Introduction

Dementia is a chronic and progressive syndrome that primarily affects cognitive function. Alzheimer’s disease (AD) is the most common form of dementia, accounting for more than 60% of all dementia cases. According to the World Health Organization, as the worldwide elderly population continues to increase, approximately 10 million new dementia cases will occur every year. Although the pathology of AD has been extensively studied and decades of data are available, the crucial trigger remains unknown [1]. A reasonable hypothesis is that the amyloid cascade underlies AD pathogenesis [2]. This hypothesis suggests that amyloid-plaques in the brain cause neuronal cell loss, leading to cognitive impairments. It is widely known that these plaques consist of amyloid-beta (Aβ) aggregates, thus suggesting that AD could be treated by inhibiting Aβ aggregation. We have isolated Aβ aggregation-inhibiting seed compounds from natural resources [3,4,5,6,7]. Using a Th-T assay-based screening approach, an extract derived from a fungus in our collection, Aspergillus sp. MPUC239, inhibited Aβ aggregation. Here, we report the isolation and structural analysis of compounds 1–11 from Aspergillus sp. MPUC239, their inhibitory effects on Aβ aggregation and β-site APP Cleaving Enzyme1 (BACE1) activity, their protective effect against Aβ toxicity, and the associated structure–activity relationships.

Results and discussion

To identify potent inhibitors useful in treating for AD, Th-T assays were performed using 70 extracts derived from 39 types of marine-derived fungi. Some of the extracts examined exhibited Aβ aggregation inhibitory activity, and an active EtOAc extract derived from Aspergillus sp. MPUC239 suppressed Aβ aggregation by 33.4% compared with the control at 10 μg/mL, whereas a CHCl3 extract showed less-potent activity of 59.9% inhibition at 10 μg/mL. The EtOAc extract derived from Aspergillus sp. MPUC239 was divided into two parts (7.2 g and 29.0 g) and separated. First, the EtOAc extract (7.2 g) was fractionated by silica gel column chromatography using n-hexane followed by CHCl3-MeOH to obtain five fractions (Fr. A–E), which were tested for Aβ aggregation suppression at 10 μg/mL (Fr. A, 4.4 g, 63.0%; Fr. B, 806.5 mg, 56.0%; Fr. C, 197.7 mg, 26.7%; Fr. D, 981.2 mg, 10.2%; Fr. E, 370.9 mg, 20.8%). Fr. C and its sub-fractions were further fractionated by silica gel column chromatography and octadecylsilyl column chromatography using bioactive-guided separation to afford compound 1. The rest of the EtOAc ext. (29.0 g) was fractionated by silica gel column chromatography (Si. C. C.) using n-hexane-CHCl3-MeOH to obtain three fractions (Fr. a–c), which were tested for Aβ aggregation suppression at 10 μg/mL (Fr. a; 8.7 g, 63.0%; Fr. b; 15.4 g, 56.0%; Fr. c; 2.3 g, 26.7%). Fr. b was fractionated and isolated using bioactive-guided separation to afford six compounds (2–7). The CHCl3 extract was also separated in the same manner, yielding four compounds (8–11) (Fig. 1).

Fig. 1
figure 1

Structures of compounds 1–11 from MPUC239 extracts

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Compound 1 was obtained as a yellow, amorphous substance, and its molecular formula was elucidated as C15H14O6 based on an HREIMS ion peak at m/z 290.0792 [M]+ (calcd for C15H14O6, 290.0790), which suggested 9 degrees of unsaturation. The 1H-NMR spectra exhibited a hydroxyl group [δH 14.24 (1H, s)], suggesting the presence of a hydrogen bond, three aromatic protons [δH 6.35 (1H, d, 0.8), 6.37 (1H, s) and 6.50 (1H, d, 0.8)], a methoxy signal [δH 3.87 (3H, s)], two methylene signals [δH 2.79 (1H, d, 1.7) and 3.07 (1H, d, 1.7)], and a methyl proton [δH 1.69 (3H, s)]. The 13C-NMR spectra revealed 10 aromatic carbon signals (δC 96.6, 101.0, 101.5, 102.5, 105.2, 142.9, 153.4, 160.7, 161.4, and 164.2), suggesting a naphthalene core in the structure. In addition to sp2 carbon signals, an acetal carbon signal (δC 100.1), a methyl carbon signal (δC 27.6), a methine carbon signal (δC 47.6), a methoxy carbon signal (δC 55.7), and a carbonyl carbon signal (δC 197.5) were revealed by the 13C-NMR spectra. These data and comprehensive 2D-NMR data indicated that compound 1 has a naphtho-γ-pyrone skeleton. There are two types of naphtho-γ-pyrone skeletons (known as linear type and angular type) [8], and compound 1 was deduced as the linear type based on the presence of a proton signal at δH 14.24 (1H, s). Thus, these data were compared with published data, and compound 1 was identified as fonsecin (1, Fig. 1) [8].

Other compounds (2–11) were also identified: TMC-256A1 (2) [9], dianhydro-aurasuperone C (3) [10], fonscecinone A (4) [10], asperpyrone D (5) [11], RF3192C (6) [12], asperpyrone A (7) [13], fonsecin B (8) [14], rubrofusarin B (9) [14], aurasperone F (10) [15], and aurasperone E (11) [16], according to previous literature. The 1D-NMR spectra for these compounds are shown in Table S1. ECD spectra of 3, 4, 5, 7, 10, and 11 were also measured to determine the absolute configurations of bis (naphtho-γ-pyrone), and all except 5 showed a negative cotton effect, indicating that compounds 3, 4, 7, 10, and 11 are (R) configuration (Figs. S1–S6) [17]. Compound 5 did not clearly show a cotton effect, so its absolute configuration remained unclear. These naphtho-γ-pyrones have been reported to show various biological activities such as anti-microbial, anti-fungal, anti-oxidant, tyrosinase inhibition, IL-4 signal transduction inhibition [18].

All of the compounds (1–11) were evaluated for their effect on Aβ aggregation, and all compounds except 11 exhibited a dose-dependent inhibition, from which IC50 values were calculated (Table 1). Compound 11 reduced Aβ aggregation by 41.9 ± 10.4% at 50 μM, 27.0 ± 11.4% at 25 μM, and 65.1 ± 9.5% at 12.5 μM as final concentrations compared with the control (100%). In terms of the structure–activity relationship (SAR), a hydroxy group at position 8 could be quite important for the activity in the case of naphtho-γ-pyrone monomers, because 1 and 2 showed stronger activity than the methylated compounds 8 and 9. In the case of dimers, the SARs remained unclear, but the methoxy group at position 8 seems to be important for the activity of compounds 4 and 7. For compounds 10 and 11, a hydroxy group at same position appears to be important, thus, further investigation is needed.

Table 1 Inhibition of Aβ aggregation and BACE1 activity by compounds 1–11
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The protective effect of the compounds against Aβ toxicity to SH-SY5Y cells was also studied. Compounds 6 at 20 μM and 10 at 10 μM significantly alleviated the toxicity of Aβ in the WST-8 assay (Figs. S7 and S8 and Tables S2 and S3). Although the SARs remain unclear, the difference between 3 and 10 suggests that the hydroxy group at position 2 is important for the activity in the case of the bis naphtho-γ-pyrones.

BACE1, an enzyme related to Aβ production from APP, also contributes to AD onset, and therefore its inhibitory activity was also studied. Although all compounds showed less BACE1 inhibitory activity than inhibitor IV as a positive control in the BACE1 FRET assay, compounds 4, 5, 7, 10, and 11 inhibited BACE1 activity with IC50 values of 14.9, 70.0, 36.5, 28.0, and 72.8 μM, respectively (Table 1). With regard to the SAR, the methoxy group at position 8 could affect the inhibitory activity of 4, 7, 10, and 11. Comparing the BACE1 inhibitory activity between 3 and 10, the hydration of the double bond at position 2 could reduce the inhibitory activity. In the case of the monomers, the SARs were unclear due to the low inhibitory activity of the compounds. Naphtho-γ-pyrones may require a dimeric structure to exhibit BACE1 inhibitory activity.

In summary, we carried out Th-T assays of extracts from our fungus collection to identify compounds exhibiting potent Aβ aggregation inhibitory activity as possible AD treatment agents. An extract derived from MPUC239 inhibited Aβ aggregation, and further assay-guided fractionations were conducted to afford eleven naphtho-γ-pyrone compounds (1–11). Compounds 1–7 were isolated from an EtOAc extract, and the other compounds were isolated from a CHCl3 extract. The IC50 values of these compounds in Thioflavin-T (Th-T) assays ranged from 2.8 (2) to 65.9 μM (8), and SAR analyses suggested that a hydroxy group at position 8 is important for the inhibitory activity in the case of the naphtho-γ-pyrone monomers, especially 1 and 8. Compounds 6 and 10 exhibited a protective effect on SH-SY5Y cells against Aβ toxicity. These results suggest that the hydroxy group at position 2 is important for the activity in the case of the bis naphtho-γ-pyrones. The bis naphtho-γ-pyrones 4, 5, 7, 10, and 11 exhibited inhibitory activity, and SAR analyses suggested that not only could the methoxy group at position 8 affect the inhibitory activity, but also that the dimeric structure could be important for their inhibitory activity. The results of the SAR analyses were, however, insufficient for understanding the mechanism of the inhibitory activity of the naphtho-γ-pyrones; therefore, further studies are needed.

Experimental

General experimental procedures

IR spectra were measured using a Thermo Scientific FT-IR Nicolet iS5 spectrometer (ATR). UV spectra were measured using a Thermo Scientific UV–Vis GENESYS 10S spectrophotometer. ECD spectra were measured with a Jasco J-820 circular dichroism spectropolarimeter (JASCO, Tokyo, Japan). 1H- and 13C-NMR spectra were recorded using a JNM-AL 400 MHz and JNM-ECZ 400 MHz spectrometer (JEOL, Tokyo, Japan). HREIMS and HRFABMS spectra were obtained using a JEOL JMS-700 spectrometer (JEOL). Column chromatography was carried out with silica gel 60 N (63–210 μm, Kanto Chemical, Tokyo, Japan) and ODS silica gel YMC-GEL ODS-A (YMC, Kyoto, Japan). MPLC was performed using a Pure C-850 FlashPrep (BUCHI, Flawil, Switzerland) and a series of Flash Cartridges (BUCHI, Flawil, Switzerland). Semi-preparative HPLC was conducted using a Jasco PU-2080 Plus pump (JASCO) and a Jasco UV-2075 Plus UV/VIS detector at 220 or 254 nm (JASCO). Inhibition of Aβ aggregation was assayed using a Synergy HTX Multi-Mode Reader (BioTek, Winooski, VT, USA), Aβ40 peptide (Peptide Institute, Osaka, Japan), thioflavin-T (FUJIFILM Wako, Osaka, Japan), 25% ammonia solution (FUJIFILM Wako), and dimethyl sulfoxide (FUJIFILM Wako).

Fungal material

The fungus was collected from an undefined seaweed off the coast of Shamijima Island, Sakaide City, Kagawa Prefecture, Japan, in August 2006. The isolate was identified as Aspergillus sp. based on analysis of the DNA sequence of the ITS region of the rRNA gene. The ITS DNA sequence of the fungus was deposited in the DNA Data Bank of Japan under accession number LC695353. The voucher specimen was deposited at the Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, as MPUC239.

Fermentation

Preliminary fermentation was carried out for 7 days in test tubes, each of which contained a slant consisting of glucose (Yoshida Pharmaceutical Co. Ltd., Tokyo, Japan), 200 mg; peptone (Kyokuto Pharmaceutical Co., Ltd., Tokyo, Japan), 100 mg; Bacto yeast extract (BD Biosciences, Franklin Lakes, New Jersey, USA), 50 mg; Bacto agar (BD Biosciences), 200 mg; and artificial seawater (Instant Ocean, Aquarium Systems), 10 mL. The cultured microbes were then suspended in artificial seawater and inoculated into Roux flasks, each containing 130 g of wheat pretreated by steeping in artificial seawater for 30 min and then filtered and 10 mL of artificial seawater. Fermentation was conducted statically at 26 °C in the dark for 14 days and cultivation flasks were manually shaken for 5 s per a day.

Extraction and isolation

Fermented wheat was extracted with CHCl3 and then EtOAc, yielding CHCl3 ext. (41.9 g) and EtOAc ext. (36.2 g), respectively. Inhibitory activity tests against Aβ aggregation using each extract at 10 μg/mL final concentration revealed Aβ aggregation rates of 59.9% (CHCl3 ext.) and 33.4% (EtOAc ext.). As the EtOAc ext. showed stronger inhibitory activity against Aβ aggregation (33.4%) than the CHCl3 extract (59.9%), a portion of the EtOAc ext. (7.2 g, 33.4%) was fractionated via Si. C. C. using n-hexane-CHCl3-MeOH (1:0:0 → 0:100:1 → 0:75:1 → 0:50:1 → 0:25:1 → 0:10:1 → 0:5:1 → 0:3:1 → 0:1:1 → 0:0:1, stepwise gradient) to obtain five fractions (Fr. A–E), which were then tested for inhibitory activity against Aβ aggregation (Fr. A, 4.4 g, 63.0%; Fr. B, 806.5 mg, 56.0%; Fr. C, 197.7 mg, 26.7%; Fr. D, 981.2 mg, 10.2%; Fr. E, 370.9 mg, 20.8%). Fr. C (197.7 mg, 26.7%) was further fractionated via octadecylsilyl silica gel column chromatography (ODS C. C.) using MeCN-H2O-CHCl3 (3:7:0 → 2:3:0 → 1:1:0 → 1:0:0 → 0:0:1, stepwise gradient) to give five fractions (Fr. C1–C5). Fr. C2 (27.6 mg) was further fractionated via ODS C. C. using MeOH-H2O (1:1 → 1:0, stepwise gradient) to afford fonsecin (1, 19.7 mg).

The remainder of the EtOAc ext. (29.0 g, 33.4%) was fractionated via Si. C. C. using n-hexane-CHCl3-MeOH (1:0:0 → 0:100:1 → 0:75:1 → 0:25:1 → 0:10:1 → 0:5:1 → 0:3:1 → 0:1:1 → 0:0:1, stepwise gradient) to obtain three fractions (Fr. a–c), which were then tested for Aβ aggregation inhibitory activity (Fr. a; 8.7 g, 63.0%; Fr. b; 15.4 g, 56.0%; Fr. c; 2.3 g, 26.7%). Fr. b (15.4 g, 56.0%) was fractionated via Si. C. C., ODS C. C., MPLC, and semi-preparative HPLC to afford TMC-256A1 (2, 6.1 mg), dianhydro-aurasperone C (3, 9.5 mg), fonsecinone A (4, 2.0 mg), and asperpyrone D (5, 1.4 mg), RF3192 (6, 64.8 mg), and asperpyrone A (7, 1.6 mg).

The CHCl3 ext. (41.6 g, 59.9%) was subjected to Si. C. C. using n-hexane-acetone-MeOH (1:0:0 → 15:1:0 → 10:1:0 → 7:1:0 → 5:1:0 → 4:1:0 → 3:1:0 → 2:1:0 → 1:1:0 → 1:2:0 → 1:3:0 → 0:1:0 → 0:0:1, stepwise gradient) to obtain eight fractions (Fr. CA, 31.2 g, 78.0%; Fr. CB, 446.3 mg, 21.5%; Fr. CC, 988.8 mg, 50.4%; Fr. CD, 842.5 mg, 20.0%; Fr. CE, 967.2 mg, 40.3%; Fr. CF, 3.4 g, 36.2%; Fr. CG, 718.2 mg, 39.3%; Fr. CH, 3.5 g, 26.4%). Fr. CD (842.5 mg, 20.0%) was fractionated via MPLC and semi-preparative HPLC to afford fonsecin B (8, 8.8 mg) and rubrofusarin B (9, 0.4 mg). Similarly, Fr. CF (3.4 g) and Fr. CE (967.2 mg) gave aurasperone F (10, 9.6 mg) and aurasperone E (11, 1.9 mg), respectively. The details of isolation procedures are described in supporting information.

Fonsecin (1): yellow amorphous; UV λmax (EtOH) nm (log ε): 287 (3.85); IR (ATR) cm−1: 3369, 2927, 1738, 1433, 1260, 1070; 1H- and 13C-NMR (Acetone-d6, 400 and 100 MHz), see Table S1; HREI-MS m/z: 290.0792 [M]+ (Calcd for C15H14O6: 290.0790).

TMC-256A1 (2): yellow amorphous; UV λmax (EtOH) nm (log ε): 277 (3.84); IR (ATR) cm−1: 3189, 2925, 2852, 1652, 1377, 1322, 1199; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; HREI-MS m/z: 272.0686 [M]+ (Calcd for C15H12O5: 272.0685).

Dianhydro-aurasperone C (3): yellow amorphous; [α]D 23 + 270.0 (c 0.08, EtOH); UV λmax (MeOH) nm (log ε): 280 (3.86), 224 (3.56); ECD (MeOH) λmax nm (∆ε): 286 (− 98.1), 271 (+ 110.9); IR (ATR) cm−1: 3350, 2963, 2160, 2032, 1615, 1411, 1261; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; HREI-MS m/z: 556.1372 [M]+ (Calcd for C31H24O10: 556.1369).

Fonsecinone A (4): yellow amorphous; [α]D 23 + 41.1 (c 0.01, MeOH); UV λmax (MeOH) nm (log ε): 281 (3.91), 248 (3.84), 225 (3.73); IR (ATR) cm−1: 2931, 2537, 2160, 2031, 1614, 1417; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; positive HRFAB-MS m/z: 571.1606 [M + H]+ (Calcd for C32H27O10: 571.1604).

Asperpyrone D (5): yellow amorphous; [α]D 23 + 25.4 (c 0.02, MeOH); UV λmax (MeOH) nm (log ε): 278 (3.71), 228 (3.55); ECD (MeOH) λmax nm (∆ε): 293 (− 31.7), 275 (+ 62.9); IR (ATR) cm−1: 2534, 2160, 2031; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; positive HRFAB-MS m/z: 557.1451 [M + H]+ (Calcd for C31H25O10: 557.1448).

RF3192C (6): orange amorphous; UV λmax (EtOH) nm (log ε): 303 (3.40), 277 (3.17), 204 (3.51); IR (ATR) cm−1: 3549, 3345, 3087, 2962, 2598, 2160, 2033, 1600, 1259; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; negative HRESI-MS m/z: 379.0449 [M − H]− (Calcd for C20H11O8: 379.0454).

Asperpyrone A (7): yellow amorphous; [α]D 23 + 29.3 (c 0.1, MeOH); UV λmax (EtOH) nm (log ε): 278 (3.72), 256 (3.57), 228 (3.55); ECD (MeOH) λmax nm (∆ε): 287 (− 31.1), 260 (+ 38.3); IR (ATR) cm−1: 2931, 2537, 2160; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; positive HRFAB-MS m/z: 557.1454 [M + H]+ (Calcd for C31H25O10: 557.1448).

Fonsecin B (8): yellow amorphous; UV λmax (EtOH) nm (log ε): 278 (4.08), 230 (4.07); IR (ATR) cm−1: 3317, 2160, 2032, 1594; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; positive HRFAB-MS m/z: 305.1028 [M + H]+ (Calcd for C16H17O6: 305.1025).

Rubrofusarin B (9): yellow amorphous; UV λmax (EtOH) nm (log ε): 275 (4.66); IR (ATR) cm−1: 2540, 2159, 2032, 1617; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; positive HRFAB-MS m/z: 287.0917 [M + H]+ (Calcd for C16H15O5: 287.0919).

Aurasperone F (10): yellow amorphous; [α]D 23 + 21.1 (c 0.04, EtOH); UV λmax (EtOH) nm (log ε): 280 (3.87), 224 (3.58); ECD (EtOH) λmax nm (∆ε): 286 (− 3.2), 271 (+ 3.5); IR (ATR) cm−1: 3350, 2963, 2160, 2032, 1615, 1411, 1261; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; positive HRFAB-MS m/z: 575.1559 [M + H]+ (Calcd for C31H27O11: 575.1553).

Aurasperone E (11): yellow amorphous; [α]D 21 − 12.0 (c 0.06, EtOH); UV λmax (EtOH) nm (log ε): 281 (5.10), 226 (4.83); ECD (EtOH) λmax nm (∆ε): 287 (− 2.9), 270 (+ 2.4); IR (ATR) cm−1: 3350, 2963, 2160, 2032, 1615, 1411, 1261; 1H- and 13C-NMR (DMSO-d6, 400 and 100 MHz), see Table S1; positive HRFAB-MS m/z: 589.1713 [M + H]+ (Calcd for C32H29O11: 589.1710).

Thioflavin-T (Th-T) assay

The activity of the compounds was expressed as Aβ aggregation rate (%) in comparison with the control group. A portion of each fraction was used for assay-guided fractionation. The assay was conducted at final concentration of 10 μg/mL with each fraction. Th-T assays were carried out according to a previous report [6]. Briefly, 80 μL of phosphate buffer containing 50 mM NaCl at pH 7.4 was added to a 500 μL test tube, followed by the addition of 10 μL of DMSO containing test sample to the phosphate buffer solution. Finally, 10 μL of 250 μM Aβ40 peptide solution dissolved in 0.02% ammonium solution was added to the phosphate buffer and DMSO mixture. All of the above procedures were conducted on ice. The test tubes were then incubated at 37 °C for 24 h, followed by cooling on ice for 5 min. Next, 300 μL of 50 mM Gly-NaOH buffer containing 5 μM Th-T was added to each test tube containing incubated and cooled solution, and then the solution was transferred to a black-bottom 96-well plate at 100 μL/well. After mixing for 30 min, fluorescence intensity was measured at an excitation wavelength of 440 nm and emission wavelength of 485 nm. The aggregation rate (%) was calculated using the following formula: [(S − B)/(C − B) × 100] (S, fluorescence of Th-T solution incubated with Aβ40 and sample; C, fluorescence of Th-T solution incubated with Aβ40 and DMSO; B, fluorescence of Th-T solution not incubated with Aβ40 and DMSO). Myricetin was used as a positive control.

Cell culture

Human neuroblastoma SH-SY5Y cells were purchased from the ATCC and cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 15 mM HEPES. The cells were cultured and maintained in T75 flasks at 37 °C in 5% CO2.

Protective effect on SH-SY5Y cells against Aβ toxicity assay

Each compound was dissolved in DMSO. The final concentration of DMSO in the culture medium was 0.1%. The assay was conducted in type I collagen-coated 96-well cell culture plates (Nippi Inc., Tokyo, Japan), and the cells were seeded at 3.0 × 104 cells/cm2. After incubation for 72 h, the usual medium was removed, and then each test sample and 250 μM Aβ42 in 0.02% aqueous ammonium solution (final concentration of Aβ42: 10 μM) was added in phenol red-free medium containing 2% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 15 mM HEPES. Another 96-well plate was also prepared with medium containing 0.02% aqueous ammonium solution and each test sample to evaluate reference absorbance, as described below. After incubation for 24 h at 37 °C in 5% CO2, the plates were centrifuged at 800 rpm for 5 min, after which 50 μL of supernatant was transferred to another 96-well plate. Cell viability was determined using the WST-8 assay (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 10 μL of WST-8 reagent was added to each well, and the plate was incubated for 3 h at 37 °C in 5% CO2, after which the absorbance at 450 nm was measured using a Synergy HTX Multi-Mode Reader. Relative cell viability was calculated by comparing the absorbance (450 nm for the plate with cells, 450 nm for the plate without cells) of each sample with that of the control without sample. LDH assays were also conducted using those supernatants and an LDH Cytotoxicity Detection kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The amount of LDH released was calculated by comparing the absorbance (490 nm for the plate with cells,  490 nm for the plate without cells) of each sample with that of the control without sample.

BACE1 FRET assay

BACE1 assays were performed in 384-well black plates using a BACE1 FRET assay kit, Red (Thermo Fisher Scientific, USA). The assay was carried out according to the supplied manual, with some modifications as described in a previous report [6].