Arylbenzofurans from the Root Bark of Morus alba as Triple Inhibitors of Cholinesterase, β-Site Amyloid Precursor Protein Cleaving Enzyme 1, and Glycogen Synthase Kinase-3β: Relevance to Alzheimer's Disease

Abstract

Cholinesterase, β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and glycogen synthase kinase-3β (GSK-3β) are the three main enzymes responsible for the early onset of Alzheimer's disease (AD). The main aim of the present study was to delineate and accentuate the triple-inhibitory potential of arylbenzofurans from Morus alba against these enzymes. Overall, the enzyme inhibition assays demonstrated the prominence of mulberrofuran D2 as an inhibitor of AChE, BChE, BACE1, and GSK-3β enzymes with IC₅₀ values of 4.61, 1.51, 0.73, and 6.36 μM, respectively. Enzyme kinetics revealed different modes of inhibition, and in silico modeling suggested that mulberrofuran D2 inhibited these enzymes with low binding energy through hydrophilic, hydrophobic, and π-cation interactions in the active site cavities. Similarly, in Aβ-aggregation assays, mulberrofuran D2 inhibited self-induced and AChE-induced Aβ aggregation in a concentration-dependent manner that was superior to reference drugs. These results suggest that arylbenzofurans from M. alba, especially mulberrofuran D2, are triple inhibitors of cholinesterase, BACE1, and GSK-3β and may represent a novel class of anti-AD drugs.

Figure 1

Structures of compounds isolated from root barks of M. alba Linn.

Figure 2

Dixon plots and Lineweaver–Burk plots for AChE (A, D), BChE (B, E), and BACE1 (C, F) inhibition, respectively, by mulberrofurans D2.

Figure 3

Dixon and Lineweaver–Burk plots for GSK-3β inhibition by mulberrofuran D2, (A, C) at constant ATP and (B, D) at constant GSM substrate, respectively.

Figure 4

Molecular docking of AChE (A) and BChE (D) binding with mulberrofuran D2 along with positive controls. Chemical structures of galantamine, donepezil, tacrine, 5i, and mulberrofuran D2 are shown as red, blue, black, pink, and green stick, respectively. Closeup of the mulberrofuran D2 binding pose in the active site of AChE (B, C) and BChE (E, F).

Figure 5

Molecular docking of BACE1 (A) binding with mulberrofuran D2 along with positive controls. Chemical structures of QUD, sinensetin, and mulberrofuran D2 are shown as red, blue, and green sticks, respectively. Closeup of the mulberrofuran D2 binding pose in the active site of BACE1 (B, C).

Figure 6

Molecular docking of glycogen synthase kinase (GSK)-3β binding with mulberrofuran D2 along with positive controls (A). Chemical structures of AMP–PNP, andrographolide, VP0.7, and mulberrofuran D2 are shown as red, blue, white, and green sticks, respectively. Magnesium ions are shown as brown spheres. Closeup of the mulberrofuran D2 binding pose in the ATP-binding site (B, C) and substrate-binding site (D, E), respectively.

Figure 7

Results from the thioflavin T assay. (A) Inhibition of Aβ_1–42 self-induced aggregation and (B) AChE-induced Aβ_1–40 aggregation by mulberrofuran D2 and reference compounds, curcumin and donepezil. The data represent the mean ± SD of three independent experiments. Different letters in graphs are significantly different with Duncan’s test at p < 0.05.