biological functions. The progress of experiments in this field may contribute to improve understanding of the basic mechanism. In this way, it will facilitate the design of new chemicals or materials to simulate natural biological structures.1 Amyloid fibrils, which are composed of many copies of aggregated proteins that usually become folded into βsheet structures and stick together into twisted or helical ribbons, are a kind of chiral supramolecular system.2 In the human body, the production, conformational transformation and aggregation of amyloid proteins are closely related to various diseases,3 such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).4-6 For general biological macromolecules, these amyloid fibrils always contain four levels of chirality.7 The first level is considered to be the stereocentre (also known as "chiral centre"), which is a stereocentre consisting of an atom holding a set of ligands (atoms or groups of atoms) in a spatial arrangement that is not superimposable on its mirror image (configurational chirality). The second level of chirality is due to the different implication of molecular conformation (conformational chirality). The third level is related to the phase structure of single crystals or helical monodomains (phase chirality). Finally, the aggregation of

helical single domains constructs macroscopic chiral objects, which belong to the highest (the fourth) level of this chirality (object chirality) (Scheme 1). Therefore, when designing amyloid fibril inhibitors, chiral selectivity has to be considered due to amyloid with a cascade of chirality. AD has become the most common form of dementia, afflicting more than 24 million people worldwide,8-10 and the β-amyloid peptide (Aβ) fibrils have been demonstrated as a critical step in the pathogenesis of AD. More importantly, Aβ fibrils also contain all the above-mentioned cascade of chirality (Aβ specifically forms left-handed helical fibrils).11 Therefore, when we design Aβ-selective targeting inhibitors, chirality has been one key issue for selectivity. On the one hand, chirality can improve the enantioselectivity and specificity of inhibitors towards their chiral biotargets.12 On the other hand, as for pharmaceuticals, chirality has been an increasingly vital concern in recent years because many drug molecules are chiral, and in most cases, just one enantiomer is pharmaceutically active, while the other is inactive or even exerts severe biological side effects. Therefore, chiral recognition is crucial for the inhibition of Aβ aggregation. Therefore, we take polyoxometalates (POMs) as an example to show how to modulate Aβ aggregation by the chirality-selected chemical method. POMs have shown great potential as biomedical agents because of their versatile bioactivities, including antibacterial, antiviral and anticancer activities.13-20 However, POM is usually a metal oxide cluster with polyatomic anions. Their interactions with biomolecules are mainly dependent on favourable

ACS Paragon Plus Environment

Journal of the American Chemical Society 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

electrostatic attractions and POM size effects, which are robust but not specific, and have no specific chiral recognition. Therefore, in consideration of decreasing POM side effects and improving targeting specificity, surface modification of POMs with synthetic organic compounds or natural chiral biomolecules may provide a solution, and this has attracted considerable attention because the organic moiety or chiral biomolecules can be a specific biorecognition unit, guide POMs to interact with target biomolecules and increase the biocompatibility.12

Scheme 1. Chirality at different-length scales: configurational (I), conformational (II), phase (III), and object (IV) chirality. The transfer of the conformational chirality to the phase chirality is dominated by intermolecular interactions via chain packing. So far, surface modification has been successfully used to synthesize a series of stable organo-functionalized POMs, such as alkoxo, amino acids, organosilyl, organophosphoryl and organometallic derivatives of polyoxotungstates,21-25 and some POM-based organohybrids have shown enhanced in vitro or in vivo bioactivity and biocompatibility.24,26 However, there are few reports of chiral POMs applied in the above-mentioned fields.27,28 For these reasons, the design and synthesis of chiral POMs with natural recognition units can be an effective way to improve specific targeting and their biocompatibility by a chirality-mediated chemical method. POMs can inhibit Aβ aggregation by binding to the cationic cluster from His 13 to Lys 16 (HHQK).17 This site has been demonstrated in an α/β discordance stretch, which is constituted by the 13-23 segment (HHQKLVFFAED) of Aβ. This region is predicted to form a β-strand during Aβ aggregation,29 and targeting this region can be a novel approach for the inhibition of Aβ aggregation.30-32 Due to the composed chirality of the α-helical/β-strand structures, Aβ is sensitive to a chiral environment, and the orientation and chirality of the Aβ strand will strongly influence its

aggregation. Therefore, chiral recognition can play a crucial role in the inhibition of Aβ aggregation. Herein, we design and synthesize a series of chiral amino acidmodified POMs that are suitable for binding to the Aβ1323 segment and show strong enantioselectivity in the inhibition of Aβ aggregation. Additionally, modification with natural amino acids can further reduce the cytotoxicity of POMs at high dosages. Based on the sequence of the Aβ13−23 segment, we rationally designed and synthesized chiral POM derivatives. This design is based on the following reasons: 1) POMs bind to the cationic cluster from His 13 to Lys 16 (HHQK); 17 2) the following sequence of Leu 17 and Val 18 (L17V18) on Aβ has been reported to interact with a hydrocarbon chain;30 3) the next following sequence is composed of two adjacent phenylalanine residues (F19F20), which can form a strong hydrophobic region and specifically interact with hydrophobic amino acids through hydrophobic interactions. Therefore, F19F20 can be useful for designing and improving POM-specific interactions with Aβ. However, it is unclear which amino acids can reveal the best effect. Furthermore, due to the chiral environment of Aβ, different enantiomers of D- and L- amino acids may have different binding preferences towards Aβ; thus, the selection of the enantiomer of amino acids is also crucial. Therefore, we synthesized a series of chiral amino acidfunctionalized POM derivatives, including positively charged amino acids (D-His and L-His), negatively charged amino acids (D-Glu and L-Glu) and hydrophobic amino acids (D-Leu, L-Leu, D-Phe and L-Phe), following previous reports by Cronin et al.22 All the chiral POM derivatives were screened by using isothermal titration calorimetry (ITC) screening system and compared with their binding affinity and enantioselectivity to Aβ (Scheme 2). To the best of our knowledge, this is the first report to show that chiral POM derivatives can inhibit protein aggregation. Intriguingly, the chiral POM derivatives can cross the blood–brain barrier and have low toxicity.18 Our work provides a new avenue for the design and synthesis of chiral inorganic metal compounds as multifunctional therapeutic agents against multifaceted AD.

Scheme 2. Rational designed D/L-amino acidfunctionalized POM derivatives. Molybdenum (Mo) was shown as brown ball in brown centrum, manganese (Mn) was shown as purple centrum and oxygen (O) was shown as red balls. The F19F20 site on Aβ can be used for designing and improving POM-specific interactions with Aβ. A series of POM derivatives were screened,10,22 and were considered as strong inhibitors or weak inhibitors for Aβ aggregation. RESULTS AND DISCUSSION

ACS Paragon Plus Environment

Page 2 of 8

Page 3 of 8 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

Journal of the American Chemical Society According to previous reports,22 we employed the Anderson-type POM with two pendant amino groups (POMNH2) as the scaffold to develop chiral inhibitors since this kind of POM has a suitable size; thus, the modified groups can interact with Aβ at the designed sites.18 Therefore, the POM-NH2 and chiral POM derivatives were synthesized and characterized as previously described (Scheme S1, Figure S1, S2, Table S1)22. Before the following experiments, the stability of the compound should be studied under physiological conditions. Upon incubation at 37 °C for 7 d, all the spectral data of the compounds measured by UV-vis, FTIR, Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) and 1H NMR did not change (Figure S3-S6), demonstrating that the compounds were stable under our experimental conditions. ITC has often been used to study ligand-protein interactions. We employed ITC to evaluate how this series of chiral POM derivatives bound to Aβ. As shown in Figures S7 and Table S2, phenylalanine (Phe)-modified POMs had much stronger binding affinity to Aβ than other amino acid-modified POM derivatives. Furthermore, the binding constant of D-phenylalanine (D-Phe)-modified POMs (POM-D-Phe) was 5.19±0.15×107 M-1, which was 8-fold stronger than that of L-phenylalanine (L-Phe)-modified POMs (POM-L-Phe) (6.70±0.12×106 M-1), showing enantioselectivity. This result was consistent with the fluorescence titration data (Figure S8, Table S2). The difference in the binding free energy change between POM-D-Phe and POM-L-Phe binding to Aβ was approximately 5.08 kJ mol-1. On the basis of the ITC and fluorescence titration screening results,17 POM-D-Phe and POM-L-Phe had much stronger binding affinities to Aβ than other derivatives. Therefore, we chose POM-D-Phe and POM-L-Phe for further studies. To explore their binding sites to Aβ, POM-D-Phe or POML-Phe binding to different Aβ fragments was first studied by using ITC. As shown in Figure S9, S10 and Table S3, the binding affinity of POM-D-Phe with Aβ1-12 (Ka POM-D-F+1-12) was much weaker than that of POM-D-Phe with Aβ1-40 (Ka POM-D-F+1-40), showing that the 1−12 segment was not the main binding site for POM-D-Phe. Moreover, the affinity of POM-D-Phe with Aβ1-16 (Ka POM-D-F+1-16) was also lower than that of Ka POM-D-F+1-40, but higher than Ka POM-D-F+1-12; therefore, the 13-16 segment might be a moiety of the binding site. When Aβ1-20 was used as the substrate, the binding constant (Ka POM-D-F+1-20) was close to Ka POM-D-F+1-40, indicating that POM-D-Phe could bind to the 13-20 segment. Subsequently, the binding constant between POM-DPhe and Aβ12-28 (Ka POM-D-F+12-28) was also close to Ka POM-DF+1-40 and Ka POM-D-F+1-20, suggesting that the 1-11 and 21-28 segments were not the main binding sites. Finally, the binding constant between POM-D-Phe and Aβ25-35 (Ka POMD-F+25-35) was determined. Ka POM-D-F+25-35 was again much smaller than Ka POM-D-F+1-40, showing that the 25-35 segment was not the central binding site. These results indicated that POM-D-Phe preferred binding to the HHQKLVFF segment, which belongs to the α/β-discordant stretch. In addition, the enantiomer, POM-L-Phe, had the same tendency as POM-D-Phe. The binding affinity of POM-D-Phe with Aβ fragments, however, was 7.8-fold higher than that of POML-Phe under the same conditions (Table S3). The binding

sites between the chiral POM derivatives and Aβ1−40 were further confirmed by 1H NMR spectroscopy (Figure S11).33 The 1H NMR signals of the aromatic moieties (F19) of Aβ140 (approximately 7.2 ppm) underwent notable changes upon the addition of POM-L-Phe or POM-D-Phe, revealing their interactions associated with these units (Figure S11A). The results indicated that the chiral inhibitors could bind to the hydrophobic core fragment of Aβ1-40 through hydrophobic interactions, π−π stacking and electrostatic interactions, which were consistent with the abovementioned results. In addition, compared with the NMR spectrum of Aβ1−40 alone, there were notable peak shifts in the signals from the amide protons from K16 (2.95 ppm) in the presence of POM-L-Phe or POM-D-Phe (Figure S11B). The results also indicated that the chiral inhibitors could bind to the cationic cluster of Aβ. All these results indicated that these chiral inhibitors bound to the Aβ central region, also known as the α/β-discordant stretch.

Figure 1. Energy-minimized average model of chiral POM derivatives-Aβ interactions. Chiral POM derivatives were shown in tube model and amino acid residues in Aβ were shown in ball- and -stick model. (A) POM-D-Phe-Aβ complex. The complex was further stabilized by two hydrogen bonds, occurring at Ser 8 and His 13, which were illustrated by yellow arrows. (B) POM-L-Phe-Aβ complex. There was no hydrogen bond formation. (C, D) Further illustration of the two hydrogen bonds formed for POM-D-Phe at Ser 8 (C) and His 13 (D), but not for POM-L-Phe.

To better understand the interaction differences between the two chiral inhibitors and monomeric Aβ1−40 peptide, molecular dynamic simulations were carried out.10,34 The NMR structure of monomeric Aβ1−40 (PDB ID: 2LFM) was used as the host structure since this structure was obtained in an aqueous solution (pH 7.3 with 50 mM NaCl). Molecular dynamic simulations were conducted using Autodock Vina (by Dr. Oleg Trott, Scripps Research Institute, USA) until a stable complex was formed.35 The best-fit obtained with this method is shown in Figure 1. For POM-D-Phe and POM-L-Phe, the binding site of POM on Aβ was identified as a cavity-like domain mainly encompassing the amino acids His13, Gln15 and Lys16. This domain formed a relatively positively charged surface (Figure S12), which allowed the interactions with the negatively

ACS Paragon Plus Environment

Journal of the American Chemical Society 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

charged POM moiety. Furthermore, the different chiral recognition of POM-D-Phe and POM-L-Phe towards Aβ was also identified. First, compared to POM-L-Phe, POM-D-Phe formed two additional hydrogen bonds with Aβ, which occurred at Ser 8 and His 13 (Figure 1A, 1B). For Ser 8, the hydrocarbon chain in POM-D-Phe was close to the hydroxyl group of Ser 8, which allowed the formation of a hydrogen bond; in contrast, the hydrocarbon chain in POM-LPhe was far from the hydroxyl group of Ser 8 and thus could not form a hydrogen bond (Figure 1C). For His 13, a similar difference between POM-L-Phe and POM-D-Phe was also observed (Figure 1D). Subsequently, the phenylalanine residues in POM-D-Phe were also closer to the two hydrophobic amino acid residues in Aβ1-40 (Phe 4 and Phe 19), which further enhanced their hydrophobic interactions and π-π stacking (Figure S13). Therefore, based on the above experimental data and simulation results, the binding mechanism and chiral discrimination of POM-LPhe and POM-D-Phe could be better understood. Since strand π-π stacking and hydrophobic interactions play vital roles in Aβ aggregation,36,37 it is predictable that POML-Phe and POM-D-Phe can inhibit Aβ aggregation by binding to the Aβ central hydrophobic α/β-discordant stretch and disrupting their strand π-π stacking and hydrophobic interactions.

Figure 2. Inhibition of Aβ aggregation and Aβ-induced cytotoxicity by chiral POM derivatives. (A) Inhibition effect of POM-D-Phe or POM-L-Phe on Aβ1−40 aggregation monitored by CD. The concentration of Aβ1−40 was 40 μM, and the chiral POM derivatives concentration was 20 μM, respectively. All the CD data of Aβ shown in figure had been corrected with the absorption of chiral POM derivative. Details were described in experimental section. (B) Aggregation kinetics of Aβ 1–40 monitored by ThT fluorescence (black square: Aβ; red square: Aβ+POM; blue square: Aβ+POM-COOH; green square: Aβ+LPFFD; black triangle: Aβ+POM-L-Asp; red triangle: Aβ+POM-D-Asp; blue triangle: Aβ+POM-L-His; green triangle: Aβ+POM-D-His; black diamond: Aβ+POM-L-Leu; red diamond: Aβ+POM-D-Leu; blue diamond: Aβ+POM-L-Phe (doubling bold, dashed line); green diamond: Aβ+POM-D-Phe(doubling bold, dashed line). The concentrations of Aβ and POMs were 40μM and 20μM, respective. (C) Blood−brain barrier penetration assay obtained from mice injected with POM-D-Phe (n = 10, dose = 25 mg POM-D-Phe/kg body weight, Test group) and without injection (n = 10, Control). Error bars indicate ±s.d. (D) Time-dependent biodistribution of POM-D-Phe in mice. Error bars are based on the standard deviation of 5 mice per group.

Page 4 of 8

For Aβ aggregation, oligomers, protofibrils and fibrils share β-sheet structure, which drives Aβ aggregation and neurotoxicity. Since POM-L-Phe and POM-D-Phe could bind to Aβ strongly, they might stabilize the Aβ monomer and prevent the conformational switch to the β-sheet structure. Therefore, we employed circular dichroism (CD) to pursue this issue.32 The results indicated that after incubation for 7 days, Aβ1–40 in the absence of inhibitors formed a βsheet structure (Figure 2A, S14). In contrast, POM-D-Phe or POM-L-Phe inhibited the structural transition from the native Aβ1−40 random coil to the β-sheet in solution, which indicated that POM-D-Phe and POM-L-Phe could prevent Aβ aggregation by binding to the α/β-discordant stretch. In this way, the chiral inhibitors might further interrupt the Aβ aggregate-mediated cellular toxicity. The successful inhibition of the β-sheet structure prompted us to examine whether POM-D-Phe and POM-LPhe could inhibit Aβ aggregation in vitro and decrease cytotoxicity in living cells. Therefore, a commonly used thioflavin T (ThT) fluorescence assay, IC50 assay and dynamic light scattering (DLS) analysis were employed.38,39 The results indicated that the formation of Aβ amyloid fibrils was suppressed (Figures 2B, S15-S17, and Table S4). To monitor the level of intracellular ROS, a dichlorofluorescein diacetate (DCFH-DA) assay was used. DCFH-DA can diffuse into cells and become fluorescent dichlorofluorescein via oxidation by intracellular ROS, and it has been used as a probe to determine intracellular ROS.40 As shown in Figure S18A, PC12 cells (rat pheochromocytoma cells) exposed to an Aβ (10 μM)-haem (5 μM) complex for 24 h resulted in an increase in ROS concentration to 351 % relative to untreated control cells. After cells were exposed to the Aβhaem complex for 6 h and then treated with 10 μM LPFFD (a classic inhibitory peptide, used as control), the level of ROS decreased to 256 % compared to that of untreated control cells. In contrast, when the cells were treated with 10 μM POM-D-Phe or POM-L-Phe, ROS had a marked decrease and decreased to 106 % and 116 %, respectively. Next, we studied whether POM-D-Phe and POM-L-Phe could decrease Aβ-mediated cellular toxicity. PC12 cells were used to test the effects of POM-D-Phe and POM-L-Phe on cells by using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Figure S18B, S18C, respectively). All the experiments were carried out under the same conditions, and the data were normalized. In the presence of POM-D-Phe or POM-L-Phe, the survival of PC12 cells was significantly increased (Figure S18B), indicating that POM-D-Phe and POM-L-Phe decreased Aβinduced cytotoxicity. All these results demonstrate that POM-D-Phe and POM-L-Phe are effective free-radical scavengers. Furthermore, POM-D-Phe showed the lowest toxicity (Figure S18C). Previous studies indicate that POMs are considered cytotoxic at high dosages, which hinder further application in biomedicine.12 Therefore, the cytotoxicities of POM-NH2, POM-L-Phe and POM-D-Phe at high dosages were determined by MTT assay. After treatment with 320 μM POMNH2 alone for 24 h, the cell viability decreased significantly to less than 50 %, showing notable cytotoxicity. When treated with 320 μM POM-L-Phe or POM-D-Phe, the cell mortality was substantially diminished, while the POM-D-

ACS Paragon Plus Environment

Page 5 of 8 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

Journal of the American Chemical Society Phe reduced the least (Figure S19). The results indicated that modification of natural amino acids on the surface of POMs could decrease cytotoxicity significantly, and the Damino acid modification was the best. The exact reason is not known. This result could be due to D-amino acids having fewer biological functions in organisms.33 Currently, a major impediment for the development of effective therapeutic agents for AD treatment is that most small molecules or peptides are difficult to cross the blood–brain barrier (BBB).34,41 Therefore, we next determined whether POM-D-Phe could cross the BBB. Wild type mice were treated first, and each mouse was administered 25 mg of POM-D-Phe (6.06 mg of Mo) per kilogram of body weight intravenously. Then, plasma and brain homogenates were analysed for the levels of Mo as the surrogate measurement of drug levels by inductively coupled plasma mass spectrometry (ICP-MS). As predicted, the levels of Mo in the plasma increased rapidly to the peak level when treated with POM-D-Phe after 5 min (approximately 5.2 mg Mo per kg, about 85.8 % for POM-D-Phe). Notably, the highest level of Mo in the brain did not appear at the same time as in the plasma but rather at 10 min after dosing (approximately 0.12 mg Mo per kg, about 1.98 % for POMD-Phe) (Figure 2C). Thereafter, the concentrations of Mo markedly decreased in both the plasma and brain. After 60 min, compared with each peak value, just 9.4 % (about 8.07% for POM-D-Phe) and 18.0 % (about 0.36% for POMD-Phe) of Mo remained in the plasma and brain, respectively. After 48 h, the concentrations of Mo in both the plasma and brain returned to the initial levels. We used mice treated with only physiological saline as controls; the concentrations of Mo in the brains were too low to measure. Finally, to understand the pathway of POM-D-Phe clearance from the mouse body, the accurate biodistribution of the contrast agent in the main organs (heart, liver, spleen, lung, kidney and brain) was measured as a function of time and detected through ICP-MS analysis. As shown in Figure 2D, markedly decreasing signals of Mo contents in all organs were observed due to stepwise clearance from the mouse body. Furthermore, POM-D-Phe was eliminated from normal tissues mainly through renal clearance, which was rapid and thorough compared to other metabolic pathways.34 To further examine the drug effects, a commonly used AD transgenic Caenorhabditis elegans (C. elegans) strain, CL2006, was employed.34 In untreated control C. elegans, the worms were almost completely paralyzed within 10 days; by comparison, the paralysis rate induced by Aβ was obviously postponed in CL2006 worms with the treatment of POM-L-Phe or POM-D-Phe. Complete paralysis occurred in 15 days with the incubation of POM-L-Phe, whereas the worms were completely paralyzed within 18 days with POM-D-Phe treatment. These data showed that when treated with chiral POM derivatives, the time period during which CL2006 worms turned to complete paralysis became longer, and POM-D-Phe was the best treatment (Figure S20).

Figure 3. The effect of chiral POM derivatives on the Aβ plaques of the transgenic strain CL2006. Representative images of Aβ plaques in the worm’s head region. (A) Bristol N2 worm (wild type). (B) Transgenic worm CL2006. (C) Transgenic worm CL2006 with the incubation of POM-L-Phe. (D) Transgenic worm CL2006 with the incubation of POM-D-Phe. Arrows indicate Aβ deposits.

Moreover, to verify whether the extension of life span was related to Aβ, thioflavin S (ThS) staining was performed to estimate the amyloid levels in the CL2006 strain.34 The N2 wild type strain worm was used as a control and did not express Aβ or paralysis. Figure 3 shows the fluorescent images of the head region. Notably, treatment with chiral POM derivatives inhibited Aβ aggregation and deposition in the CL2006 strain compared with the untreated CL2006 worms. These results demonstrate that the life span of C. elegans extended by chiral POM derivatives is due to attenuating Aβ-induced toxicity. CONCLUSION In summary, amyloid fibrils are a self-assembled supramolecular system formed by β-sheet aggregates. Therefore, chirality can be a key issue for selectively chemically targeting amyloid aggregates. Several D- or L-amino acidmodified POM derivatives were designed and screened. Among these series, D-phenylalanine-modified POM (POMD-Phe) showed the best inhibition effect by specifically targeting the Aβ central hydrophobic α/β-discordant stretch and demonstrating a novel enantioselectivity of Dand L-amino acids on Aβ inhibition. Furthermore, the biocompatibility of POM-D-Phe improved at high dosage. In vivo studies indicated that the chiral POM derivatives crossed the blood–brain barrier and extended the life span of the AD transgenic Caenorhabditis elegans CL2006 strain.

ASSOCIATED CONTENT

Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (21431007, 21533008, 21820102009, 91856205, 21871249) and the Frontier Science Key Program of CAS (QYZDJ-SSW-SLH052).

REFERENCES

ACS Paragon Plus Environment

Journal of the American Chemical Society 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

(1) Nyström, G.; Arcari, M.; Mezzenga, R. Confinementinduced liquid crystalline transitions in amyloid fibril cholesteric tactoids. Nat. Nanotechnol. 2018, 13, 330-336. (2) Dobson, C. M. Protein folding and misfolding. Nature 2003, 426, 884. (3) Glenner, G. G. Amyloid Deposits and Amyloidosis - The β-Fibrilloses. N. Engl. J. Med. 1980, 302, 1283-1292. (4) Maddelein, M. L.; Dos Reis, S.; Duvezin-Caubet, S.; Coulary-Salin, B.; Saupe, S. J. Amyloid aggregates of the HET-s prion protein are infectious. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 74027407. (5) Barnhart, M. M.; Chapman, M. R. In Annu. Rev. Microbiol. 2006; Vol. 60, p 131-147. (6) Maji, S. K.; Perrin, M. H.; Sawaya, M. R.; Jessberger, S.; Vadodaria, K.; Rissman, R. A.; Singru, P. S.; Nilsson, K. P. R.; Simon, R.; Schubert, D.; Eisenberg, D.; Rivier, J.; Sawchenko, P.; Vale, W.; Riek, R. Functional Amyloids As Natural Storage of Peptide Hormones in Pituitary Secretory Granules. Science 2009, 325, 328332. (7) Li, C. Y.; Cheng, S. Z. D.; Weng, X.; Ge, J. J.; Bai, F.; Zhang, J. Z.; Calhoun, B. H.; Harris, F. W.; Chien, L.-C.; Lotz, B. Left or Right, It Is a Matter of One Methylene Unit. J. Am. Chem. Soc. 2001, 123, 2462-2463. (8) Ke, P. C.; Sani, M.-A.; Ding, F.; Kakinen, A.; Javed, I.; Separovic, F.; Davis, T. P.; Mezzenga, R. Implications of peptide assemblies in amyloid diseases. Chem. Soc. Rev. 2017, 46, 6492-6531. (9) Rauk, A. The chemistry of Alzheimer's disease. Chem. Soc. Rev. 2009, 38, 2698-2715. (10) Li, M.; Howson, S. E.; Dong, K.; Gao, N.; Ren, J.; Scott, P.; Qu, X. Chiral Metallohelical Complexes Enantioselectively Target Amyloid beta for Treating Alzheimer's Disease. J. Am. Chem. Soc. 2014, 136, 11655-11663. (11) Rubin, N.; Perugia, E.; Goldschmidt, M.; Fridkin, M.; Addadi, L. Chirality of amyloid suprastructures. J. Am. Chem. Soc. 2008, 130, 4602-4603. (12) Sun, T.; Cui, W.; Yan, M.; Qin, G.; Guo, W.; Gu, H.; Liu, S.; Wu, Q. Target Delivery of a Novel Antitumor Organoplatinum(IV)Substituted Polyoxometalate Complex for Safer and More Effective Colorectal Cancer Therapy In Vivo. Adv. Mater. 2016, 28, 7397-7404. (13) Fluetsch, A.; Schroeder, T.; Gruetter, M. G.; Patzke, G. R. HIV-1 protease inhibition potential of functionalized polyoxometalates. Bioorg. Med. Chem. Lett. 2011, 21, 1162-1166. (14) Rhule, J. T.; Hill, C. L.; Judd, D. A. Polyoxometalates in medicine. Chem. Rev. 1998, 98, 327-357. (15) Judd, D. A.; Nettles, J. H.; Nevins, N.; Snyder, J. P.; Liotta, D. C.; Tang, J.; Ermolieff, J.; Schinazi, R. F.; Hill, C. L. Polyoxometalate HIV-1 protease inhibitors. A new mode of protease inhibition. J. Am. Chem. Soc. 2001, 123, 886-897. (16) Wu, Q.; Wang, J.; Zhang, L.; Hong, A.; Ren, J. S. Molecular recognition of basic fibroblast growth factor by polyoxometalates. Angew. Chem. Int. Ed. 2005, 44, 4048-4052. (17) Geng, J.; Li, M.; Ren, J.; Wang, E.; Qu, X. Polyoxometalates as Inhibitors of the Aggregation of Amyloid beta Peptides Associated with Alzheimer's Disease. Angew. Chem. Int. Ed. 2011, 50, 4184-4188. (18) Gao, N.; Sun, H.; Dong, K.; Ren, J.; Duan, T.; Xu, C.; Qu, X. Transition-metal-substituted polyoxometalate derivatives as functional anti-amyloid agents for Alzheimer's disease. Nat. Commun. 2014, 5, 3422. (19) Yu, H.; Ru, S.; Dai, G.; Zhai, Y.; Lin, H.; Han, S.; Wei, Y. An Efficient Iron(III)-Catalyzed Aerobic Oxidation of Aldehydes in Water for the Green Preparation of Carboxylic Acids. Angew. Chem. Int. Ed. 2017, 56, 3867-3871. (20) Luo, J.; Chen, K.; Yin, P.; Li, T.; Wan, G.; Zhang, J.; Ye, S.; Bi, X.; Pang, Y.; Wei, Y.; Liu, T. Effect of Cation–π Interaction on Macroionic Self-Assembly. Angew. Chem. Int. Ed. 2018, 57, 40674072.

Page 6 of 8

(21) Dolbecq, A.; Dumas, E.; Mayer, C. R.; Mialane, P. Hybrid Organic-Inorganic Polyoxometalate Compounds: From Structural Diversity to Applications. Chem. Rev. 2010, 110, 6009-6048. (22) Yvon, C.; Surman, A. J.; Hutin, M.; Alex, J.; Smith, B. O.; Long, D. L.; Cronin, L. Polyoxometalate Clusters Integrated into Peptide Chains and as Inorganic Amino Acids: Solution-and SolidPhase Approaches. Angew. Chem. Int. Ed. 2014, 53, 3336-3341. (23) Miras, H. N.; Vila-Nadal, L.; Cronin, L. Polyoxometalate based open-frameworks (POM-OFs). Chem. Soc. Rev. 2014, 43, 5679-5699. (24) Wang, X. H.; Liu, J. F.; Li, J. X.; Yang, Y.; Liu, J. T.; Bin, L.; Pope, M. T. Synthesis and antitumor activity of cyclopentadienyltitanium substituted polyoxotungstate CoW11O39(CpTi) (7-) (CP=eta(5)-C5H5). J. Inorg. Biochem. 2003, 94, 279-284. (25) Landsmann, S.; Wessig, M.; Schmid, M.; Colfen, H.; Polarz, S. Smart Inorganic Surfactants: More than Surface Tension. Angew. Chem. Int. Ed. 2012, 51, 5995-5999. (26) Yang, H. K.; Cheng, Y. X.; Su, M. M.; Xiao, Y.; Hu, M. B.; Wang, W.; Wang, Q. Polyoxometalate-biomolecule conjugates: A new approach to create hybrid drugs for cancer therapeutics. Bioorg. Med. Chem. Lett. 2013, 23, 1462-1466. (27) Chen, L.; Chen, W.-L.; Wang, X.-L.; Li, Y.-G.; Su, Z.-M.; Wang, E.-B. Polyoxometalates in dye-sensitized solar cells. Chem. Soc. Rev. 2019, 48, 260-284. (28) Wu, J.; Wang, X.; Wang, Q.; Lou, Z.; Li, S.; Zhu, Y.; Qin, L.; Wei, H. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II). Chem. Soc. Rev. 2019, 10.1039/C8CS00457A. (29) Mahmoudi, M.; Quinlan-Pluck, F.; Monopo, M. P.; Sheibani, S.; Vali, H.; Dawson, K. A.; Lynch, I. Influence of the Physiochemical Properties of Superparamagnetic Iron Oxide Nanoparticles on Amyloid beta Protein Fibrillation in Solution. ACS Chem. Neurosci. 2013, 4, 475-485. (30) Nerelius, C.; Sandegren, A.; Sargsyan, H.; Raunak, R.; Leijonmarck, H.; Chatterjee, U.; Fisahn, A.; Imarisio, S.; Lomas, D. A.; Crowther, D. C.; Stromberg, R.; Johansson, J. alpha-Helix targeting reduces amyloid-beta peptide toxicity. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 9191-9196. (31) Toeroek, B.; Sood, A.; Bag, S.; Kulkarni, A.; Borkin, D.; Lawler, E.; Dasgupta, S.; Landge, S.; Abid, M.; Zhou, W.; Foster, M.; LeVine, H., III; Toeroek, M. Structure-Activity Relationships of Organofluorine Inhibitors of beta-Amyloid Self-Assembly. ChemMedChem 2012, 7, 910-919. (32) Yu, H.; Li, M.; Liu, G.; Geng, J.; Wang, J.; Ren, J.; Zhao, C.; Qu, X. Metallosupramolecular complex targeting an alpha/beta discordant stretch of amyloid beta peptide. Chem. Sci. 2012, 3, 3145-3153. (33) Kaminski, H. M.; Feix, J. B. Effects of D-Lysine Substitutions on the Activity and Selectivity of Antimicrobial Peptide CM15. Polymers 2011, 3, 2088-2106. (34) Guan, Y.; Du, Z.; Gao, N.; Cao, Y.; Wang, X.; Scott, P.; Song, H.; Ren, J.; Qu, X. Stereochemistry and amyloid inhibition: Asymmetric triplex metallohelices enantioselectively bind to Aβ peptide. Sci. Adv. 2018, 4, eaao6718. (35) Li, Y.; Su, M.; Liu, Z.; Li, J.; Liu, J.; Han, L.; Wang, R. Assessing protein–ligand interaction scoring functions with the CASF-2013 benchmark. Nat. Protoc. 2018, 13, 666. (36) Lee, S.; Zheng, X.; Krishnamoorthy, J.; Savelieff, M. G.; Park, H. M.; Brender, J. R.; Kim, J. H.; Derrick, J. S.; Kochi, A.; Lee, H. J.; Kim, C.; Ramamoorthy, A.; Bowers, M. T.; Lim, M. H. Rational Design of a Structural Framework with Potential Use to Develop Chemical Reagents That Target and Modulate Multiple Facets of Alzheimer's Disease. J. Am. Chem. Soc. 2014, 136, 299-310. (37) Qing, G.; Zhao, S.; Xiong, Y.; Lv, Z.; Jiang, F.; Liu, Y.; Chen, H.; Zhang, M.; Sun, T. Chiral Effect at Protein/Graphene Interface: A Bioinspired Perspective To Understand Amyloid Formation. J. Am. Chem. Soc. 2014, 136, 10736-10742. (38) Yoo, S. I.; Yang, M.; Brender, J. R.; Subramanian, V.; Sun, K.; Joo, N. E.; Jeong, S.-H.; Ramamoorthy, A.; Kotov, N. A. Inhibition

ACS Paragon Plus Environment

Page 7 of 8 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

Journal of the American Chemical Society of Amyloid Peptide Fibrillation by Inorganic Nanoparticles: Functional Similarities with Proteins. Angew. Chem. Int. Ed. 2011, 50, 5110-5115. (39) Gao, N.; Sun, H.; Dong, K.; Ren, J.; Qu, X. GoldNanoparticle-Based Multifunctional Amyloid-beta Inhibitor against Alzheimer's Disease. Chem. Eur. J. 2015, 21, 829-835. (40) Chen, Z.; Li, Z.; Wang, J.; Ju, E.; Zhou, L.; Ren, J.; Qu, X. A Multi-synergistic Platform for Sequential Irradiation-Activated High-Performance Apoptotic Cancer Therapy. Adv. Funct. Mater. 2014, 24, 522-529. (41) Geng, J.; Li, M.; Wu, L.; Ren, J.; Qu, X. Liberation of Copper from Amyloid Plaques: Making a Risk Factor Useful for Alzheimer's Disease Treatment. J. Med. Chem. 2012, 55, 9146-9155.

ACS Paragon Plus Environment

Journal of the American Chemical Society 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

SYNOPSIS TOC: A novel enantioselectivity of D- and L-amino acids on Aβ inhibition was reported. Phenylalanine modified POMs show better inhibition effect on Aβ aggregation than other amino acid-modified POMs, and Dphenylalanine modified POM has 8-fold stronger inhibition effect on Aβ aggregation than L-phenylalanine modified POM.

ACS Paragon Plus Environment

Page 8 of 8