Study design
In vitro anti-Aβ screening assays utilizing Thioflavin T (ThT) fluorescence were performed to identify small molecules that could dissociate aggregates of Aβ. Animal studies using 5XFAD and APP/PS1 transgenic AD mouse models were performed to assess amyloid burden reduction, amelioration of AD pathology, and cognitive improvement. BBB-specific in vitro parallel artificial membrane permeability assay (PAMPA) and ADME assessments were performed to assess druggability. Fragment-based peptide mapping assays and constrained docking simulations predicted target-ligand binding conformations.
Chemical syntheses of small molecules
Unless specified, all reagents and starting materials were purchased from commercial sources and used as received without purification. “Concentrated” refers to the removal of volatile solvents via distillation using a rotary evaporator. “Dried” refers to pouring onto, or passing through, anhydrous magnesium sulfate followed by filtration. Flash chromatography was performed using silica gel (230–400 mesh) with hexanes, ethyl acetate, and dichloromethane as the eluents. All reactions were monitored by thin-layer chromatography on 0.25-mm silica plates (F-254) visualized with UV light. Melting points were measured using a capillary melting point apparatus. 1H and 13C NMR spectra were recorded on a 400 MHz NMR spectrometer and were described as chemical shifts, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constant in hertz (Hz), and number of protons. HRMS were measured with an electrospray ionization (ESI) and Q-TOF mass analyzer.
General procedure for the synthesis of YIAD001
To a stirred solution of epichlorohydrin (1.3 equiv) in acetonitrile (7 mL) were added 2,4-di-tert-amylphenol (2.13 mmol, 1 equiv) and K2CO3 (2.0 equiv) at room temperature. After being stirred at reflux for 24 h (monitored by TLC), the reaction mixture was concentrated under reduced pressure, extracted with dichloromethane (3 × 5 mL), and washed with water. The organic layer was dried over MgSO4 and concentrated in vacuo to afford the crude product which was used for the next step without further purification. To a mixture of the crude residue 1 in water (7 mL) was added morpholine (1.5 equiv) at room temperature. After being stirred at 80°C for 16 h (monitored by TLC), the reaction mixture was extracted with ethyl acetate (3 × 5 mL) and washed with water. The organic layer was dried over MgSO4 and concentrated in vacuo to afford the crude product which was purified by silica gel column chromatography (hexane/EtOAc/dichloromethane = 3:1:1) to give 2 (692.6 mg, 86%). Then, 1M HCl in Et2O (2 mL) was added to a solution of 2 in Et2O (10 mL) on ice to give a salt form of 2, YIAD001. Characterization data of the newly synthesized small molecules are listed as follows:
4-(3-(2,4-Di-tert-pentylphenoxy)-2-hydroxypropyl)morpholin-4-ium chloride (YIAD001)
White solid, mp: 192.5–193.1°C; 1H NMR (400 MHz, CDCl3) δ 12.29 (s, 1H), 7.17 (s, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 7.6 Hz, 1H), 5.39 (s, 1H), 4.69–4.79 (m, 1H), 4.26–4.39 (m, 2H), 4.09–4.15 (m, 1H), 3.94–4.04 (m, 3H), 3.67–3.76 (m, 2H), 3.24–3.36 (m, 2H), 3.00–3.11 (m, 1H), 2.89–2.99 (m, 1H), 1.72–1.80 (m, 2H), 1.55–1.62 (m, 2H), 1.33 (s, 6H), 1.25 (s, 6H), 0.60–0.68 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 154.1, 141.8, 134.9, 126.1, 124.3, 111.4, 69.0, 64.1, 63.7, 63.6, 54.7, 52.9, 38.5, 37.4, 37.0, 33.8, 28.5, 28.1, 28.1, 9.5, 9.1; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for C23H39NNaO3 400.2822, found 400.2825.
1-(3-(2,4-Di-tert-pentylphenoxy)-2-hydroxypropyl)pyrrolidin-1-ium chloride (YIAD002)
White solid, mp: 179.8–180.7°C; 1H NMR (400 MHz, CDCl3) δ 11.78 (s, 1H), 7.16 (s, 1H), 7.09 (dd, J = 2.0, 8.4 Hz, 1H) 6.76 (d, J = 8.4 Hz, 1H) 5.41 (s, 1H), 4.54–4.63 (m, 1H), 4.09–4.14 (m, 1H), 3.99–4.05 (m, 1H), 3.93–3.99 (m, 2H), 3.35–3.41 (m, 2H), 2.93–30.1 (m, 1H), 2.83–2.91 (m, 1H), 2.21–2.30 (m, 2H), 2.05–2.14 (m, 2H), 1.71–1.81 (m, 2H), 1.55–1.62 (m, 2H), 1.32 (s, 6H), 1.24 (s, 6H), 0.59–0.66 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 154.2, 141.6, 134.8, 126.0, 124.3, 111.4, 69.1, 65.8, 61.1, 55.9, 54.4, 38.5, 37.4, 37.0, 33.7, 28.5, 28.1, 28.0, 23.3, 23.1, 9.5, 9.1; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C23H40NO2 362.3054, found 362.3061.
4-(2-Hydroxy-3-(4-methoxyphenoxy)propyl)morpholin-4-ium chloride (YIAD003)
White solid, mp: 160.2–160.5°C; 1H NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 6.85–6.99 (m, 4H), 5.98 (s, 1H), 4.42 (s, 1H), 3.83–4.01 (s, 6H), 3.73 (s, 3H), 3.44–3.60 (m, 2H), 3.10–3.36 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 154.0, 152.7, 116.0, 115.0, 71.1, 64.0, 63.5, 59.5, 55.8, 55.8; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C14H22NO4 268.1543, found 268.1551.
1-(2-Hydroxy-3-(4-methoxyphenoxy)propyl)piperidin-1-ium chloride (YIAD004)
Pale yellow solid, mp: 169.3–169.9°C; 1H NMR (400 MHz, CDCl3) δ 6.75–6.84 (m, 4H), 4.52–4.65 (m, 1H), 4.04–4.11 (m, 1H), 3.82–3.89 (m, 1H), 3.75 (s, 3H), 2.86–3.50 (m, 6H), 1.90–2.20 (m, 4H), 1.46–1.80 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 154.3, 152.0, 115.4, 114.7, 69.6, 64.2, 62.6, 55.7, 55.6, 22.8, 21.9; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for C15H23NNaO3 288.1570, found 288.1571.
4-(3-(4-Bromophenoxy)-2-hydroxypropyl)morpholin-4-ium chloride (YIAD005)
White solid, mp: 172.1–172.7°C; 1H NMR (400 MHz, CDCl3) δ 12.11 (s, 1H), 7.37 (d, J = 7.6 Hz, 2H), 6.76 (d, J = 7.6 Hz, 2H), 5.39 (s, 1H), 4.72 (s, 1H), 4.21–4.36 (m, 2H), 4.00–4.13 (m, 2H), 3.86–3.99 (m, 2H), 3.62–3.81 (m, 2H), 3.23–3.34 (m, 2H), 2.90–3.12 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 156.9, 132.5, 116.2, 113.9, 69.3, 63.9, 63.7, 62.5, 54.6, 52.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C13H19BrNO3 316.0543, found 316.0539.
1-(3-(4-Bromophenoxy)-2-hydroxypropyl)piperidin-1-ium chloride (YIAD006)
Pale yellow solid, mp: 178.1–178.6°C; 1H NMR (400 MHz, CDCl3) δ 11.28 (s, 1H), 7.36 (d, J = 7.6 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 5.59 (s, 1H), 4.58–4.70 (m, 1H), 4.06–4.14 (m, 1H), 3.84–3.93 (m, 1H), 3.64–3.76 (m, 2H), 3.11–3.32 (m, 2H), 2.65–2.90 (m, 2H), 2.17–2.40 (m, 2H), 1.86–1.94 (m, 2H), 1.75–1.86 (m, 1H), 1.35–1.53 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 157.0, 132.4, 116.2, 113.7, 69.2, 64.1, 62.5, 56.2, 54.3, 22.7, 21.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C14H21BrNO2 314.0750, found 314.0761.
1-(3-(4-Bromophenoxy)-2-hydroxypropyl)pyrrolidin-1-ium chloride (YIAD007)
Pale green solid, mp: 176.9–177.7°C; 1H NMR (400 MHz, CDCl3) δ 11.70 (s, 1H), 7.37 (d, J = 6.8 Hz, 2H), 6.77 (d, J = 5.6 Hz, 2H), 4.44–4.70 (m, 1H), 4.07–4.17 (m, 1H), 3.86–4.03 (m, 3H), 3.22–3.47 (m, 2H), 2.73–3.13 (m, 2H), 2.20–2.32 (m, 2H), 2.04–2.18 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 157.0, 132.5, 116.3, 113.8, 69.3, 65.5, 60.3, 56.0, 54.7, 23.4, 23.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C13H19BrNO2 300.0594, found 300.0606.
1-(3-(4-(Tert-butyl)phenoxy)-2-hydroxypropyl)piperidin-1-ium chloride (YIAD008)
White solid, mp: 201.7–202.2°C; 1H NMR (400 MHz, CDCl3) δ 11.32 (s, 1H), z7.29 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 4.55–4.70 (m, 1H), 4.09–4.18 (m, 1H), 3.88 (t, J = 8.8 Hz, 1H), 3.70 (t, J = 13.8 Hz, 2H), 3.21–3.31 (m, 1H), 3.09–3.20 (m, 1H), 2.66–2.90 (m, 2H), 2.20–2.40 (m, 2H), 1.81–1.95 (m, 3H), 1.39–1.50 (m, 1H), 1.28 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 155.6, 144.3, 126.4, 113.9, 68.8, 64.2, 63.0, 56.3, 54.2, 34.1, 31.5, 22.6, 21.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C18H30NO2 292.2271, found 292.2262.
4-(3-(4-(Tert-butyl)phenoxy)-2-hydroxypropyl)morpholin-4-ium chloride (YIAD009)
White solid, mp: 197.9–198.3°C; 1H NMR (400 MHz, CDCl3) δ 12.20 (s, 1H), 7.30 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.4 Hz, 2H), 5.29 (s, 1H), 4.80(s, 1H), 4.25–4.37 (m, 2H), 4.09–4.18 (m, 1H), 3.99 (t, J = 11.8 Hz, 2H), 3.87–3.94 (m, 1H), 3.69 (t, J = 11.0 Hz, 2H), 3.20–3.35 (m, 2H), 2.91–3.11 (m, 2H), 1.29 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 155.5, 144.4, 126.4, 113.9, 68.9, 64.0, 63.7, 63.7, 62.9, 54.5, 52.8, 34.1, 31.5; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H28NO3 294.2064, found 294.2066.
1-(3-(4-(Tert-butyl)phenoxy)-2-hydroxypropyl)pyrrolidin-1-ium chloride (YIAD010)
Pale yellow solid, mp: 139.9–140.4°C; 1H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 7.6 Hz, 2H), 6.82 (d, J = 7.6 Hz, 2H), 5.30 (s, 1H), 4.55 (s, 1H), 4.09–4.18 (m, 1H), 3.83–4.00 (m, 2H), 3.24–3.44 (m, 2H), 1.98–2.32 (m, 4H), 1.57–1.81 (m, 2H), 1.29 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 155.6, 144.3, 126.4, 113.9, 68.8, 65.6, 60.5, 34.1, 31.5, 23.3; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for C17H27NNaO2 300.1934, found 300.1939.
1-(2-Hydroxy-3-phenoxypropyl)piperidin-1-ium chloride (YIAD011)
Pale yellow solid, mp: 151.2–151.9°C; 1H NMR (400 MHz, CDCl3) δ 7.26–7.32 (m, 2H), 6.97 (t, J = 7.4 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 4.54–4.64 (m, 1H), 4.10–4.17 (m, 1H), 3.91 (t, J = 8.6 Hz, 1H), 2.94–3.37 (m, 6H), 1.95–2.11 (m, 4H), 1.51–1.76 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 158.0, 129.6, 121.4, 114.4, 68.9, 64.3, 62.6, 55.1, 23.0, 22.1; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C14H22NO2 236.1645, found 236.1624.
Preparation of Aβ peptides and tau fragments
Synthesized monomeric Aβ(1–42), DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA, Aβ(1–40), DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV, Aβ(pE3-42), pEFRHDSGYEV HHQKLVFFAE DVGSNKGAII LMVGGVVIA, Tau(244–274) repeat 1 (R1), QTAPVPMPDL KNVKSKIGST ENLKHQPGGG K, Tau(275–305) R2, VQIINKKLDL SNVQSKCGSK DNIKHVPGGG S, Tau(306–336) R3, VQIVYKPVDL SKVTSKCGSL GNIHHKPGGG Q, and Tau(337–368) R4, VEVKSEKLDF KDRVQSKIGS LDNITHVPGG GN, were acquired by Fmoc solid phase peptide synthesis as previously described [11]. Recombinant tau K18 fragments, QTAPVPMPDL KNVKSKIGST ENLKHQPGGG KVQIINKKLD LSNVQSKCGS KDNIKHVPGG GSVQIVYKPV DLSKVTSKCG SLGNIHHKPG GGQVEVKSEK LDFKDRVQSK IGSLDNITHV PGGGNKKIE, cloned from full-length human (hTau40) were purified from Escherichia coli BL21 (DE3) cells [12, 13].
ThT fluorescence assay
ThT fluorescence was used to assess the in vitro inhibition of Aβ(1–42) and disaggregation of Aβ(1–42), Aβ(1–40), Aβ(pE3-42), recombinant tau K18, and tau repeats R1, R2, R3, R4 by YIAD compounds. In house synthetic Aβ(1–42) was dissolved in DMSO (1 mM, 100% DMSO) and diluted to 20-fold in deionized water to make 50 μM Aβ(1–42) stocks (5% DMSO). YIAD compounds were first dissolved in DMSO (100 mM, 100% DMSO) and serially diluted with deionized water to make YIAD stocks with concentrations of 1, 10, 100, and 1000 μM (10% DMSO). For the Aβ(1–42) inhibition assay, monomeric Aβ(1–42) stocks and YIAD stocks were co-incubated at 37°C for 3 days. For Aβ(1–42) disaggregation assay, monomeric Aβ(1–42) stocks were preincubated at 37°C for three days to form Aβ(1–42) aggregates, and subsequently, YIAD stocks were added for an additional 3 days. For Aβ(1-40) disaggregation assays, Aβ(1–40) (50 μM, 5% DMSO) was preincubated at 37°C for 3 days to induce aggregation, and co-incubated with YIAD001 and YIAD002 for an additional 3 days. For Aβ(pE3-42) disaggregation assays, in house synthetic Aβ(pE3-42) (50 μM, 5% DMSO) was preincubated at 37°C for 1 day and co-incubated with YIAD002 for an additional 3 days. For tau K18 disaggregation assay, recombinant tau K18 fragments (35 μM) were dissolved in PBS with 0.1 mg/mL of heparin and 100 μM of DTT, then pre-aggregated at 37°C for 3 days. Then, YIAD stocks were added to aggregated K18 fragments for an additional 3 days. For tau repeat disaggregation assays, tau repeats (R1, R2, R3, R4) (35 μM) were dissolved in PBS with 0.1 mg/mL of heparin and 100 μM of DTT, then pre-aggregated at 37°C for 1 day. Then, YIAD002 was added to aggregated repeats for an additional 3 days. Prior to fluorescence intensity measurement, ThT solution (5 μM) was prepared by dissolving ThT, purchased from Sigma-Aldrich (MO, USA) in 50 mM glycine buffer (pH 8.5). To quantify the effect of inhibition and disaggregation, 25 μL of all samples and 75 μL of ThT solution were loaded into a half-area 96-well black plate in triplicate. Fluorescence intensity was measured at 450 (excitation) and 485 (emission) using the TECAN Infinite 200 PRO microplate reader.
SDS-PAGE with PICUP and silver staining
SDS-PAGE with photo-induced cross-linking of unmodified proteins (PICUP) and silver staining was used to evaluate the disaggregating effects of YIAD001 and YIAD002 by visualizing the size distributions and band intensity of Aβ(1–42). Samples were prepared under the same conditions as the Aβ(1–42) disaggregation ThT fluorescence assay. Amyloid proteins in the samples from the aforementioned disaggregation assay were subjected to cross-linking, initiated by irradiation (three exposures, each for 1 second) in the presence of tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(Bpy)) (1 mM) and 1 μL of ammonium persulfate (20 mM). Reactions were immediately quenched with β-mercaptoethanol and boiled for 5 min at 95°C. After cross-linking the samples, we separated amyloid species by SDS-PAGE on a 15% tris-tricine gel and performed silver staining according to the PlusOne Silver Staining kit protocol (GE Healthcare, USA).
BBB-PAMPA
Commercially available Double-Sink PAMPA (Pion® Double sink) assay was performed using a two compartment 96-well microtiter plate and protocols specified by the manufacturer. Progesterone and lidocaine were used as positive controls and ranitidine was used as a negative control. Stock solutions of controls and YIAD002 were dissolved in DMSO (10 mM) and subsequently diluted to 200-fold in pION buffer (pH 7.4) to 50 μM. The donor plate of the PAMPA sandwich was loaded with 50 μM solutions, and after wetting the artificial membrane with BBB lipid solution, the acceptor plate was loaded with acceptor sink buffer. Then, the acceptor plate was placed on the donor plate and incubated at 25°C for 4 h. Compound concentration in the reference, donor, and acceptor plates were measured on a UV plate.
CYP450 inhibition assay
Incubation mixtures for Cytochrome P450s (CYPs) inhibition assay contained human liver microsomes (0.25 mg/ml), 0.1 M phosphate buffer (pH 7.4), 10 μM of YIAD002, and an enzyme substrate cocktail (phenacetin 50 μM, diclofenac 10 μM, S-mephenytoin 100 μM, dextromethorphan 5 μM, and midazolam 2.5 μM). The mixture was pre-incubated at 37°C for 5 min and 1 mM NAPDH was added to initiate reactions. After 15 min of incubation, acetonitrile with terfenadine was added to stop reactions and microsomal solutions were centrifuged at 14,000 rpm at 4°C for 5 min. For LC-MS/MS analysis, supernatants were injected on a Kinetex C18 column (2.1 × 100 mm, 2.6 μm particle size, Phenomenex, USA). The composition of the mobile phase was 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B).
Plasma stability
To assess the plasma stability of YIAD002, YIAD002 stock was dissolved at a concentration of 40 μM in DMSO. 5 μL of YIAD002 stock was added to 195 μL of human plasma or 195 μL of rat plasma and incubated at 37°C for the following time points: 0, 30, and 120 min. At the end of incubation, acetonitrile with chlorpropamide was added to stop reactions. The solutions were centrifuged at 14,000 rpm at 4°C for 5 min and the supernatant was analyzed with LC-MS/MS. High performance liquid chromatography was carried out on a Kinetex C18 column (2.1 × 100 mm, 2.6 μm particle size, Phenomenex, USA). The composition of the mobile phase was 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B).
Microsomal stability
To evaluate the rate of drug metabolism, we tested YIAD002 stability in liver microsomes of human, rat, and mouse. Microsomal concentration was 0.5 mg/ml in 0.1 M phosphate buffer (pH 7.4). 1 μM of YIAD002 was added to microsome solutions and was prewarmed at 37°C for 5 min. NADPH-regenerating solution was added to microsomes and incubated at 37°C for 30 min. At the end of incubation, acetonitrile with chlorpropamide was added to stop reactions and microsomal solutions were centrifuged at 14,000 rpm at 4°C for 5 min. For LC-MS/MS analysis, supernatants were injected on a Kinetex C18 column (2.1 × 100 mm, 2.6 μm particle size, Phenomenex, USA). The composition of the mobile phase was 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B).
Animals
5XFAD mice (strain name: B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/-Mmjax) expressing the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations in APP and the M146L and L286V mutations in PSEN1) and wild-type mice (C57BL/6 x SJL) were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Heterozygous transgenic mice were maintained through cross-breeding with the wild-type mice, and the genotype of all mice was confirmed via PCR analysis of tail DNA following the standard PCR condition from the Jackson Laboratory. All mice were housed in a laboratory animal room and were maintained under controlled temperature, humidity, and an alternating 12-h light–dark cycle. Access to food and water were available ad libitum. Protocols were approved by Institutional Animal Care and Use Committee (IACUC) of the Yonsei Laboratory Animal Research Center.
APP/PS1 (strain name: B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax) expressing chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed in a laboratory animal room and were maintained under controlled temperature, humidity, and an alternating 12-h light–dark cycle. Access to food and water were available ad libitum. Protocols were approved by the IACUC of NDIC Inc. (Kyunggido, Korea; approval No. P191114).
Oral administration of YIAD001 and YIAD002 to 5XFAD mice
To test the in vivo efficacy of YIAD001 and YIAD002, female 5XFAD mice (n = 25) and female wildtype mice (n = 7) were assessed at the age of 4.5 months. YIAD compounds were dissolved in DMSO and diluted in drinking water to a final concentration of 50 mg/kg/day (5% DMSO, 5% Tween) for oral administration. The three compounds, YIAD001, YIAD002, and YIAD003, were freely administered via drinking water to 4.5-month-old 5XFAD mice (n = 6 for each group) for 5 weeks. As a control, 5% DMSO, 5% Tween in drinking water was provided ad libitum to littermate 5XFAD mice (n = 7) and wildtype mice (n = 7) for 5 weeks.
After the administration period, the mice were deeply anaesthetized with 4% avertin through intraperitoneal injection and transcardially perfused with 0.9% saline. Subsequently, the mice were sacrificed for brain excision. Each collected brain was divided through the mid-sagittal section; the hippocampal and cortical regions were dissected from one half and stored at −80°C for subsequent brain lysates, while the other half was immersed in 4% paraformaldehyde (pH 7.4) for 24 h and 30% sucrose for 48 h at 4°C and subsequently deep-froze at −80°C for immunohistochemistry.
Oral administration of YIAD002 to APP/PS1 mice
To test whether YIAD002 could reduce amyloid plaques in another AD mouse model, APP/PS1 transgenic mice (n = 24) and wildtype mice (n = 5) were assessed at the age of 7.5 months. YIAD002 was dissolved in PBS (0.5% methylcellulose) at two different concentrations (10 and 30 mg/kg/day) and administrated daily to APP/PS1 mice (n = 8 for each group) via oral gavage for 8 weeks. As a control, vehicle solutions were orally administrated to littermate APP/PS1 mice (n = 8) and wildtype mice (n = 5).
Morris water maze
The Morris water maze was used to evaluate whether YIAD002 could improve cognitive function in APP/PS1 mice during the tenth week of administration. A circular stainless pool with a radius of 90 cm and height of 50 cm with water kept at 22±1°C. Nontoxic opaque white paint was used to hide a platform with a radius of 9 cm hidden 1 cm beneath the surface. The training phase consisted of 5 days, in which the mice were given 60 s to find the platform. On circumstances where a mouse could not reach the platform within 60 s, it was placed on the platform where it had to remain for 10 s. The probe test took place on the sixth day, in which the platform was removed and the mice swam freely for 60 s. Swimming trajectories were recorded and analyzed using SMART VIDEO TRACKING Software (Panlab, USA). To assess cognitive improvement in YIAD002-treated mice groups, latency to target (seconds) and the number of target crossings were compared to vehicle-treated APP/PS1 mice. After the probe trial, the mice were sacrificed and the collected brains were fixed in formalin.
Immunohistochemical staining
Coronal hippocampal sections of 5XFAD mice brains (35 μm) were cut with a Cryostat (Leica, CM1860) and mounted onto glass slides. Antigen retrieval was performed by submerging slides in 1% SDS and unspecific binding was blocked through incubation in 20% horse serum in PBS for 1 h at RT. Aβ deposits were visualized through immunolabeling by anti-6E10 antibody (1:200 in PBS with 5% horse serum, Biolegend 803003) and detection by fluorescent secondary antibody (1:200 in PBS, IgG Alexa 488). Brain sections were additionally stained with Hoechst 33342. Fluorescent images of the brain sections were taken on a Leica DM2500 fluorescence microscope. Amyloid plaques were quantified using Image-J software (NIH).
Formalin-fixed APP/PS1 mice brains were infiltrated with paraffin using a tissue processor (LEICA ASP300S, Germany). Blocks of tissues were produced with a tissue embedding center and sectioned into thin slices (4 μm), which were mounted onto glass slides. To prepare for 6E10 staining, slides were deparaffinized, rehydrated, and blocked with 0.03% H2O2 to remove endogenous peroxidase. For antigen retrieval, slides were boiled in Tris/EDTA solution (pH 9.0) using a pressure cooker. Unspecific binding was blocked through incubation in 4% BSA at RT for 30 min. Slides were immunolabeled with anti-6E10 antibody (1:2000, Novus Biologicals NBP2-62566) at RT for 1 h and HRP-labeled secondary antibody (Dako K4003) for 30 min and subsequently stained with DAB (3,3′-Diaminobenzidine) and counterstained with Mayer’s hematoxylin. Stained slides were scanned using ZEISS Axio scan.Z1 microscope. Amyloid plaques were quantified using Image-J software (NIH).
Lysis of 5XFAD mouse brain tissues
Isolated cortical and hippocampal tissue samples were homogenized in ice-cold RIPA buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail. Lysed tissues were incubated in ice for 30 min and subsequently centrifuged at 14,000 rpm at 4°C for 30 min. The supernatants of the brain lysates were collected and soluble protein concentrations were quantified with the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA).
Dot blot assay
To analyze total and oligomeric amyloid levels, protein samples (12 μg in 2 μL) and prepared in vitro disaggregation assay samples (2 μL) were directly blotted on nitrocellulose membranes. After blocking with 5% skim milk in TBS-T, the membranes were probed with anti-6E10 antibody (1:1,000, Biolegend 803003) and anti-A11 antibody (1:2000, Invitrogen AHB0052) at 4°C overnight. HRP-conjugated goat anti-mouse antibody (1:10,000, Bethyl Laboratories A90-116P) and anti-rabbit secondary antibody (1:10,000, Bethyl Laboratories A120-101P) were used for 6E10 and A11, respectively. Proteins were detected using an ECL kit (Thermo Fisher Scientific, USA). Membranes were washed with TBS-T in between steps, three times for 10 min.
Molecular weight cut-off filtration of soluble lysates
To exclusively investigate soluble Aβ levels in cortical lysates, guanidinium hydrochloride (GdnHCl) in a lysis buffer (5.0 M GdnHCl, 50 mM Tris-HCl, pH 8.0) was utilized to solubilize and denature endogenous Aβ aggregates prior to filtration. The homogenates were incubated with agitation for 3 h at RT. The incubated samples were then quantified to prepare protein samples (10 μg in 4 μL) and were filtered through a 100 kDa molecular weight cut-off (MWCO) filter, centrifuged at 14,000 rpm at RT for 30 min, to remove APP. For subsequent dot blot assay, total 8 μL of the filtrate was spotted onto a nitrocellulose membrane and probed with anti-6E10 antibody (1:1000, Biolegend 803003).
Western blot
Western blot analysis was performed to assess YIAD002-induced alterations of protein biomarkers in 5XFAD mice brain lysates. Twenty micrograms of lysate samples were loaded onto 12% tris-tricine gels, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked by 5% skim milk or 5% BSA in TBS-T. The following primary antibodies were used for immunoblotting: anti-6E10 for APP (1:1000, Biolegend 803003), anti-AT8 (1:1000, Invitrogen MN1020), anti-Tau (1:1000, Santa Cruz SC390476), anti-ionized calcium-binding adapter molecule (Iba1) (1:1000, CST, 17198S), anti-glial fibrillar acidic protein (GFAP) (1:2000, Milipore AB5541), anti-postsynaptic density protein 95 (PSD95) (1:1000, CST 3450S), anti-synaptophysin (1:1000, Milipore MAB5258), and anti-β-Actin (1:10,000, Milipore MAB1501). Membranes were incubated with primary antibodies at 4°C overnight and probed with HRP-conjugated IgG antibodies (1:10,000) in RT for an hour. Proteins were detected using an ECL kit (Thermo Fisher Scientific, USA). Membranes were washed with TBS-T in between steps, three times for 10 min.
Mapping assay of Aβ(1–42)
Aβ fragments made up of six overlapping amino acid residues and a cysteine residue at the C-terminal were acquired by Fmoc solid phase peptide synthesis as previously described [11]. Full length Aβ(1–42) with an additional cysteine residue at the C-terminal was used for comparison. The peptides were dissolved in DMSO (1.5 M) and diluted in binding buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 10 mM EDTA; pH 7.2) to prepare 50 μg/mL peptide solutions. To immobilize the Aβ fragments and monomers to maleimide-activated microplates, 100 μL of peptide solutions (50 μg/mL) was added and incubated at RT for 2 h while shaking. Cysteine solution (10 μg/mL with binding buffer) was added to cap unreacted maleimide groups. Flamma 552-conjugated Aβ(1–42) was prepared in deionized water (10% DMSO) (10 μM) and incubated in the wells at 37°C for 2 h. To define the fluorescence baseline for each well, fluorescence intensity was measured at 555 (excitation) and 580 (emission). YIAD002 (500 μM in binding buffer, 7.5% DMSO) was added to each well and incubated at RT for 24 h while shaking. Fluorescence intensity was remeasured at 555 (excitation) and 580 (emission) and baseline fluorescence was subtracted to quantify the change in fluorescence intensity caused by YIAD002. The plate was washed with 200 μL of wash buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 0.05% Tween®-20 Detergent; pH 7.2) per well three times in between steps.
Docking models against Aβ and tau
The three-dimensional conformers for YIAD002 were prepared and separately docked to the multiple Aβ and tau oligomer structures (2BEG, 5KK3, 2NAO, 2MXU for Aβ and 2V5B, 5V5C, 2ON9 for tau) representing structural polymorphism (Table S1). The conformer generation procedure resulted in 129 YIAD002 conformers. For each oligomer structure, the binding site was initially predicted by global docking search with PatchDock. In Aβ, the binding site was confined near the Aβ(16–21) and Aβ(32–37) fragments in edge strands of β-sheets. The top 50 docking conformations for each oligomer structure were used to estimate potential binding sites for the subsequent local docking refinement by Autodock vina. Finally, all docking conformations were pooled together for Aβ and tau, respectively, and the docking model that showed the lowest binding energy was selected for the structural analysis.
Statistical analysis
Graphs were obtained with GraphPad Prism 7 and statistical analyses were performed with Student’s unpaired t-tests or one-way analysis of variance followed by Bonferroni’s post hoc comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; other comparisons were not significant). The error bars represent the SEMs.