A Novel multifunctional 5,6-dimethoxy-indanone-chalcone-carbamate hybrids alleviates cognitive decline in Alzheimer’s disease by dual inhibition of acetylcholinesterase and inflammation

doi:10.21203/rs.3.rs-1467420/v1

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A Novel multifunctional 5,6-dimethoxy-indanone-chalcone-carbamate hybrids alleviates cognitive decline in Alzheimer’s disease by dual inhibition of acetylcholinesterase and inflammation

https://doi.org/10.21203/rs.3.rs-1467420/v1

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Backgrounds: Alzheimer's disease (AD) is a multifactorial neurodegenerative disease. The treatment of AD through multiple pathological targets may generate therapeutic efficacy better. The multifunctional molecules that simultaneously hit several pathological targets have been of great interest in intervention of AD.

Methods: Here we combined the chalcone scaffold with carbamate moiety and 5,6-dimethoxy-indanone moiety to generate a novel multi-target-directed ligand (MTDL) molecule(E)-3-((5,6-dimethoxy-1-oxo-1,3-dihydro-2H-inden-2-ylidene)-methyl)phenylethyl(methyl) carbamate (named AP5). In silico approaches were used to virtually predict the binding interaction of AP5 with AChE, the drug-likeness and BBB penetrance, and later validated by evaluation of pharmacokinetics (PK) in vivo by LC-MS/MS. Moreover, studies were conducted to examine the potential of AP5 for inhibiting AChE and AChE-induced amyloid-β (Aβ) aggregation, attenuating neuroinflammation and providing neuroprotection in the APP/PS1 model of AD.

Results: We found that AP5 can simultaneously bind to the peripheral and catalytic sites of AChE by molecular docking. AP5 exhibited desirable pharmacokinetics (PK) characteristics including oral bioavailability (67.2%), > 10% brain penetrance and favorable drug-likeness. AP5 inhibited AChE activity and AChE-induced Aβ aggregation in vivo and in vitro. Further, AP5 lowered Aβ plaque deposition and insoluable Aβ levels in APP/PS1 mice. Moreover, AP5 exerted anti-inflammatory responses by switching microglia to a disease-associated microglia (DAM) phenotype and preventing A1 astrocytes formation. The phagocytic activity of microglial cells to Aβ was recovered upon AP5 treatment. Importantly, chronic AP5 treatment significantly prevented neuronal and synaptic damage and memory deficits in AD mice.

Conclusion: Together, our work demonstrated that AP5 inhibited the AChE activity, decreased Aβ plaque deposition by interfering Aβ aggregation and promoting microglial Aβ phagocytosis, and suppressed inflammation, thereby rescuing neuronal and synaptic damage and relieving cognitive decline. Thus, AP5 can be a new promising candidate for the treatment of AD

Alzheimer’s disease

Multi-target-directed ligands

Acetylcholinesterase

Amyloid-β

Neuroinflammation

Alzheimer’s disease (AD) is the primary cause of dementia characterized by progressive memory decline and cognitive dysfunction, often manifested pathologically by deposition of extracellular amyloid-β (Aβ) plaques, the formation of intraneuronal neurofibrillary tangles and neuroinflammation [1, 2].With exponentially increasing incidence as the global population ages, AD has imposed enormous burden on health care systems [3]. Although progress has been made on the mechanism of AD pathology, effective treatment is still limited[4].

AD is a progressive multifactorial neurodegenerative disease in which multiple causative factors lead to cognitive decline and disease deterioration [5, 6]. Therefore, targeting any single factor (e.g. Aβ, tau, neuroinflammation etc.), even if successful, is not efficacious to reverse the disease escalation. Therefore, an effective cure may require a multipronged approach to combat multiple detrimental features in parallel. The multi-target-directed ligands (MTDLs), designed to modulate several molecule targets in parallel with a hybrid molecule, may be effective at lower doses due to additive or synergistic effects [7]. Several MTDLs for AD have been developed and currently in clinical trials, including ladostigil tartrate [8], eltoprazine, bexarotene, dextromethorphan, and so on.

In order to reach the desired dual or multiple effects, the molecule targets of the MTDL drugs need to be charily selected. Acetylcholinesterase inhibitors (AChEIs) are usually taken into consideration for their symptomatic ameliorations [9]. AChE is a pivotal enzyme in the hydrolysis of neurotransmitter acetylcholine (ACh).It has been shown that AChE possesses two active binding sites, including the peripheral anionic site (PAS) at the edge of the gorge and catalytic anionic site (CAS) at the bottom [10]. AChE catalyzes the hydrolysis of ACh through its CAS. Accumulating evidences have demonstrated that PAS of AChE, responsible for non-cholinergic functions, greatly accelerates the assembly of Aβ into fibrils and promotes Aβ deposition [11-13]. Blockade of PAS is effective for the prevention of Aβ plaque deposition by inhibiting Aβ assembling and consequently promoting Aβ clearance [12]. AChEIs aiming at both PAS and CAS opened the prelude of AChEI-based MTDLs [13]. It is well known that the carbamate fragment in rivastigmine is the inhibitory pharmacophore, which could covalently interact with CAS [14]. In addition, the 5,6-dimethoxy-indanone moiety of donepezil was directed toward PAS of AChE [15].

Neuroinflammation is not merely a consequence of disease progression but also exacerbate the pathology of AD and accelerate cognitive impairment [16]. Inflammation is linked with increases in Aβ generation, aggregation and tau phosphorylation. Co-inhibition of inflammation and AChE has shown potential as a preventive treatment [17]. Chalcone (α-phenyl-β-benzoylethylene) is a simple molecule of many naturally existing compounds, such as flavonoids and isoflavonoids. Chalcone and its derivatives possess a diversity of pharmacological activities, including antioxidant activity, anti-inflammatory, and neuroprotective properties [18].Therefore, we designed the a novel MTDL molecule (E)-3-((5,6-dimethoxy-1-oxo-1,3

-dihydro-2H-inden-2-ylidene)-methyl)phenylethyl(methyl)carbamate (thereafter named AP5) to fuse a carbamate moiety as CAS binding unit and a dimethoxy-indanone moiety as PAS binding unit with chalcone scaffold, hoping the molecule possess AChE inhibitory effect, anti-Aβ aggregation and anti-inflammatory properties. The objective of this investigation is to determine whether ACh hydrolysis, Aβ deposition and neuroinflammation can be resolved simultaneously by this MTDL and to demonstrate the effect of this novel MTDL on cognitive deficits in AD model mice.

Mouse

The APP/PS1 (APPswe/PSEN1dE9) double-transgenic mice of ages 6 months and age-, gender-matched nontransgenic control mice were bred at the Jiangsu ALF Biotechnology Co. (Nanjing, China). The C57BL/6J mice of ages 6 months for pharmacokinetics analysis were obtained from the Hunan SJA laboratory animal Co. (Changsha, China). Only male animals were used to avoid the influence of gender and minimize variability. All mice were fed with food and water available ad libitum on a 12-h light/dark cycle.

The multi-target-directed ligand drug

The novel MTDL molecule (E)-3-((5,6-dimethoxy-1-oxo-1,3-dihydro-2H-inden-2-ylidene) -methyl) phenylethyl (methyl) carbamate (AP5) were synthesized at department of medicinal chemistry (Hainan University, Haikou, China), using the methods reported in the patent cooperation treaty. The chemical structure of AP5 was validated by nuclear magnetic resonance at 400 MHz ¹H NMR and 101 MHz ¹³C NMR (Supplementary Figure 1). ¹H NMR (400 MHz, Chloroform-d) δ 7.59 (d, J = 2.1 Hz, 1H), 7.52-7.41 (m, 3H), 7.36 (s, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.02 (s, 1H), 4.04-3.95 (m, 8H), 3.57-3.42 (m, 2H), 3.13 (s, 1H), 3.05 (s, 1H), 1.27 (dt, J = 23.0, 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 193.03, 155.51, 151.86, 149.68, 144.99, 136.90, 136.13, 131.64, 131.03, 129.61, 127.57, 123.22, 122.79, 107.27, 105.12, 56.33, 56.20, 44.18, 34.35, 33.91, 32.02, 13.30, 12.51.

Molecular docking assay

Autodock Vina version 4.2 ( Scripps research institute, Jupiter, FL, USA) was employed to carry out the molecule docking between AChE (PDB ID 2WHQ) and AP5. Affinity grids of docking wrapped the whole AChE protein, and 200 conforms were searched. The conformation with the lowest binding energy is chosen as the optimal conformation. All pictures were observed and produced using PyMOL (DeLano Scientific, San Carlos, CA) and Discovery Studio 2019 (Biova, Waltham, MA, USA).

Pharmacokinetics (PK) analysis in mice

To determine the pharmacokinetics of AP5 in mice, mice were administered a single dose AP5 by i.v. and oral gavage dose routes. The plasma and brain homogenate were detected by UPLC-TSQ/MS(Thermo Fisher Scientific, Waltham, MA, USA). The plasma drug concentrations were analyzed for PK data by DAS (Drug and Statistics) Version 3.0 (BioGuider Corporation, Shanghai, China). The extent of brain penetration was measured by brain-to-plasma distribution ratio (B/P). The B/P value was calculated as: AUC_{INF brain}/ AUC_{INF plasma}×100. Bioavailability (F%) after oral administration was calculated as: AUC_{INF p.o.}/ AUC_{INF i.v.} / 4×100.

Drug treatment

APP/PS1 mice of ages 6 months were randomly divided into vehicle (APP/PS1, n=10) and treatment groups (AP5, n=10), which intragastrically administered with AP5 (40 mg/kg) once daily for consecutive 5 months. AP5 was dissolved in distilled water containing 0.2% DMSO.

Estimation of acetylcholinesterase (AChE) and acetylcholine (ACh)

The brain samples were weighed, homogenized in PBS solution and centrifuged for 20 minutes at 25,000 g at 4°C. Amplex® Red acetylcholinesterase / acetylcholine assay kit (A12217, Thermo Fisher Scientific) was used to detect AChE activity and ACh levels in the supernatant in accordance with the manufacturer’s instructions.

Amyloid-β aggregation and disaggregation assay

The effects of AP5 on Aβ fibril formation and pre-formed Aβ fibrils disaggregation were determined using a previously described method of Thioflavin T (ThT, T3516, Sigma Aldrich, St. Louis, MO, USA) fluorometry [19].

Dot blot

Aβ40 or Aβ42 oligomers formation was monitored by dot blot with oligomer-specific antibody. Briefly, the resulting Aβ film was dissolved in ddH₂O to form a 100 μM solution, and then the oligomerization reaction [20] was initiated in absence or presence of different concentrations of AP5. An aliquots (2 μL) of each solution was dripped on a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in TBS for 1 h, incubated with the anti-β amyloid (MOAB-2, NBP2-13075SS, Novus Biologicals, Colorado, USA), anti-amyloid oligomers (A11, NBP1-97930, Novus Biologicals) or anti-amyloid fibrils (OC, NBP1-97930, Novus Biologicals) antibodies overnight at 4°C. The membrane was then incubated using HRP-conjugated secondary antibodies at room temperature for 1 hand developed with ECL.

Behavioral tests

To investigate whether AP5 treatment play a key role in cognition improvement of APP/PS1 mice models, the five different behavioral tests were sequentially carried out as the following order: open field, novel object recognition task, spatial object-location task, Y-maze and Morris water maze (MWM). AP5 was also administered daily during the test periods. A set of video analysis systems (TopScan, CleverSys, USA) was used throughout behavioral tests.

Preparation of brain samples

After behavioral tests, all animals were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) and decapitated. The left hemisphere were fixed in 4% paraformaldehyde for 48 h. The right hemispheric hippocampus and cortex were collected separately, snap frozen in liquid nitrogen, and then stored at -80°C until the analyses of protein and gene expression.

Immunohistochemistry (IHC) and immunofluorescence (IF)

The fixed hemibrains were paraffin-embedded and sectioned in the coronal plane. The slices were deparaffinized by xylene and rehydrated by a series of gradient alcohol. Antigen retrieval through microwave boiling was performed with 10mM sodium citrate solution at pH 6.0.The slices were cooled down to room temperature and then rinsed in PBS solution. Then the slices were incubated in 10% goat serum blocking solution for 1 h, followed by primary antibodies overnight at 4°C. The next day, the slides were labeled with biotinylated or fluorescent secondary antibodies. Slices were washed three times with PBS before being mounted. Images were captured using a SpinSR10 scanner (Olympus, Tokyo, Japan).

Thioflavin S staining

The hemisphere slices were stained with 0.5% fluorescent amyloid dye Thioflavin S (ThS, T1892, Sigma Aldrich) in the dark for 10 min, followed by three washes with 50% ethyl alcohol and a final wash in PBS. Image analysis of the number and size of cortical and hippocampal ThS positive plaques was carried out on Image J FIJI software with ‘analyze particles’ function.

Enzyme-linked immunosorbent assay(ELISA)

For detecting the pathological soluble or insoluble Aβ in mice, sequential extractions were performed as follows: the half of right hippocampus and cortex from three groups were homogenized in TBS solution containing 5 mM EDTA, phosphatase inhibitor

(P1050, Beyotime, Shanghai, China), protease inhibitor cocktail (P1010, Beyotime) followed by centrifugation at 25,000g at 4°C for 1 h. The supernatant was collected as TBS fraction, and the resulting pellet was solubilized in 70% formic acid (FA), sonicated until the lysis buffer was clear, followed centrifugation at 25,000g at 4°C for 1 h. The supernatant was collected as the FA fraction and neutralized (1:20) in neutralization buffer (1 M tris base, 0.5 M Na₂HPO₄, 0.05% NaN₃). Human Aβ40 and Aβ42 were determined by ELISA kits (E-EL-H0452c and E-EL-H0453c, Elabscience, WuHan, China) in accordance with the manufacturer’s instructions. In addition, the levels of IL-1β (88-7013-88, Invitrogen), IL-6 (EK206HS-96, Multi sciences, HangZhou, China) and TNF-α (88-7324-88, Invitrogen) in TBS fraction were also determined by ELISA.

Western blotting

Frozen hippocampus and cortex were homogenized in pre-cooling RIPA buffer (P0013B, Beyotime) supplemented 1 mM phenylmethanesulfonyl fluoride (PMSF) and cocktail of protease and phosphatase inhibitors. Protein samples was separated in 10% or 12% SDS-PAGE and then transferred to PVDF membranes. The membranes were incubated with 5% fat-free milk blocking solution for 1 h at room temperature, and then probed with primary antibodies overnight at 4°C. The membranes were incubated with the corresponding HRP-conjugated secondary antibodies at room temperature for 1 h, and developed with ECL. Densitometric analysis was performed using Quantity One software (Bio-Rad, Berkeley, CA, USA).

RNA-sequence

Hippocampal specimen from WT, APP/PS1 and AP5 mice stored in dry ice were sent to Beijing Genomics Institute (Wuhan, China), and total RNA was extracted for RNA sequencing on BGISEQ-500 system. Moreover, the differentially expressed genes between APP/PS1 and AP5 were analyzed after RNA sequencing.

Statistical analysis

Prism 8.3.0 Software (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analyses. Data are expressed as mean ± standard error of mean (SEM). The statistical difference between two independent groups was analyzed with the two-tailed unpaired t-test. Samples among more than two groups were compared using parametric one- or two-way ANOVA followed by the Turkey's post-hoc test. Post-hoc tests were conducted when the F value achieved the necessary level (p < 0.05) and there was no significant variance inhomogeneity. p values less than 0.05 were considered statistically significant.

Design, drug-likeness and pharmacokinetic evaluation of 5,6-dimethoxy-indanone-

chalcone-carbamate hybrids as a novel MTDL

We designed and generated a new MTDL molecule (E)-3-((5,6-dimethoxy-1-oxo-1,3-

dihydro-2H-inden-2-ylidene)-methyl)phenylethyl(methyl)carbamate (named AP5) by binding 5,6-dimethoxy-indanoneand carbamate moiety with chalcone molecular scaffolds (Figure1a). Then, we first explored the binding potential of AP5 with the active pocket of AChE (PDB ID 2WHQ) through molecular docking. The molecular docking result showed that AP5 had moderate binding affinity with AChE binding pocket (Figure 1b) with lowest binding energy of -10.64 kcal/mol, compared to donepezil (-11.94kcal/mol) and rivastigmine (-8.55kcal/mol). The carbamate moiety of AP5 was buried deep into the catalytic site of AChE, however 5,6-dimethoxy

-indanone moiety contacted the peripheral sites at the edge of gorge (Figure 1b). Based on active amino acids in the vicinity of CAS and PAS of AChE [21], the binding of AP5 was stabilized by the hydrogen bond interaction of Phe295 and Arg296 of CAS, Tyr337 and Trp86 of PAS (Figure 1c). The other hydrophobic interaction was observed between 5,6-dimethoxy-indanone and side-chain Tyr124 located in PAS binding region. The carbamate group contacted the Trp286 and Leu289 in CAS region by hydrophobic interaction. AP5 also exhibited van der Waals interactions with Asp74, Gly120, Ser125 of PAS, and Ile294, Phe338 of CAS. Taken together, the docking analysis suggests that AP5 fits well in the active pocket of AChE by interacting with the peripheral and catalytic site residues simultaneously, which indicates its dual inhibition and high potency.

Combining several pharmacophores may lead to large molecule which may compromise drug-likeness properties [22]. Drug-likeness assessment in silicoby Lipinski's rule of five [23] showed that AP5 complied with the Lipinski's rule of five (Supplementary Table 1). Moreover, in silico assessment of blood-brain barrier (BBB) penetration showed that BBB-score >0.02 [24](Supplementary Table 1). These data indicate that AP5 holds appropriate drug-likeness properties and BBB permeability.

We next determine the pharmacokinetic profile of AP5 in male C57BL/6J mice given by a single intravenous (i.v.) dose of 10 mg/kg or single per os (p.o.) dose of 40 mg/kg, respectively. After a single oral dose, AP5 concentration in plasma reached the peak (2088 ng/mL) at 1.5 h by LC-MS/MS analysis. Importantly, we determined AP5 in the brain homogenate with the highest concentration of 95.3 ng/mL at 2h (Figure 1d-e). PK parameters revealed oral AP5 holds low systemic clearance (Cl =2.756 L/hr/kg), moderate volume of distribution (Vss=18.79 L/kg), highly favorable oral bioavailability F = 67.2% and adequate oral brain penetration (B/P = 10.23%) (Figure 1d-e,Supplementary Table 2). Taken together, AP5 showed adequate BBB permeability, suitable PK properties and drug-likeness properties, which supports the potential to develop AP5 as a promising candidate for studies in AD model.

AP5 inhibits AChE activity and blocks Aβ oligomerization or fibrillization

As result of carbamate pharmacophore of AP5 binding to CAS of AChE, we further examined the effects of AP5 on AChE activity in APP/PS1 mice. Consistent with previous study[25], our data showed that AChE activity in the cortex and hippocampus was markedly increased in APP/PS1 mice (Figure 2a), which led to reduction of ACh level compared to WT mice (Figure 2b). AP5-treated mice exhibited decreased activity of AChE compared with APP/PS1 mice in cortex (F_2,15= 7.730 p = 0.0049) and hippocampus (F_2,15= 29.21, p < 0.0001), and elevated levels of ACh in cortex (F_2,15= 7.886, p = 0.0046) and hippocampus (F_2,15= 22.51, p < 0.0001) (Figure 2a-b).

It is reported that Aβ could bind to AChE through PAS and induces its fibrillization [10, 11]. Propidium and fasciculin, PAS inhibitors, are capable of preventing the effect of AChE on Aβ fibril aggregation process [11]. As 5,6-dimethoxy-indanone moiety of AP5 binds AChE at PAS, we next evaluated whether AP5 exerted an inhibitory effects on Aβ fibrillogenesis induced by AChE. Firstly, we analyzed the inhibition of AP5 on Aβ fibril formation induced by AChE using 24h kinetic ThT fluorescence assay. As shown in Figure 2c, incubation of Aβ with AChE resulted in an increase in fluorescence intensity, confirming that AChE was able to promote Aβ assembly into fibrils. Conversely, co-incubation of AP5 and Aβ40with AChE substantially lowered the ThT intensity in a dose-dependent manner, indicating that AP5 inhibited the enhancement of Aβ fibrillization triggered by AChE. Besides, the effects of AP5 on disaggregation of pre-formed Aβ40 or Aβ42 fibrils was determined. Aβ40 or Aβ42 was firstly pre-aggregated with AChE for 2 days before co-incubation with AP5.In the presence of AP5, Aβ fibrils were disaggregated as shown by a marked decline of ThT fluorescence intensity in Aβ40 (F_3,8= 49.64, p < 0.0001) and Aβ42 (F_3,12= 43.32, p < 0.0001) (Figure 2d). This observation was further confirmed by dot blot assay with the amyloid fibrils-specific antibody (Aβ40, F_4,10= 93.92, p < 0.0001) (Aβ42, F_4,10= 161.9, p < 0.0001) (Figure 2e-f).

To study whether AP5 could inhibit Aβ self-oligomerization, dot blot assay was carried out with the amyloid oligomer-specific antibody. Monomeric Aβ40 or Aβ42 was incubated with or without AP5 under conditions that led to oligomerization. As indicated in Figure 2g-h, AP5 decreased the levels of oligomeric Aβ40 (F_3,8= 34.91, p < 0.0001) and Aβ42 (F_3,8= 13.92, p = 0.0015).This result was further corroborated by immunoblot detection that discriminated between Aβ oligomers and monomers. The synthetic oligomer preparations comprised a mixture of low molecular weight oligomers and high molecular weight (HMW) oligomers.HMW Aβ oligomers were observed as a smear on SDS-PAGE with typically ranging in molecular weight from 50-150 kDa [26]. As shown in Figure 2i, a 5 kDa Aβ monomer band, a 10 kDa Aβ dimer band as well as a smear were detected. In the presence of AP5, the intensity of the Aβ dimers and smear were decreased, demonstrating that AP5 specifically blocked Aβ oligomerization. Taken together, these data suggested that AP5 might not only elevated ACh level but also interfered toxic Aβ aggregation.

AP5 reduces Aβ deposition without altering APP processing in APP/PS1 mice

We next evaluated the overall effect of AP5 on Aβ pathology. Brain slices were stained with Thioflavin S (ThS) to detect dense-core fibrillar amyloid plaques. Indeed, we observed there was a pronounced reduction in the load (cortex, F_2,5= 86.47, p < 0.0001) (hippocampus, F_2,15= 44.26, p < 0.0001) and number (cortex, F_2,15= 246.6, p < 0.0001) (hippocampus, F_2,15= 150.8, p < 0.0001) of ThS-positive plaque in APP/PS1mice treated with AP5 (Figure 3a-b). Further this reduction was most pronounced for larger ( > 50μm² in area) plaques in cortex (F_2,15= 253.2, p < 0.0001) and hippocampus (F_2,15= 251.6, p < 0.0001). Similarly, immunofluorescent staining of Aβ peptide by MOAB-2 antibody, which recognizes the unaggregated, oligomeric and fibrillar form of Aβ, indicated that the area occupied by unaggregated and aggregated Aβ was also decreased in cortex (F_2,15= 82.42, p < 0.0001) and hippocampus (F_2,15= 64.06, p < 0.0001) of the AP5-administered AD mice(Figure 3c).

To further detect Aβ aggregation in these mice, we performed a serial extraction of cortex and hippocampus.TBS fraction contains more soluble Aβ species while the 70% FA fraction contains insoluble Aβ aggregates.APP/PS1 mice had significantly higher Aβ40 and Aβ42 levels in TBS- and FA-fractions (Figure 3d-e). Furthermore the majority of Aβ in APP/PS1 mice was fractioned in the FA-soluble fractions (Figure 3e), indicating its deposition in the amyloid plaque. In addition, as shown in Figure 3e, the concentrations of insoluble Aβ40 (cortex, F_2,15= 82.42, p < 0.0001) (hippocampus, F_2,15= 46.30, p < 0.0001) and Aβ42 (cortex, F_2,15= 132.4, p < 0.0001) (hippocampus, F_2,15= 112.2, p < 0.0001) were markedly decreased in FA-fractions, but no marked differences were determined in TBS-fraction after AP5 administration (Figure 3d).

Next, to clarity the mechanism that contributed to the reduction in Aβ following AP5 administration, we assessed APP processing by western blot. The results showed there wereno significant changes in the expression levels of the amyloid precursor protein (APP full length,APPfl), CTFs (C-terminal fragments, CTFβ), β-site-APP cleaving enzyme (BACE) and catalytic subunit of the gamma-secretase complex (presenilin 1), between APP/PS1 and AP5-treated APP/PS1 mice (Figure 5f-g). These data suggest that AP5 did not alter APP expression or processing. Overall, these results demonstrated that AP5 administration mitigates Aβ load without influencing Aβ production.

AP5 alleviates neuroinflammation of APP/PS1 mice

On the basis of the structure properties of AP5,which contains anti-inflammatory and antioxidative chalcone structure, we examined the effects of AP5 on neuroinflammation in APP/PS1 mice. Firstly, the brain slices were immunostained with ionized calcium-binding adapter molecule 1(Iba1) antibody and glial fibrillary acidic protein (GFAP) to investigate the effects of AP5 on microgliosis and astrogliosis, respectively. We morphologically classified Iba1⁺microglia to quantify microglia activation state, and observed that microglial cells in APP/PS1 mic eexhibited a significantly elevated proportion of intermediate, amoeboid or round, showing an increase in microglia activation. For the APP/PS1 mice treated with AP5, we found a marked decrease in the proportion of activated phenotype (F_2,15= 10.71, p = 0.0013) (Figure 4a-b). Consistent with previous study [27], quantitative analysis of individual reactive astrocyte morphology using Sholl analysis showed that elevated ramification index (F_2,15= 44.12, p < 0.0001) and the sum of intersects (F_2,15= 82.97, p < 0.0001) in APP/PS1 mice. Similar to microglia, these reactive phenotypes of astrocytes were attenuated by AP5 treatment (Figure 4c-d). However, the ending radius, an indicator of astrocyte territory, was not changed in APP/PS1 mice.

Next, we determined the levels of several proinflammatory cytokines (TNF-α, IL-6 and IL-1β) implicated in AD. A greatly increased secretion of TNF-α (F_2,15= 107.0, p < 0.0001), IL-6 (F_2,15= 54.31, p < 0.0001) and IL-1β (F_2,15= 25.01, p < 0.0001) were detected in APP/PS1 mice, which were markedly reduced by AP5 administration (Figure 4e).Together, these results show AP5 attenuated neuroinflammatory responses in AD mice model.

It has been reported that microglia clustered in the vicinity of Aβ plaque form a protective barrier which compacts amyloid fibrils and reduces their toxicity [28]. Thus, we quantified the Aβ plaque-associated microglia by immunofluorescent co-staining with MOAB-2 and Iba1.The increased clustering of microglia surrounding a plaque was observed in brains of AP5-treated APP/PS1 mice (Figure 4f-g). Microglia clustering around plaques facilitate extracellular Aβ clearance [29]. Thus, we further investigated whether the increased microglia recruitment in the vicinity of plaques upon AP5 treatment can promote Aβ phagocytosis. As shown in Figure 4j-k, the area of CD68 (a marker of phagocytic activity of microglia) within microglia was increased, suggesting that AP5 treatment enhances microglial response to Aβ plaques and phagocytic activity. These results revealed that AP5 decreased microglia activation and enhanced the clustering of microglia around Aβ plaque, thereby diminishing plaque-associated neurotoxicity and promoting microglia phagocytosis and clearance of Aβ. Besides, we found there was no statistically difference in the number of astrocytes around Aβ plaque (Figure 4h-i).

AP5 rescues the neuron and synapse damage in the APP/PS1 mice

Aβ aggregation in the brain is a key initiating step in the pathogenesis of AD, which induces many downstream detrimental events such as oxidative stress, neuroinflammation, synaptic impairment, neuronal degeneration, and eventual cognitive defects [2]. Therefore, we next examined whether the protective effects of AP5 against Aβ-induced neurotoxicity was reflected at the neuronal and synaptic level. Firstly, to determine the effects of AP5 on neuron loss and degeneration in APP/PS1 mice, we carried out co-immunostaining with NeuN and MAP2. Remarkably, we detected that the average number of NeuN⁺ cells was reduced (F_2,15= 21.55, p < 0.0001), and MAP2 signals were also significantly disrupted (F_2,15= 30.17, p < 0.0001) in hippocampal CA1 pyramidal layer of APP/PS1 mice, but these changes were restored in AP5-treated APP/PS1 mice (Figure 5a-b). Consistent with these results, the protein expression of NeuN and MAP2 was markedly increased in hippocampus of AP5-treated mice (Supplementary Figure 2a-b).

Next, we investigated the efficacy of AP5 against synaptic pathology in APP/PS1 mice.

synaptophysin and PSD95 are two membrane protein markers located in the presynaptic and postsynaptic cells, respectively. Co-immunofluorescence of synaptophysin with PSD95 revealed reduced pre- (F_2,15= 16.1, p = 0.002), post- (F_2,15= 20.04, p < 0.0001), and double-positive

-synaptic puncta (F_2,15= 37.82, p < 0.0001) in APP/PS1 mice, and the synapse damage was markedly recovered by AP5 (Figure 5c-d).Western blot further indicated that the expression levels of synaptophysin and PSD95 in hippocampus of AP5-treated mice were more than that in the APP/PS1 mice, respectively (Supplementary Figure 2a-b).

These results suggested that AP5 prevented or reversed neuronal and synaptic damage, which might play a pivotal role in ameliorating downstream cognitive defects.

Chronic dosing AP5 from the early stages of β-amyloidosis alleviates cognitive deficits of APP/PS1 mice

We next assessed whether the beneficial effects of AP5 in decreasing Aβ plaque deposition and neuroinflammation are accompanied by cognitive improvement of APP/PS1 mice. Firstly, we evaluated general locomotor activity of WT, APP/PS1 and AP5 mice through the open field test, and no difference in the total distance traveled (F_2,27= 0.6091, p = 0.5511) was observed among groups, indicating that AP5 administration did not elicit any influence on general locomotor activity of the mice (Figure 5e). Furthermore, we employed Y-maze to evaluate spatial recognition memory. AP5-treated mice demonstrated a marked elevation of their preference index (F_2,27= 11.07, p = 0.0003) relative to APP/PS1 mice after 3 h ’s training, suggesting the improvement of short-term spatial recognition memory (Figure 5f).

Moreover, we used the novel object recognition task and the spatial object location task to evaluate recognition memory. APP/PS1 mice demonstrated impaired short- and long-term recognition memory by a significant decline of its discrimination index at 1h and 24h compared with WT mice (Figure 5g-h). AP5-treated APP/PS1 mice performed significantly better than APP/PS1 mice without treatment in the novel object recognition task (F_2,27= 8.215, p = 0.0016) and the spatial object location task (F_2,27= 11.05, p = 0.0003) at 24h (Figure 5g-h). In addition, the discrimination index in AP5-treated AD mice for novel object recognition was significantly higher (F_2,27= 9.417, p = 0.0008) than APP/PS1 mice at 1h (Figure 5g). These data indicate an improvement of short- and long-term recognition memory upon AP5 treatment.

We examined the reference memory through Morris water maze (MWM). As indicated in Figure 5i, APP/PS1 mice exhibited marked spatial learning and memory decline by longer escape latency in the successive platform learning trials, less time spent in the target quadrant and lower platform crossover number in the probe trail (Figure 5k). The swimming velocity during the successive training period were indistinguishable (F_2,27= 0.7216, p = 0.4951) between each group (Figure 5j). After 5 months’ treatment of AP5, the mice performed much better in escape latency including the time effect (F_2.797,75.51= 66.46, p < 0.0001) and group effect (F_2,27= 79.06, p < 0.0001) by two-way ANOVA, platform crossing number (F_2,27= 8.695, p = 0.0012) and time spent in the target quadrant (F_2,27= 9.236, p = 0.0009) (Figure 5k), suggesting that AP5 slows cognitive decline in AD mice.

AP5 regulates gene transcription in APP/PS1 mice

Given the multitargeted effects of AP5 that we observed, we next explore the underlying molecular mechanism by high-throughput bulk RNA sequencing (RNA-seq) of the hippocampal tissues. A total of 17981 genes were identified in the hippocampi from above WT, APP/PS1 and AP5 mice. Volcano plots depicted that 933 genes were differentially expressed, of which 425 genes were induced and 508 genes were suppressed in APP/PS1 mice treated with AP5 treatment compared with APP/PS1 mice without treatment (Figure 6a). Gene ontology (GO) term analysis demonstrated that, in addition to regulation of gene expression, inflammation-related features were enriched, including microglial activation involved in immune response, astrocyte activation, synapse pruning and complement-mediated synapse pruning as well as processes responsible for phagocytosis, engulfment and Aβ clearance (Figure 6b). In agreement with previous reports[30], deposition of Aβ in AD patients is associated with the innate immune system activation. Together, these data suggested that AP5 is of great importance to regulate inflammation in disease progression.

Because microglia and astrocyte are the main cell types exerting inflammatory effects in the brain, we carried out heatmaps of disease-associated microglia (DAM) genes from hippocampal RNA-seq data. We analyzed gene expression profile in the hippocampus and observed an increase in DAM gene expression in APP/PS1 mice with AP5 treatment compared with APP/PS1 mice without treatment (Figure 6c), further suggesting that microglial cells acquired a typical DAM phenotype. The ability of microglia to switch to a DAM phenotype appears essential to limit Aβ plaque deposition and inflammation, thereby removing the neuronal damage in AD [31]. Moreover, CD11c (encoded by Itgax) has recently been identified as a marker for DAM [31]. We found CD11c-positive microglia were accumulated around Aβ plaques. Notably, CD11c-positive area within activated microglia was significantly elevated with AP5 treatment (Figure 6d-e), further confirming that AP5 skewed microglia to a DAM phenotype and contributed to the decreased deposition of Aβ and inflammation.

At the same time, we compared the transcriptional profile of reactive astrocytes with that of astrocytes subpopulations recently identified pan-reactive, A1 and A2 specific astrocytes [32]. A1 specific astrocytes, induced by neuroinflammation, are powerfully neurotoxic, where their presence may well account for neurodegeneration and drive disease deterioration. However, A2 astrocytes upregulate many neurotrophic factors, and therefore are protective. As expected, astrocytes in the hippocampus of APP/PS1 mice displayed an increased gene expression of pan-reactive and A1specific astrocytes signatures. AP5 reduced the expression of most genes linked to pan-reactive and A1 astrocytes (Figure 6f), suggesting that AP5 could prevent or revert A1-specific astrocytes formation. No differences among groups were detected in the expression of signature genes of A2-specific astrocytes. Since complement C3 (C3) is one of the most distinctive and highly elevated genes in A1 specific astrocytes and is not expressed by A2 specific astrocytes [33], we performed immunofluorescence to identify whether C3-expressing A1 astrocytes are decreased following AP5 treatment. We found C3-positive area within astrocytes was markedly reduced after AP5 treatment (Figure 6g-h), which further confirming AP5 could prevent and reverse A1specific astrocyte formation. Taken together, our data demonstrated that regulation of astrocyte and microglia phenotype transition by AP5 may be an important mechanism to control inflammation in AD.

While it is clear that the fundamental pathology of AD is driven by Aβ toxicity,

neurofibrillary tangles and neuroinflammation, recent clinical results suggest that addressing individual component of AD pathology may not be efficacious to treat the majority of AD[1, 4]. Therefore, the MTDLs aiming at several underlying causes of neurodegenerative disease in parallel presents an opportunity for successful AD treatment [5, 7].To this end, we utilized the chalcone scaffold for the integration of carbamate moiety and dimethoxy-indanone moiety to generate a novel MTDL—AP5.AP5 was able to span the active gorge of AChE and exhibits high affinity for the CAS and PAS. Furthermore, AP5 displayed favorable drug-likeness properties and desirable PK characteristics including oral bioavailability (67.2%) and >10% brain penetrance.

AP5 could markedly inhibit AChE-induced Aβ aggregation in vitro and brain AChE catalytic activity in AD mice, which was consistent with the molecular docking result showing that AP5 could bind AChE through PAS and CAS. More importantly, AP5 was also capable of reducing Aβ deposition by influencing Aβ aggregation but not through APP processing in APP/PS1 mice. Yet, these findings may be contradictory with a prior report that increased levels of ACh could agonize M1 muscarinic acetylcholine receptor in brain, and consequently result in the decrease of APP processing [34]. However, Nitsch and co-workers subsequently found chronic treatment with M1-selective agonists AF102B markedly lowered cerebrospinal fluid (CSF) Aβ levels in AD patients, while physostigmine (an AChEI) did not show a significant effect on CSF Aβ in the same study as AF102B [35].This finding is consistent with prior preclinical study [36], the study showed physostigmine did not change APP processing in rat brain. Moreover, stimulation of M1 and M3 muscarinic receptors increases α-secretase APP processing, but simultaneous stimulation of M2 receptors blocks the M1 effect [37]. In this context, although AP5 elevates Ach level, the effect is non-specific and extends to all muscarinic receptors. Thus, AP5 did not alter APP processing.

The compounds based on inhibition of AChE cholinergic and non-cholinergic function, dual inhibitor of AChE, may exert dual therapeutic effects in symptom-relieving and disease-modifying manners [38].The most potent dual AChEI reported so far is a dimer of indol and 6-chlorotacrine pharmacophore, named NP-61, that has a marked inhibition effect on AChE catalytic activity and great inhibition of AChE-induced Aβ fibrillization in vitro. NP-61 has been shown to display not only an cognitive improvement but also lead to a decrease of the plaque load in mice models [39]. Similar to the effect of NP-61, the present investigations demonstrated that it is possible to mitigate Aβ pathology through targeting AChE by AP5 in vivo. The above results strongly imply that AP5 targeting simultaneously dual binding site of AChE offers an additive or synergistic therapeutic effect for AD.

Neuroinflammation is a prevalent feature across neurodegenerative disease and implicated in the progression of neurodegeneration [16]. Hence, MTDLs simultaneously against AChE and neuroinflammation are expected to have more desired therapy potency [17]. To this end, we furthermore focused our study on dual acting drugs that link AChE inhibitory and anti-inflammatory response in a hybrid molecule. In our work, the inhibitory effect of AP5 on inflammatory effect is confirmed by reducing microglial and astrocyte activation-triggered morphological changes of and secretion of pro-inflammatory cytokines.

Microglia clustered around the Aβ plaque not only form a protective barrier but also promote clearance of Aβ through phagocytosis and degradation [28]. Likewise, suppression of pro-inflammatory cytokines secretion restores microglial phagocytosis toward Aβ in APP/PS1 mice brain [40]. Just recently, there is a growing recognition that microglia constitute intercellular networks for enhanced clearance of intake pathological aggregation in order to control and alleviate microglial inflammatory effects and cytotoxicity [41]. In line with previous studies, our data demonstrated that AP5 enhanced microglia clustering around Aβ plaque, thereby diminishing plaque-associated neurotoxicity and promoting microglia Aβ phagocytosis and degradation in the brain of APP/PS1 mice. The transcription data further indicated a conversion of activated microglia towards a DAM phenotype following AP5 treatment. Hence, our results revealed that AP5 elicited morphological, functional, and transcriptional changes of microglia to endow them with enhanced capacity to clear Aβ aggregates and decrease neuroinflammation.

Reactive astrocytes are a cellular component of gliosis in neurodegenerative disorders. In AD, astrocytes could also ingest Aβ peptides to induce degradation pathways such as autophagy [42]. However, reactive astrocytes are more involved in the neuron support and the modulation of neuronal signaling relative to its phagocytic activities or degrading functions [43]. Thus, there was no marked change in the number of astrocytes around Aβ plaque. Moreover, AP5 could prevent or revert A1 astrocytes formation, which may ultimately lead to a more favorable neuronal milieu and improved neuronal and synaptic loss in the AD brain.

The absence of cognitive benefit in clinical trials targeting Aβ in late stages of AD may account for the fact that neurodegeneration, neuronal death and brain lesion(s) are too serious to be relieved [44]. Hence, the most effective therapeutic strategy is likely to be prevention. To evaluated whether AP5 has the capacity to slow the memory deficits in AD mice models, we chronically administered AP5 to 6-month-old APP/PS1 mice, which were at the early stage of β-amyloidosis, for 5 consecutive months. By a series of behavioral tests, we found AP5 exhibited the amelioration of cognitive decline. Overall, AP5 exerts multiple beneficial effects by targeting AChE and neuroinflammation from the early stage of AD.

Limitations

Here, there are also some limitations which warrant attention. Firstly, multi-target molecular AP5 exerts biological activity targeting AChE by molecular docking and pharmacological activity detection in vitro and in vivo. Confirming the inhibition effects

of AP5 on AChE using additional tests would strengthen our conclusions.In future studies, the amino acids sites of AChE that may interact with AP5 should be mutated and then detected by surface plasmon resonance method to confirm the action site of AP5. Secondly, we found that AP5 exerted an anti-inflammatory effect by inhibiting phenotype transition of microglia and astrocytes, but the molecular mechanism is not well understood. Leeet al. consider that chalcones molecular scaffolds demonstrate NF-κB inhibitory activity by the covalent modification of the IKK proteins via the α,β-unsaturated ketone with the Michael-type activity [45]. Kumar et al. reported a novel chalcone derivatives induce Nrf2 through the covalent modification of the Keap1 cysteines, resulting in a conformational change that facilitates the process of Nrf2 escaping from the KEAP1−Nrf2 interaction [46]. Whether AP5 also targets these molecules and thus mediates its inhibition of microglia and astrocytes phenotype transformation needs further study.

In conclusion, this study identifies a novel MTDL candidate AP5 and demonstrate that AP5 is a dual inhibitor of AChE to constrain the hydrolysis of ACh and Aβ plaque deposition in APP/PS1 mice model. In addition to AChE inhibitory activity, AP5 can suppress neuroinflammation by regulating microglia and astrocyte phenotype transition, which may account for the rescue of neuron and synapse loss, and cognitive deficits. Together, our findings shed light on the therapeutic potency of AP5 for multifactorial disorders and provide a new solution to the treatment of AD.

AD: Alzheimer’s disease; MTDL: multi-target-directed ligand; AChE: acetylcholinesterase; ACh: acetylcholine; PAS: peripheral anionic site; CAS: catalytic anionic site; BBB: blood-brain barrier; Aβ: Amyloid-β; ThS: Thioflavin S; ThT: Thioflavin T; APP: amyloid precursor protein; CTFs: C-terminal fragments; BACE: β-site-APP cleaving enzyme; Iba1:ionized calcium-binding adapter molecule 1; GFAP: glial fibrillary acidic protein; DAM: disease-associated microglia; PK: pharmacokinetics; MWM: Morris water maze.

Acknowledgements

We thank ShanYang, DaidiLi, Xianyu Huang, Taotao Liu and YingyinLi for their technical supports in instrument operation and data collection.

Funding

This work was funded by the National Natural Science Foundation of China [U1812403] and the Pharmacy National First-class Discipline Construction Project of Guizhou Province of China [GNYL (2017-006)], and Guizhou Provincial Department of Education Tutor Studio [99-050].

Author contributions

C.L. performed the experiments and analyzed the data and prepared the manuscript. Y.Q. and J.S.S. provided intellectual support in experiment design, supervised the studies and reviewed the manuscript. Z.P.S. designed and synthesized the novel MTDL drug. H.P. detected the novel MTDL concetrations in plasma and brain homogenates by UPLC-TSQ/MS. Q.W. provided the valuable resource of instrument acquisition.

Availability of data and materials

The data supporting the findings of this study are available on reasonable request.

Ethics approval and consent to participate

The experimental procedures were performed in accordance with the Institutional Animal Care and Use Committees of ZunYi medical university.

Consent for publication

All authors read and approved the final manuscript.

Competing interests

The Authors declare that they have no conflict of interest.

Author details

¹Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. ²Shanghai Universities Collaborative Innovation Center for Translational Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.³Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Hainan University, Haikou, Hainan, 570228, China. ⁴Joint International Research Laboratory of Ethnomedicine of Ministry of Education and Key Laboratory of Basic Pharmacology of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou,560003,China.

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A Novel multifunctional 5,6-dimethoxy-indanone-chalcone-carbamate hybrids alleviates cognitive decline in Alzheimer’s disease by dual inhibition of acetylcholinesterase and inflammation

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