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Minimalistic Principles for Designing Small Molecules with Multiple Reactivities against Pathological Factors in Dementia
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Minimalistic Principles for Designing Small Molecules with Multiple Reactivities against Pathological Factors in Dementia
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  • Mingeun Kim
    Mingeun Kim
    Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
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  • Juhye Kang
    Juhye Kang
    Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
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  • Misun Lee
    Misun Lee
    Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
    Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
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  • Jiyeon Han
    Jiyeon Han
    Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
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  • Geewoo Nam
    Geewoo Nam
    Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
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  • Eunyoung Tak
    Eunyoung Tak
    Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of Korea
    Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
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  • Min Sun Kim
    Min Sun Kim
    Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of Korea
    Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
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  • Hyuck Jin Lee
    Hyuck Jin Lee
    Department of Chemistry Education, Kongju National University, Gongju 32588, Republic of Korea
  • Eunju Nam
    Eunju Nam
    Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
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  • Jiyong Park
    Jiyong Park
    Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
    Center for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS), Daejeon 34141, Republic of Korea
    More by Jiyong Park
  • Soo Jin Oh
    Soo Jin Oh
    Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of Korea
    Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
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  • Ji-Yoon Lee
    Ji-Yoon Lee
    Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of Korea
    Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
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  • Joo-Yong Lee*
    Joo-Yong Lee
    Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of Korea
    Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
    *[email protected]
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  • Mu-Hyun Baik*
    Mu-Hyun Baik
    Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
    Center for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS), Daejeon 34141, Republic of Korea
    *[email protected]
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  • Mi Hee Lim*
    Mi Hee Lim
    Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
    *[email protected]
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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2020, 142, 18, 8183–8193
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https://doi.org/10.1021/jacs.9b13100
Published April 1, 2020

Copyright © 2020 American Chemical Society. This publication is licensed under these Terms of Use.

Abstract

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Multiple pathogenic elements, including reactive oxygen species, amyloidogenic proteins, and metal ions, are associated with the development of neurodegenerative disorders. We report minimalistic redox-based principles for preparing compact aromatic compounds by derivatizing the phenylene moiety with various functional groups. These molecular agents display enhanced reactivities against multiple targets such as free radicals, metal-free amyloid-β (Aβ), and metal-bound Aβ that are implicated in the most common form of dementia, Alzheimer’s disease (AD). Mechanistic studies reveal that the redox properties of these reagents are essential for their function. Specifically, they engage in oxidative reactions with metal-free and metal-bound Aβ, leading to chemical modifications of the Aβ peptides to form covalent adducts that alter the aggregation of Aβ. Moreover, the administration of the most promising candidate significantly attenuates the amyloid pathology in the brains of AD transgenic mice and improves their cognitive defects. Our studies demonstrate an efficient and effective redox-based strategy for incorporating multiple functions into simple molecular reagents.

Copyright © 2020 American Chemical Society

Introduction

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Reactive oxygen species (ROS) play a crucial role in many aspects of cellular metabolism, (1) and due to their intrinsically high reactivity, they must be tightly regulated through biological scavenging mechanisms utilizing a range of enzymes and antioxidants. (2) Under normal conditions, the production and removal of ROS are balanced in cellular processes such as signal transduction and gene transcription. (3) A shift in balance resulting in excess amounts of free radicals can damage lipids, proteins, and DNA, leading to oxidative stress. (4,5) A substantial amount of ROS is produced in the brain because of the heightened O2 metabolism, high metal ion content, and insufficient antioxidant capacity. (6,7) As neurons are particularly vulnerable to oxidative stress, (8) ROS are thought to be involved in neurodegeneration in Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis. (6,9,10) Clinical efforts to prevent oxidative damage with natural antioxidants have failed, (11,12) demonstrating that treating AD requires a more sophisticated solution than simply reducing oxidative stress using antioxidants. A potential strategy is to design agents that can engage ROS, amyloid-β (Aβ), and metal ions simultaneously to regulate oxidative stress, impact the amyloid cascade, and control the metal ion availability. (6,13−16) Following a similar strategy, multifunctional molecules were previously developed employing structure- or mechanism-based approaches that suggested a correlation between target recognition, redox properties, and chemical reactivities toward pathological factors. (9,17−27)
We questioned whether redox-active aromatics carrying Lewis basic functional groups such as hydroxyls, amines, and carboxylates might interact strongly enough with metal-free and metal-bound Aβ species to have a notable impact on their aggregation behavior. In principle, Aβ should offer numerous interaction points for small molecules to modulate or even disrupt its aggregation, but very limited compact aromatic compounds have been reported to be effective so far. Thus, a series of redox-active aromatic reagents shown in Figure 1a was selected and tested against multiple pathological factors, namely, free radicals, metal-free Aβ, and metal-bound Aβ (metal–Aβ), as illustrated in Figure 1b. In addition to assessing the redox potentials and chemical reactivities, the in vivo efficacy of the most promising candidate to reduce Aβ accumulation and ameliorate cognitive function in AD transgenic mice was evaluated. Overall, our studies demonstrate that aromatic molecules of surprisingly simple structural composition can display multiple desirable reactivities against pathogenic factors in neurodegenerative diseases, including AD.

Figure 1

Figure 1. Overview of a rational strategy of designing compact aromatic molecules with multiple reactivities against pathological factors found in the AD-affected brain and the chemical series studied in this work. (a) Structures of 14 (Group-I) and 510 (Group-II). 1, benzene-1,4-diamine; 2, 4-aminophenol; 3, N1,N1,N4,N4-tetramethylbenzene-1,4-diamine; 4, 4-(dimethylamino)phenol; 5, aniline; 6, 4-aminobenzoic acid; 7, pyridine-4-amine; 8, N,N-dimethylaniline; 9, 4-(dimethylamino)benzoic acid; 10, N,N-dimethylpyridine-4-amine. (b) Summary of the multiple targets and the desired effects in vitro and in vivo.

Results and Discussion

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Redox-active compounds have recently been reported as multifunctional molecules capable of targeting and regulating multiple factors implicated in the pathology of AD, (22,23,25,28) suggesting that redox activity may be a necessary characteristic. Therefore, aromatic compounds 110, as shown in Figure 1a, were tested against free radicals, metal-free Aβ, and metal–Aβ found in the AD-affected brain. The test set can be divided into two groups: Group-I (14) contains electron-rich aromatics carrying two electron-donating functionalities such as amino, dimethylamino, or hydroxy groups. The aromatics in Group-II (510), on the other hand, are relatively electron-poor.
Cyclic voltammetry measurements were conducted to establish the redox properties of all molecules considered in this study. After sampling several solvents, acetonitrile (CH3CN) with 1% v/v DMSO was chosen to optimize the solubility of 110 (especially, 2, 4, 7, and 9). As expected from previous studies, (29−31) these experiments revealed reversible and quasi-reversible redox behaviors for 14 (Figure S1) with half-wave potentials (E1/2) in the range of −193 to −5 mV vs Ag/Ag(I), as illustrated in Figure 2. Unfortunately, compounds 510 displayed irreversible oxidations, as presented in Figure S2 and Table S1, making it challenging to obtain E1/2 values. (32) The anodic peak potentials of 510 were generally found to be shifted by roughly +700 mV, compared to those of 14. Although these peak potentials must not be confused with standard redox potentials, these data strongly support the conclusion that the molecules in Group-II are more difficult to oxidize than 14, in good agreement with the expectation that electron-donating groups promote the stabilization of the cationic radical. (33,34) To contrast the performance of the aniline species to another molecule that is redox-active, but displays an entirely different molecular structure, the redox property of l-ascorbic acid (vitamin C) was also examined. Not surprisingly, we found that vitamin C underwent irreversible oxidation with an anodic peak potential at −347 mV (Figure 2b). Thus, it is more easily oxidized than the molecules in Group-I.

Figure 2

Figure 2. Redox behaviors of 110 and vitamin C measured by cyclic voltammetry. (a) Cyclic voltammograms of 14 in CH3CN with 1% v/v DMSO. (b) Values of Epa1 and E1/2 at the scan rate of 250 mV/s. aQuasi-reversible redox behavior was indicated for 2. Conditions: [compound] = 1 mM; [TBAPF6] = 100 mM (for supporting electrolyte and reference electrode); [AgNO3] = 10 mM (for reference electrode); N2(g); scan rates = 25, 50, 100, 150, 200, and 250 mV/s; three electrodes: glassy carbon working electrode, Ag/Ag(I) reference electrode, and platinum counter electrode; room temperature.

Scavenging Free Organic Radicals

To assess the ability of 110 and vitamin C for quenching free radicals, the Trolox equivalent antioxidant capacity (TEAC) assay (35) was performed in a medium containing the lysates of murine neuroblastoma Neuro-2a (N2a) cells. We employed the cationic radical form of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) as the organic radical substrate. As shown in Figure 3, 1, 2, 4 and vitamin C displayed TEAC values close to 1.0, indicating that their radical scavenging capacities are similar to that of Trolox, a water-soluble vitamin E analogue. Interestingly, 3 was more than two times more effective than Trolox, possibly due to its rapid kinetics of oxidation. (36,37) As anticipated from the redox activities mentioned above, compounds 510 showed no measurable activity in the TEAC assay.

Figure 3

Figure 3. Scavenging capability of 110 and vitamin C against free organic radicals determined by the TEAC assay in cell lysates. The TEAC values are relative to that of an analogue of vitamin E, Trolox (6-hydroxy-2,5,7,8-tetramethlychroman-2-carboxylic acid). The error bars indicate the standard error from four independent experiments. *TEAC values of 510 were not obtained because they showed no measurable capacity to quench free radicals.

Impact on Aβ Aggregation

Aggregation and accumulation of Aβ peptides can induce toxicity in the brain and, thus, Aβ is considered a pathogenic hallmark of AD. (13,15,38,39) In addition, binding of metal ions such as Cu(II) and Zn(II) to Aβ can alter its aggregation pathways to different degrees depending on the metal-to-Aβ stoichiometry. (40−43) Therefore, modulating the aggregation of metal-free Aβ and metal–Aβ by small molecules presents a promising avenue for controlling their toxicity. (22,44,45) The molecular weight (MW) distribution and morphology of both metal-free Aβ and metal–Aβ aggregates after exposure to 110 were analyzed by gel electrophoresis with Western blot (gel/Western blot) using an anti-Aβ antibody (6E10) and transmission electron microscopy (TEM), as depicted in Figures 4 and S3–S9. Inhibition and disaggregation experiments were carried out employing Aβ40 and Aβ42, two major isoforms of Aβ. (13) Aβ species with a molecular mass of ca. 4–270 kDa can appear on the gel/Western blot as smearing bands. Larger Aβ aggregates that cannot penetrate the gel matrix are not detected in the gel/Western blot, but they may be visualized by TEM.

Figure 4

Figure 4. Effects of 15 on the formation of metal-free or metal-treated Aβ40 aggregates. (a) Scheme of the inhibition experiments. (b–d) Gel/Western blots (an anti-Aβ antibody, 6E10) of the Aβ40 species generated in the (b) absence and (c and d) presence of metal ions. Lanes: (c) Aβ40; (1) Aβ40 + 1; (2) Aβ40 + 2; (3) Aβ40 + 3; (4) Aβ40 + 4; (5) Aβ40 + 5. (e) Quantification of Aβ40 species visualized in the gel by the ImageJ software. The intensity of the gel from the sample was normalized to that from the corresponding control (ISample/IControl). (f) TEM images of the samples obtained from (b) metal-free Aβ40 and Aβ40 with 1 equiv of (c) Cu(II) and (d) Zn(II). Conditions: [Aβ40] = 25 μM; [Cu(II) or Zn(II)] = 12.5, 25, and 50 μM; [compound] = 50 μM; 20 mM HEPES, pH 7.4 [for metal-free or Zn(II)-containing samples] or pH 6.6 [for Cu(II)-added samples], 150 mM NaCl; 37 °C; 24 h; constant agitation. Scale bar = 200 nm.

As illustrated in Figure 4, the inhibition experiments with metal-free Aβ40 showed increased smearing bands in the presence of 1, 3, and 4 in the range of 4–270 kDa. Upon treatment of 2, the intensity of the bands ranging from 15 to 70 kDa was slightly increased, but the intensity of the bands below ca. 15 kDa was lowered, compared to Aβ40 only (control). In particular, 2 reduced the level of Aβ40 monomers and dimers. These results suggest that the treatment of 14 could produce more of the smaller Aβ40 assemblies capable of penetrating the gel matrix. In the presence of Cu(II) and Zn(II), the changes in the MW distributions of Aβ40 treated with 14 became more prominent. At the 1:1 metal-to-Aβ stoichiometry, the change in metal–Aβ40 aggregation was noticeably observed by incubation of 14 in the inhibition experiments, as presented in Figure 4c and d. Upon treatment of 1 to Aβ40 added with 1 equiv of Cu(II) or Zn(II), the intensity of the smearing bands was increased in the range of ca. 4 to 270 kDa, indicating a notable shift in the MW distribution of metal–Aβ40 species, relative to compound-free metal–Aβ40 species (controls). In the case of Cu(II)–Aβ40 incubated with 24, the signal intensities of monomeric Aβ40 at ca. 4 kDa were diminished, while new bands between ca. 16 and 80 kDa appeared. Under Zn(II)-present conditions, such distinct reactivities of 24 were also observed. 2 induced the MW distribution of Aβ40 species in the presence of Zn(II) in the range of ca. 4 to 270 kDa, while 3 and 4 altered the MW distribution of Aβ40 to produce peptide species in the range of ca. 4 to 40 kDa, along with larger Aβ40 aggregates (≥ ca. 240 kDa). Furthermore, modulative effects of the compounds toward metal–Aβ40 aggregation in the presence of sub- and supra-equimolar metal concentrations were identified (Figure 4). Compounds 14, which were capable of modulating metal–Aβ40 aggregation at a 1:1 metal-to-Aβ ratio, also exhibited notable reactivity toward Aβ40 aggregation in the presence of 0.5 and 2 equiv of Cu(II) or Zn(II), as presented in Figure 4c and d. Conversely, 510 did not discernibly alter the MW distributions of both metal-free Aβ40 and metal–Aβ40 under all conditions used for testing 14, as indicated in Figures 4 and S3. To quantify the amounts of Aβ40 species visualized in the gel/Western blots, the signal intensity of the gels from metal-free and metal-bound Aβ40 samples was analyzed as a measure of Aβ40 concentration. As presented in Figures 4e and S3e, the treatment of 14 to metal-free and metal-treated Aβ40 led to varying amounts of Aβ40 aggregates analyzed by gel/Western blot, compared to those of compound-free metal-free and metal-treated Aβ40 (controls) as well as those from the samples incubated with compounds 510 lacking reactivity.
TEM studies revealed the abnormal conformations of metal-free Aβ40 and metal–Aβ40 aggregates for the samples that were exposed to 14, as visualized in Figure 4f. More specifically, smaller Aβ40 fibrils were observed, along with amorphous aggregates, presenting a notable conformational contrast from mature Aβ40 fibrils produced from the samples of compound-free or 5-treated Aβ40 in the absence and presence of metal ions. Specifically, such amorphous aggregates have been previously reported to be less toxic than the structured aggregates of Aβ. (22,23) Moreover, the modulative reactivity of vitamin C against Aβ40 aggregation was further evaluated to test the notion that redox activity is a critical parameter in a molecule’s ability to alter Aβ aggregation. In these experiments, no noticeable change in the aggregation of Aβ40 with and without metal ions was observed with the treatment of vitamin C, as depicted in Figure S10, indicating that low oxidation potential alone does not sufficiently signify a molecule’s ability to impact the aggregation behavior of metal-free and metal-bound Aβ.
As shown in Figures S4 and S5, the disaggregation experiments employing Aβ40 displayed similar trends to the inhibition experiments. Compounds 14 altered the MW distribution of preformed metal-free Aβ40 and metal–Aβ40 aggregates and their morphologies, while 510 did not exhibit such reactivity. Furthermore, both the inhibition and disaggregation experiments with Aβ42 treated with 110 showed results that were nearly identical to what was obtained with Aβ40, as summarized in Figures S6–S9. In addition, the aggregation of metal-free Aβ and metal–Aβ incubated with our compounds in both inhibition and disaggregation experiments employing Aβ40 and Aβ42 was quantitatively monitored by the turbidity assay (Figure S11). Compounds 4 and 5, lacking any interference within the detection window of the turbidity assay were selected as the representative compounds of Group-I and Group-II, respectively. The turbidity values of the 4-treated metal-free Aβ and metal–Aβ samples were noticeably distinct from those of the compound-free and 5-treated samples in both inhibition and disaggregation experiments. As expected from the gel/Western blot and TEM, the modulating reactivity of 4 toward the aggregation of both metal-free and metal-treated Aβ was verified.
Taken together, our in vitro experiments illustrate that 14 can modulate the aggregation of both metal-free Aβ and metal–Aβ and disassemble the corresponding preformed aggregates, whereas 510 and vitamin C did not exhibit such reactivity. It can be inferred from the gel/Western blot data that the redox activity of 14 is closely correlated with their ability to impact Aβ aggregation in the absence and the presence of metal ions at varying concentrations. On the basis of the current understanding of the toxicity induced by Aβ aggregates (in particular, structured oligomers), (38) the altered aggregation profiles by treatment of 14 observed by gel/Western blot and TEM may generate less toxic metal-free Aβ and metal–Aβ aggregates. Overall, these observations confirm the suspected relation between the redox activity of the small molecules and their ability to impact the aggregation of metal-free Aβ and metal–Aβ, suggesting that the easily oxidized phenylene moiety is critical for such reactivity.

Oxidative Reactions

To better understand the connection between the reactivity toward metal-free and metal-treated Aβ aggregation and the redox properties, the chemical transformations of 110 were monitored with and without Aβ40 and Cu(II) or Zn(II) by ultraviolet–visible (UV–vis) spectroscopy, as shown in Figures 5a and S12. In the absence of Aβ40 and metal ions, the optical spectrum of 1 exhibited an increase in absorption at 530 nm, resulting in the appearance of a broad band, as depicted in Figure S12a, indicative of the formation of a cationic radical and subsequent dimerization and trimerization. (29,46−48) Furthermore, a hypsochromic shift from 300 to 250 nm was accelerated by the addition of Cu(II), along with the optical enhancement at 530 nm, implying the oxidative transformation of 1. (47,48)

Figure 5

Figure 5. Analyses of 1’s transformation and interactions with metal-free Aβ40 or Cu(II)-added Aβ40. (a) Oxidative transformation of 1 in the presence of Aβ40 with or without Cu(II) detected by UV–vis spectroscopy. Conditions: [Aβ40] = 25 μM; [Cu(II)] = 25 μM; [1] = 50 μM; 20 mM HEPES, pH 7.4 (for metal-free samples) or pH 6.6 [for Cu(II)-added samples], 150 mM NaCl; 37 °C; 0–24 h; no agitation. (b–d) Interactions of 1 with metal-free Aβ40 and Cu(II)-treated Aβ40 monitored by ESI-MS, ESI-MS2, and MALDI-MS. Aβ40 monomer incubated with 1 in the (b and d) absence and (d) presence of Cu(II) was analyzed by (b) ESI-MS or (d) MALDI-MS. The oxidized Aβ40 and the BQ–Aβ40 adduct are indicated with red and blue circles, respectively. The covalent bond with Aβ40 (green circle) was only observed from 1-treated samples. (c) ESI-MS2 spectrum of the singly oxidized Aβ403+ produced upon addition of 1. Conditions (for ESI-MS studies): [Aβ40] = 50 μM; [1] = 100 μM; 1 mM ammonium acetate, pH 7.4; 37 °C; 24 h; constant agitation. The samples were diluted by 10-fold with ddH2O before injection to the mass spectrometer. Conditions (for MALDI-MS measurements): [Aβ40] = 25 μM; [Cu(II)] = 25 μM; [1] = 50 μM; pH 7.4 (for metal-free samples) or pH 6.6 [for Cu(II)-added samples]; 37 °C; 24 h; constant agitation.

Notable optical changes of 1 were observed in the presence of Aβ40 with and without metal ions, as illustrated in Figures 5a and S12a. Upon incubation with metal-free Aβ40 or Zn(II)–Aβ40, two optical bands at 350 and 530 nm were subject to enhancement. (47,48) These spectral changes suggest the oxidative transformation of 1 and the formation of covalent adducts between transformed 1 and metal-free Aβ40 or Zn(II)–Aβ40, as previously reported with a similar compound. (22) The optical spectra of 1 exhibited several distinct changes with Cu(II)–Aβ40: (i) significant sequential increase and decrease of the absorption band at 250 nm; (ii) band intensification at 350 nm that was notably larger than those found without any metal ions and with Zn(II); (iii) absence of the broad peak at 530 nm that was detected under metal-free conditions. These observations indicate that both the cationic radical formation and dimerization or trimerization are significantly inhibited, as a consequence of the compound’s oxidation to benzoquinonediimine (BQDI) or p-benzoquinone (BQ) in the presence of Cu(II)–Aβ40, followed by the generation of covalent adducts between BQDI or BQ and Aβ.
In addition to 1, compounds 24 were optically analyzed in the absence and presence of Aβ40 and metal ions. As presented in Figure S12b–d, the oxidative transformation of each compound was detected without Aβ40, as previously reported. (29,46,49,50) When the compounds were incubated with Aβ40, the optical spectra showed that 2 and 4 were oxidized to give BQ and generate covalent adducts with Aβ40, as depicted in Figure S12b and d. Note that such spectral changes of 3 were undetectable, as indicated in Figure S12c, due to interference from its radical species. In contrast to 14, compounds 510 did not present notable spectral alterations throughout the measurement period under our experimental conditions, as shown in Figure S12e–j. This observation suggests that the redox activity of the small molecules is critical for undergoing oxidative transformation to yield products such as BQ capable of forming covalent adducts with both metal-free Aβ and metal–Aβ. Collectively, our optical studies suggest a potential mechanism for the reactivities of 14 with metal-free Aβ or metal–Aβ involving oxidative transformations.

Interactions with Metal-Free Aβ or Metal–Aβ

Potential interactions between 15 and Aβ40 were first visualized by AutoDock Vina (51) employing a previously reported NMR structure of Aβ40 monomer (PDB 2LFM (52)). Docking studies showed that 15 were situated in a pocket near the self-recognition site, as depicted in Figure S13. The binding energies of these interactions were ranged from −4.7 to −4.0 kcal/mol. Based on our in vitro results, in which 14 showed modulation reactivity toward Aβ aggregation and 5 did not show such reactivity, redox activity would be critical over the regional contacts with Aβ.
To identify the direct interactions of 15 with metal-free Aβ40 or metal–Aβ40 at the molecular level, we employed two types of mass spectrometry, i.e., electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). ESI-MS studies were first conducted with the samples containing 15 and metal-free Aβ40, as shown in Figures 5b and S14a. In the presence of 14, the addition of 104 Da to the +3-charged-Aβ40 monomer corresponding to BQ covalently linked to Aβ40 (BQ–Aβ40) (22) was detected. Furthermore, the samples of metal-free Aβ40 exposed to 14 indicated the peaks at 1449 and 1484 m/z that were assigned as [Aβ40 + O + 3H]3+ and [Aβ40 + O + 104 + 3H]3+, respectively, indicating the oxidation of both Aβ40 and the BQ–Aβ40 adduct. To determine an oxidation site of Aβ40, the peak at 1449 m/z was analyzed by tandem MS (ESI-MS2), as illustrated in Figure 5c. Fragmental analysis of the peak through collision-induced dissociation suggested Met35 as a possible oxidation site. (14,53) As expected, the ESI-MS analysis of the Aβ40 sample to which 5 was added exhibited neither formation of a covalent bond with Aβ40 nor oxidation of Aβ40.
To further investigate the interactions of 1 with metal-free Aβ40 and metal–Aβ40, MALDI-MS was carried out, as presented in Figures 5d and S14b. Upon addition of 1 to metal-free Aβ40, a new peak at 4435 m/z, assigned to [Aβ40 + 104 + H]+, was observed, indicative of a covalent adduct of BQ with Aβ40, which was shown in ESI-MS studies. The covalent bond formation was also detected in the presence of Cu(II) and Zn(II). In the sample containing Cu(II)–Aβ40 and 1, the peaks corresponding to the addition of one or two oxygen atoms to Aβ40 monomer at 4346 and 4362 m/z, respectively, or the BQ–Aβ40 adduct at 4451 and 4467 m/z, respectively, were indicated.
Our MS data provide mechanistic details of Group-I’s modulative reactivity toward the aggregation of metal-free Aβ and metal–Aβ: covalent adduct formation and peptide oxidation. The proposed mechanisms depend on the redox properties of the molecules. Oxidative transformations of 14 can (i) generate BQ, which is capable of covalently binding to Aβ; (22) (ii) form the cationic radicals that could abstract a hydrogen atom from Aβ followed by reaction with O2 to yield a peroxyl radical and subsequent oxidation of Aβ; (14,54) (iii) produce ROS (e.g., superoxide anion radical and hydrogen peroxide) that are able to oxidize Aβ. (14,55,56) Together, our MS studies evince the significance of redox properties as a critical parameter for designing small aromatic molecules with the reactivity against metal-free Aβ and metal–Aβ.

Biological Applicability

The cell viability, metabolic stability, and brain uptake of 14 capable of regulating free radicals, metal-free Aβ, and metal–Aβ were assessed prior to in vivo studies. First, the toxicity of 14 was determined in N2a cells by the MTT assay [MTT = 3-(4,5-dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide], compared to the viability of compound- and vehicle (i.e., 1% v/v H2O)-untreated cells, as depicted in Figure S15. N2a cells were treated with 14 for 24 h in a range of concentrations from 5 to 50 μM. The cells incubated with 1 showed greater than ca. 80% viability at up to 50 μM. In contrast, the cells exhibited ca. 50%, 60%, and 30% survival upon incubation with 50 μM of 2, 3, and 4, respectively. Second, the half-life (t1/2) and intrinsic clearance (CIint) of 14, which are representative indicators for microsomal degradation of the molecules, (57) were monitored as a measure of their metabolic stability. To alleviate the saturation effect in determining the metabolic stability of 14 against human liver microsomes, a low concentration (1 μM) of the compounds was used. (58)1, 2, and 4 presented t1/2 greater than 60 min and CIint of ca. 20 mL/min/mg, indicating their relatively modest metabolic stability. On the other hand, 3 presented poor metabolic stability in comparison (t1/2 = 19 min; CIint = 74 mL/min/mg protein). Lastly, the biodistribution of 1, the least toxic candidate with relatively modest metabolic stability, was examined in vivo. Biodistribution studies of 1 in male wild-type littermates of 5×FAD mice [10 mg/kg; intraperitoneal (i.p.) injection] revealed that the compound was detected in plasma (5.8%), brain (7.8%), liver (1.5%), kidney (9.0%), and intestine (10%), as indicated in Table S2. With the exception of the liver, the organ-to-plasma ratio of 1’s concentration was greater than 1.0 in other organs. Detection of 1 in the brain following i.p. and oral administration (Tables S2 and S3) supports the molecule’s ability to cross the blood–brain barrier and availability in the central nervous system. Our evaluation of Group-I’s biological applicability prompted 1 as a suitable candidate for further histochemical and behavioral investigations in vivo.

In Vivo Efficacies

Compound 1 was evaluated, along with BQ as a product of 1’s oxidative transformation, for the efficacies in 5×FAD transgenic mice. 5×FAD is a transgenic mouse model of AD overexpressing mutant human APP695 [Swedish (K670N/M671L), Florida (1716 V), and London (V7171)] and PS1 (M146L and L286V) that exhibits the onset of AD pathology and cognitive decline. (22,23,59) 5×FAD mice were subjected to treatment of the compounds that were dissolved in 20 mM HEPES, pH 7.4, and 150 mM NaCl with 1% v/v DMSO; vehicle in this work indicates the buffered solution (20 mM HEPES, pH 7.4, and 150 mM NaCl) containing 1% v/v DMSO. First, the weight, physical appearance, and behavior of 5×FAD mice were examined every day for the duration of compound administration. In comparison with the vehicle- and 1-treated (1 mg/kg/day; i.p. injection) groups, which showed no death among 19 mice tested and 14% mortality (2 deaths among 14 mice), respectively, mice receiving BQ (1 mg/kg/day; i.p.) exhibited higher mortality rates (40%; 6 deaths among 15 mice). As summarized in Table S4, administering the vehicle and 1 yielded a general increase in weight, whereas BQ resulted in a perceivable decrease and a slight increase in the weights of males and females among the survivors, respectively. Furthermore, BQ-treated 5×FAD mice manifested hypomotility and reduced task performance capacity compared to the vehicle- or 1-treated mice (data not shown). Gross murine necropsy revealed the abnormal morphology of i.p. organs, including lesions at the injection site and severe internal adhesions accompanied by chronic inflammation in a majority of BQ-treated 5×FAD mice. Such detriments were not observed in the vehicle- or 1-added mice (data not shown). Long-term i.p. administration of BQ induced significant toxicity in 5×FAD mice at 1 mg/kg/day and, at times, resulted in the termination of the test subject. In contrast, 1 was sufficiently tolerated by 5×FAD mice throughout the period of repeated treatments, reinforcing its in vivo safety.
The effects of 1 and BQ on amyloid deposition in the brains of 5×FAD mice were verified in the surviving population of test subjects (n = 12 of 14 and 9 of 15 for 1 and BQ, respectively). 1 prominently attenuated the amyloid pathology in the brains of 5×FAD mice. All fractions of Aβ42 (soluble, insoluble, and total Aβ42) and oligomeric Aβ from 1-treated 5×FAD mice were significantly reduced in comparison with vehicle-treated 5×FAD mice by 28%, 27%, 28%, and 28%, respectively, obtained via the enzyme-linked immunosorbent assay (ELISA), as depicted in Figure 6a. Compound 1 also diminished the deposition of 4G8-immunoreactive amyloid aggregates and compact congophilic amyloid plaques by 28% and 17%, respectively, as shown in Figure 6b and c. Moreover, the repeated administration of 1 significantly improved the cognitive performance of 5×FAD mice. As indicated in Figure 7a, during the trial period of the Morris water maze (MWM) test for 5 days, 1-treated 5×FAD mice found the escape platform more easily and quickly than the vehicle- or BQ-treated ones, which supports that 1 ameliorates their spatial learning ability. In the subsequent probe test, which examines the long-term spatial memory of the mice, (60)1-added 5×FAD mice took shorter paths to the target zone and less time to reach the target location, more frequently traversed the target location, and spent more time in the target quadrant, as illustrated in Figure 7b–f. These results prove that 1 can effectively improve the spatial learning and memory of 5×FAD mice.

Figure 6

Figure 6. Analysis of the amounts of Aβ species in 1- or BQ-treated 5×FAD mice. (a) Levels of soluble Aβ42, insoluble Aβ42, total Aβ42, and oligomeric Aβ measured in triplicate per sample by ELISA. Soluble phosphate buffered saline (PBS)- and sodium dodecyl sulfate (SDS)-soluble fractions (for soluble Aβ42), formic acid (FA)-soluble fractions (for insoluble Aβ42), and the sum of PBS-, SDS-, and FA-soluble fractions (for total Aβ42) were analyzed. Lanes: (Vehicle) 5×FAD + vehicle; (BQ) 5×FAD + BQ; (1) 5×FAD + 1. (b) Loads of amyloid deposits and plaques in the brain expressed as the percent area of 4G8-immunoreactive deposits or the number of congophilic plaques per mm2 of a region of interest, which was taken from hippocampal (hip), cortical (ctx), and thalamic (tlm) areas. (c) Representative images of 4G8-immunoreactive (1st row) or Congo red-positive (2nd and 3rd rows) amyloid deposits or plaques in hip and ctx (1st and 3rd rows) or tlm (2nd row) regions in the brains of vehicle- (1st column), BQ- (2nd column), or 1-treated (3rd column) 5×FAD mice are shown. Congo red-stained brain sections were also counter-stained with hematoxylin to differentiate the nuclei of neural cells (2nd and 3rd rows). Subiculum (sub), corpus callosum (cc), and fornix (fx). Scale bars = 500 μm (white) or 200 μm (black). The measurements were performed in five sagittal sections taken every 200 μm from midline per animal. Bars denote mean ± standard errors of mean (s.e.m.) (animal numbers; n = 19 for vehicle-treated 5×FAD mice; n = 9 for BQ-treated 5×FAD mice; n = 12 for 1-treated 5×FAD mice). *P < 0.05 or **P < 0.01 by unpaired two-tail t-test.

Figure 7

Figure 7. Measurement of spatial learning and memory improvements in 1- or BQ-administrated 5×FAD mice. (a) Escape latency time daily assessed for 5 days from the day of the 30th compound treatment in the MWM test. From the second training trial to the fifth trial, the latency time became significantly shorter in nontransgenic wild-type mice [WT; P = 0.012 by one-way analysis of variance (ANOVA) with Student–Newman–Keuls post hoc test] or 1-treated 5×FAD mice (1; P = 0.0017) but not in vehicle- (Vehicle; P = 0.054) or BQ-treated (BQ; P = 0.40) 5×FAD mice. (b) After the MWM test, the probe trials were performed in the same water pool without the escape platform. All images present the representative paths of the mice to search for the previous platform location [the small circle area in the gray, northwest (NW) target quadrant] for 60 s (from point S to point E). (c–f) In the probe test, we recorded (c) the path distance to first enter the target quadrant, (d) the latency time to touch the previous location of the platform, (e) the crossing frequency to traverse across the target platform, and (f) the times spent in the target quadrant to search for the platform. Lanes: (WT) wild-type; (Vehicle) 5×FAD + vehicle; (BQ) 5×FAD + BQ; (1) 5×FAD + 1. The statistical comparisons were performed between vehicle-treated 5×FAD and their wild-type littermate mice (*) or between vehicle- and 1-treated 5×FAD mice (#). Bars denote mean ± s.e.m. Animal number: n = 17 for wild-type mice; n = 19 for vehicle-treated 5×FAD mice; n = 9 for BQ-treated 5×FAD mice; n = 12 for 1-treated 5×FAD mice. *,#P < 0.05, **,##P < 0.01, or ***,###P < 0.001 by unpaired two-tail t-test.

In the brains of BQ-treated 5×FAD mice, the levels of soluble, insoluble, and total Aβ42 as well as oligomeric Aβ species were reduced by 20%, 7.4%, 15%, and 26%, respectively, compared to those of vehicle-treated 5×FAD mice; however, these differences were not deemed statistically significant, as presented in Figure 6a. 4G8-immunohistochemical evaluations exhibited a 31% decrease in the level of amyloid deposits by BQ with borderline statistical significance. Additionally, BQ was able to lower the deposition of compact congophilic amyloid plaques by only 7.2%, as shown in Figure 6b and c. Note that BQ’s toxicity likely poses a potential interference to the in vivo evaluation regarding the molecule’s ability to curb the Aβ pathology in 5×FAD mice. Additional studies are warranted to elucidate the dose-dependent efficacy and toxicity of BQ and evaluate its effects on the behavior and cognitive function in vivo. Overall, our comparative evaluation of 1 and BQ in vivo demonstrates that although both molecules can reduce the cerebral and hippocampal load of Aβ species, 1 presents significantly lower toxicity and is much more efficient in reducing the Aβ pathology and recovering the cognitive ability of AD transgenic mice.

Conclusions

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Difficulty in understanding the pathology of AD stems from its multifactorial nature. Ensuing inadequacy in comprehending the causes of neurodegeneration in AD severely impedes the development of therapeutics capable of halting the progression of the disease. To advance our understanding of AD pathology, small molecules capable of targeting multiple pathogenic factors have been designed as investigative chemical tools with therapeutic potential. As a recently emerging concept, the structure-mechanism-based molecular design has presented the feasibility of controlling a molecule’s reactivity toward various pathological factors of AD by tuning its electronic property. To consolidate this design strategy, we rationally selected compact aromatic molecules (110) by adjusting the electronic distribution of the phenyl, phenylene, or pyridyl moiety to impart redox-dependent reactivities against multiple pathological factors of AD, i.e., free radicals, metal-free Aβ, and metal–Aβ. Our biochemical and biophysical studies demonstrate the redox-dependent multiple reactivities of the small molecules 14 toward free radicals, metal-free Aβ, and metal–Aβ. Further spectroscopic and spectrometric results indicate the mechanisms involved in their regulatory effects against metal-free Aβ and metal–Aβ: covalent adduct formation and peptide oxidation. As the most promising candidate molecule with suitable biological applicability such as low cytotoxicity, moderate metabolic stability, and potential BBB permeability, 1 is able to significantly reduce cerebral and hippocampal Aβ deposits and produce statistically significant improvements in the cognitive function of 5×FAD transgenic mice. Taken together, our multidisciplinary studies establish that redox properties of small molecules, along with an aromatic backbone, are a critical parameter to consider when designing compounds with multiple reactivities against pathological factors in AD, as proven through our minimalist redox-based design strategy of modifying the electronic properties of benzene. Our overall approaches and findings can assist in elucidating the uncovered relationships among intertwined pathogenic factors and contribute toward finding effective chemical reagents against neurodegenerative disorders, including AD.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.9b13100.

  • Experimental Section, Tables S1–S4, and Figures S1–S15 (PDF)

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Author Information

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  • Corresponding Authors
    • Joo-Yong Lee - Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of KoreaDepartment of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea Email: [email protected]
    • Mu-Hyun Baik - Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of KoreaCenter for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS), Daejeon 34141, Republic of KoreaOrcidhttp://orcid.org/0000-0002-8832-8187 Email: [email protected]
    • Mi Hee Lim - Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of KoreaOrcidhttp://orcid.org/0000-0003-3377-4996 Email: [email protected]
  • Authors
    • Mingeun Kim - Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
    • Juhye Kang - Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
    • Misun Lee - Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of KoreaDepartment of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
    • Jiyeon Han - Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
    • Geewoo Nam - Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
    • Eunyoung Tak - Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of KoreaDepartment of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
    • Min Sun Kim - Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of KoreaDepartment of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
    • Hyuck Jin Lee - Department of Chemistry Education, Kongju National University, Gongju 32588, Republic of KoreaOrcidhttp://orcid.org/0000-0001-8769-2967
    • Eunju Nam - Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea
    • Jiyong Park - Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of KoreaCenter for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS), Daejeon 34141, Republic of KoreaOrcidhttp://orcid.org/0000-0002-3225-4510
    • Soo Jin Oh - Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of KoreaDepartment of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
    • Ji-Yoon Lee - Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of KoreaDepartment of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
  • Author Contributions

    M.K., J.K., and M.L. contributed equally to this work.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research is supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government [NRF-2017R1A2B3002585 (to M.H.L); NRF-2017R1D1A1B03030567 (to J.-Y.L.)]; the Institute for Basic Science (IBS-R010-A1) in Korea (to M.-H.B.); Asan Institute for Life Sciences, Asan Medical Center [Seoul, Korea; 2019-396 (to J.-Y.L.)]. We thank the DMPK core facility at the Convergence Medicine Research Center (CREDIT), Asan Medical Center, for support and instrumentation.

References

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  • Abstract

    Figure 1

    Figure 1. Overview of a rational strategy of designing compact aromatic molecules with multiple reactivities against pathological factors found in the AD-affected brain and the chemical series studied in this work. (a) Structures of 14 (Group-I) and 510 (Group-II). 1, benzene-1,4-diamine; 2, 4-aminophenol; 3, N1,N1,N4,N4-tetramethylbenzene-1,4-diamine; 4, 4-(dimethylamino)phenol; 5, aniline; 6, 4-aminobenzoic acid; 7, pyridine-4-amine; 8, N,N-dimethylaniline; 9, 4-(dimethylamino)benzoic acid; 10, N,N-dimethylpyridine-4-amine. (b) Summary of the multiple targets and the desired effects in vitro and in vivo.

    Figure 2

    Figure 2. Redox behaviors of 110 and vitamin C measured by cyclic voltammetry. (a) Cyclic voltammograms of 14 in CH3CN with 1% v/v DMSO. (b) Values of Epa1 and E1/2 at the scan rate of 250 mV/s. aQuasi-reversible redox behavior was indicated for 2. Conditions: [compound] = 1 mM; [TBAPF6] = 100 mM (for supporting electrolyte and reference electrode); [AgNO3] = 10 mM (for reference electrode); N2(g); scan rates = 25, 50, 100, 150, 200, and 250 mV/s; three electrodes: glassy carbon working electrode, Ag/Ag(I) reference electrode, and platinum counter electrode; room temperature.

    Figure 3

    Figure 3. Scavenging capability of 110 and vitamin C against free organic radicals determined by the TEAC assay in cell lysates. The TEAC values are relative to that of an analogue of vitamin E, Trolox (6-hydroxy-2,5,7,8-tetramethlychroman-2-carboxylic acid). The error bars indicate the standard error from four independent experiments. *TEAC values of 510 were not obtained because they showed no measurable capacity to quench free radicals.

    Figure 4

    Figure 4. Effects of 15 on the formation of metal-free or metal-treated Aβ40 aggregates. (a) Scheme of the inhibition experiments. (b–d) Gel/Western blots (an anti-Aβ antibody, 6E10) of the Aβ40 species generated in the (b) absence and (c and d) presence of metal ions. Lanes: (c) Aβ40; (1) Aβ40 + 1; (2) Aβ40 + 2; (3) Aβ40 + 3; (4) Aβ40 + 4; (5) Aβ40 + 5. (e) Quantification of Aβ40 species visualized in the gel by the ImageJ software. The intensity of the gel from the sample was normalized to that from the corresponding control (ISample/IControl). (f) TEM images of the samples obtained from (b) metal-free Aβ40 and Aβ40 with 1 equiv of (c) Cu(II) and (d) Zn(II). Conditions: [Aβ40] = 25 μM; [Cu(II) or Zn(II)] = 12.5, 25, and 50 μM; [compound] = 50 μM; 20 mM HEPES, pH 7.4 [for metal-free or Zn(II)-containing samples] or pH 6.6 [for Cu(II)-added samples], 150 mM NaCl; 37 °C; 24 h; constant agitation. Scale bar = 200 nm.

    Figure 5

    Figure 5. Analyses of 1’s transformation and interactions with metal-free Aβ40 or Cu(II)-added Aβ40. (a) Oxidative transformation of 1 in the presence of Aβ40 with or without Cu(II) detected by UV–vis spectroscopy. Conditions: [Aβ40] = 25 μM; [Cu(II)] = 25 μM; [1] = 50 μM; 20 mM HEPES, pH 7.4 (for metal-free samples) or pH 6.6 [for Cu(II)-added samples], 150 mM NaCl; 37 °C; 0–24 h; no agitation. (b–d) Interactions of 1 with metal-free Aβ40 and Cu(II)-treated Aβ40 monitored by ESI-MS, ESI-MS2, and MALDI-MS. Aβ40 monomer incubated with 1 in the (b and d) absence and (d) presence of Cu(II) was analyzed by (b) ESI-MS or (d) MALDI-MS. The oxidized Aβ40 and the BQ–Aβ40 adduct are indicated with red and blue circles, respectively. The covalent bond with Aβ40 (green circle) was only observed from 1-treated samples. (c) ESI-MS2 spectrum of the singly oxidized Aβ403+ produced upon addition of 1. Conditions (for ESI-MS studies): [Aβ40] = 50 μM; [1] = 100 μM; 1 mM ammonium acetate, pH 7.4; 37 °C; 24 h; constant agitation. The samples were diluted by 10-fold with ddH2O before injection to the mass spectrometer. Conditions (for MALDI-MS measurements): [Aβ40] = 25 μM; [Cu(II)] = 25 μM; [1] = 50 μM; pH 7.4 (for metal-free samples) or pH 6.6 [for Cu(II)-added samples]; 37 °C; 24 h; constant agitation.

    Figure 6

    Figure 6. Analysis of the amounts of Aβ species in 1- or BQ-treated 5×FAD mice. (a) Levels of soluble Aβ42, insoluble Aβ42, total Aβ42, and oligomeric Aβ measured in triplicate per sample by ELISA. Soluble phosphate buffered saline (PBS)- and sodium dodecyl sulfate (SDS)-soluble fractions (for soluble Aβ42), formic acid (FA)-soluble fractions (for insoluble Aβ42), and the sum of PBS-, SDS-, and FA-soluble fractions (for total Aβ42) were analyzed. Lanes: (Vehicle) 5×FAD + vehicle; (BQ) 5×FAD + BQ; (1) 5×FAD + 1. (b) Loads of amyloid deposits and plaques in the brain expressed as the percent area of 4G8-immunoreactive deposits or the number of congophilic plaques per mm2 of a region of interest, which was taken from hippocampal (hip), cortical (ctx), and thalamic (tlm) areas. (c) Representative images of 4G8-immunoreactive (1st row) or Congo red-positive (2nd and 3rd rows) amyloid deposits or plaques in hip and ctx (1st and 3rd rows) or tlm (2nd row) regions in the brains of vehicle- (1st column), BQ- (2nd column), or 1-treated (3rd column) 5×FAD mice are shown. Congo red-stained brain sections were also counter-stained with hematoxylin to differentiate the nuclei of neural cells (2nd and 3rd rows). Subiculum (sub), corpus callosum (cc), and fornix (fx). Scale bars = 500 μm (white) or 200 μm (black). The measurements were performed in five sagittal sections taken every 200 μm from midline per animal. Bars denote mean ± standard errors of mean (s.e.m.) (animal numbers; n = 19 for vehicle-treated 5×FAD mice; n = 9 for BQ-treated 5×FAD mice; n = 12 for 1-treated 5×FAD mice). *P < 0.05 or **P < 0.01 by unpaired two-tail t-test.

    Figure 7

    Figure 7. Measurement of spatial learning and memory improvements in 1- or BQ-administrated 5×FAD mice. (a) Escape latency time daily assessed for 5 days from the day of the 30th compound treatment in the MWM test. From the second training trial to the fifth trial, the latency time became significantly shorter in nontransgenic wild-type mice [WT; P = 0.012 by one-way analysis of variance (ANOVA) with Student–Newman–Keuls post hoc test] or 1-treated 5×FAD mice (1; P = 0.0017) but not in vehicle- (Vehicle; P = 0.054) or BQ-treated (BQ; P = 0.40) 5×FAD mice. (b) After the MWM test, the probe trials were performed in the same water pool without the escape platform. All images present the representative paths of the mice to search for the previous platform location [the small circle area in the gray, northwest (NW) target quadrant] for 60 s (from point S to point E). (c–f) In the probe test, we recorded (c) the path distance to first enter the target quadrant, (d) the latency time to touch the previous location of the platform, (e) the crossing frequency to traverse across the target platform, and (f) the times spent in the target quadrant to search for the platform. Lanes: (WT) wild-type; (Vehicle) 5×FAD + vehicle; (BQ) 5×FAD + BQ; (1) 5×FAD + 1. The statistical comparisons were performed between vehicle-treated 5×FAD and their wild-type littermate mice (*) or between vehicle- and 1-treated 5×FAD mice (#). Bars denote mean ± s.e.m. Animal number: n = 17 for wild-type mice; n = 19 for vehicle-treated 5×FAD mice; n = 9 for BQ-treated 5×FAD mice; n = 12 for 1-treated 5×FAD mice. *,#P < 0.05, **,##P < 0.01, or ***,###P < 0.001 by unpaired two-tail t-test.

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