A Novel Multifunctional 5,6-Dimethoxy-Indanone-Chalcone-Carbamate Hybrids Alleviates Cognitive Decline in Alzheimer’s Disease by Dual Inhibition of Acetylcholinesterase and Inflammation

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 the 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 pharmacokinetic (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 insoluble 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.

evaporation to form film. Then Aβ film was dissolved in anhydrous DMSO to make a 100 μM fibril solution.
To study the kinetics of Aβ-AChE fibril complex formation, 10 μM Aβ was mixed with AChE (R005282, Roche, Basel, Switzerland) and different concentrations AP5 and incubated with 10 μM ThT and 50 mM glycine-NaOH (pH 8.5) at 37°C, the ThT fluorescence was continually measured for 24 h at 30 min intervals. For the disaggregation of pre-formed Aβ-AChE fibril complex, the Aβ and AChE (Aβ final concentration 10 µM, Aβ/AChE molar ratio 100:1) solution was incubated at 37°C for 48 h. AP5 at various concentrations was then added to the pre-aggregated Aβ solution and incubated for another 48 h at 37°C. In ThT-binding measurements, mix 80 µl of Aβ-AChE plus AP5 fraction with 10 µl of 100 µM ThT and 10 µl of 500 mM glycine-NaOH. Fluorescence emission at 485 nm was measured on a Spectra MAX GeminiXS fluorometerat an excitation wavelength of 450 nm. For each determination, the fluorometer was calibrated by ThT reagent alone.

Open field test
To assess locomotor activity, mice were individually tested in an open-field box (38 cm × 38 cm × 25 cm). The movement of the mouse was tracked for 5 min with open field video analysis system, and the total move distances was automatically recorded as a direct measure of locomotor activity. Between each trial, the walls and floor were wiped with 75% ethanol.

Novel object recognition, spatial object location
For the novel object recognition task, mice were individually exposed to the configuration of two identical objects (A+A) in open field during a 5 min acquisition trial. Exactly 1 h or 24 h later, during the retention sessions, mice were placed back into the same arena where they were exposed to one familiar object A and to a novel object B (after 1 h) and object C (after 24 h). The chamber was cleaned with 75% ethyl alcohol to eliminate odor before the next test. The discrimination index was calculated and defined as: (time spent exploring the new object-time spent exploring the familiar object) / total exploration time for the novel and familiar object.
For the spatial object location task (an object moved to a new location), mice first explored the two objects placed in two corners of the open field for 5 min in training period. After 1 h or 24 h, the mice received a 5 min retention trial with a new spatial configuration with an object moved from one corner to the opposite corner of the open field. The discrimination index was calculated and defined as: (time spent exploring the displaced object-time spent exploring nondisplaced objects) / total exploration time for the two objects during the 5 min retention trial.

Y-maze
Work on spatial recognition memory was tested by Y-maze. The symmetrical Y-maze consisted of three arms joined in the middle to form a Y shape. During the training trial, one of the arms (novel arm) was closed, and each mouse was placed in the start arm and allowed to explore the start arm freely for 5 min. One hour later, the testing trial was performed, and all three arms were open. Mice were allowed to explore the three arms for 5 min. The ratio between the distance spent in the novel arm and the total distance travelled in the maze was quantified as preference index.

Morris water maze (MWM)
The effect of AP5 on spatial learning and memory performance of mice was tested by the MWM. Briefly, during the training period, the mice were allowed to freely swim for 60 s to search for the platform (10 cm in diameter, 2 cm beneath the water surface), which was fixed in the center of one of the four quadrants. Mice went through three trials each day and were alternated among three random quadrants for five consecutive days. Mice that failed to find the platform within 60 s were manually guided to platform and allowed to stay on it for 30 s. The time (escape latency) for each mouse to find the way and reach the target platform was analyzed. For the probe trial, 24 h after the last training session, the platform was removed and the mice were allowed to swim for 60 s. The parameters measured during the probe trial included number of platform crossing and time spent in the target quadrant.
Signaling Technology, Danvers, MA, USA), anti-Complement Component C3d (1:100, BAF2655, R&D, Minneapolis, MN, USA). The slides were rinsed three times in PBS. Then the slides were labeled with fluorescent secondary antibodies as follows: goat anti-rabbit IgG H&L Alexa Fluor® 488 (ab150077, Abcam), goat anti-rabbit IgG H&L Alexa Fluor® 594 (ab150080, Abcam) or goat anti-mouce IgG H&L Alexa Fluor® 594 (ab150116, Abcam). Slides were washed three times with PBS before being mounted with DAPI fluoromount. Images were captured using an Olympus SpinSR10 scanner. For the quantification of Aβ plaque-associated microglia and astrocyte, Iba1-and GFAP-immunopositive cells around plaques were counted. On each slide, ten plaques were randomly determined and the microglia (nuclei) in the immediate vicinity (two times the plaque diameter) of each plaque were counted. For colocalization of PSD95 and synaptophysin, images were analyzed with the puncta analyzer plugin with a minimum pixel specification of 4 in Image J and thresholding was consistent for every image.

Western blotting
Protein samples was separated in 10% or 12% SDS-PAGE and then transferred to PVDF membranes. The membranes were incubated with 5% non-fat milk blocking solution for 1 h at room temperature, and then probed with primary antibodies overnight at 4°C, including anti-amyloid precursor protein full length (APPfl, These analyses were normalized to β-tubulin (1:1000, 10494-1-AP, Proteintech). volume of distribution at steady state (Vss),and clearance (Cl). The extent of brain penetration were measured by brain-to-plasma distribution ratio (B/P).The oral B/P value was calculated as: AUCINF brain/AUCINF plasma ×100.

Supplementary Table1 Predicted physicochemical values for oral absorption and blood-brain barrier penetration with Lipinski's rule of five and BBB score in silico
Bioavailability (F%) after oral administration was calculated as: AUCINF P.O./AUCINF i.v. /4 ×100. Quantity One software. *P < 0.05 compared with WT mice, # P < 0.05, ## P < 0.01 compared with APP/PS1 group by one-way ANOVA followed by a Turkey's post-hoc test, n = 6 mice per group.