Assessing the acetylcholinesterase inhibitory potential of novel pharmacophores for potential treatment of Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is the predominant form of dementia which primarily affects the elderly. To date the aetiology of AD has not been fully elucidated, though may include: loss of cholinergic transmission; excessive accumulation of amyloid-beta (Aβ) aggregates extracellularly in the brain; accumulation of tau protein intracellularly; glutamate excitotoxicity and oxidative stress. Current AD therapeutic approaches rely on the administration of acetylcholinesterase inhibitors (AChEIs), memantine and monoclonal antibodies. However, these drugs provide only symptomatic relief and do not prevent progressive neurodegeneration.

Among the above-mentioned approved drugs, AChEIs are suggested to have the ability to inhibit the synthesis, deposition and aggregation of toxic Aβ proteins and tau-protein phosphorylation. For these reasons, AChE has received much attention as the prime target in drug discovery and development for the treatment of AD. However, an overall attrition rate of more than 95% has been noted for novel drugs targeting neurodegenerative diseases, and this is mainly due to a lack of efficacy, blood-brain barrier (BBB) permeability, multifactorial nature and/or toxicity. This reinforces the rationale for assessing a broad spectrum of effects of pharmacophores that show potential for therapeutic use at a preclinical level to reduce the high attrition rate. The aim of the study was to identify novel pharmacophores with the potential to be developed further as treatments for AD.

An in-house miniaturised AChE assay was used to assess the AChEI activity of 1,453 synthetic compounds from a commercial library (Charles River Laboratory/BioFocus, UK) housed at the Council for Scientific and Industrial Research (CSIR). The selected compounds had been previously identified through in silico screening of the compound library within the active site of the electric eel and human AChE enzyme. The Swiss Institute for Bioinformatics pharmacokinetic prediction tool, SwissADME, was used to assess the compound’s BBB permeability and pharmacokinetics. Combinational AChEI effects of active pharmacophores and donepezil were assessed using a checkerboard assay. The sulforhodamine B assay was employed to determine the active compounds and synergistic combinations cytotoxic potential after 72 h in SH-SY5Y neuroblastoma and brain endothelial (bEnd.5) cells. Michaelis-Menten kinetics of active in silico BBB-permeable compounds were determined using the miniaturised AChE assay to elucidate its inhibitory mechanism. Ultra-performance liquid chromatography (UPLC) coupled to a mass spectrometry (MS) was used for quality control analysis to ensure the chemical integrity of compounds. Cellular AChEI activity of active in silico BBB-permeable predicted compounds was determined using an SH-SY5Y AChE-based assay. An in vitro BBB model was used to assess the effect of compounds on the integrity of the bEnd.5 monolayer, as well as the permeation of compounds using UPLC-MS.

The miniaturised assay’s validation parameters were within acceptable ranges (coefficient of variation ≤ 20%, Z’-factor ≥ 0.5 but < 1, and signal window ≥ 2). Only six compounds (A8, A51, A73, A136, A175 and A176) from the primary screen of subset A (n = 400) were regarded as possessing AChEI activity (≥ 60% inhibition at 5 µM). For the secondary screen, two subsets of structurally similar compounds, subset B (n = 508) and subset C (n = 545) were selected and evaluated. The AChEI activity for a further six compounds (C33, C43, C53, C82, C129 and C189) were regarded as positive and ranged between 62 and 72%. Of the 12 active compounds, compound A51 had the lowest half maximal inhibitory concentration (IC50) of 0.20 µM, albeit less potent than donepezil (IC50 = 0.03 µM). The SwissADME pharmacokinetic tool predicted that compounds A8, A73, A136, C53 and C129 were BBB-permeable. Synergism (combination index [CI] < 1) was observed between: ¼IC50 donepezil and ½IC50 A136 (CI = 0.61); ¼IC50 donepezil and IC50 A136 (CI = 0.81); ½IC50 donepezil and ½IC50 A136 (CI = 0.81); ½IC50 donepezil and IC50 A136 (CI = 0.69); ¼IC50 donepezil and ¼IC50 C53 (CI = 0.82). Compounds A8 and A73 were not cytotoxic (IC50 >100 µM), whereas A136 (IC50 = 4.99 µM) and C129 (IC50 = 13.64 µM) possessed greater cytotoxicity than donepezil and C53 (IC50 = 43.00 and 40.45 µM, respectively). The combination of A136 and donepezil eradicated all cell growth, while moderate cytotoxicity (20% cell density reduction) was observed for the combination of C53 and donepezil. Furthermore, no compounds were found to induce cytotoxicity in the bEnd.5 cells (IC50 >100 µM).

Donepezil displayed mixed competitive and non-competitive inhibition, while A8 indicated uncompetitive inhibition, and A73 and C53 mixed inhibition. No decomposition was observed for the active in silico BBB-permeable non-cytotoxic compounds. Compound A73 exhibited dose-dependent AChEI activity in SH-SY5Y neuroblastoma cells, whereas A8 and C53 did not. At the IC50, 61% AChEI activity was observed for C53, with the combination of C53 and donepezil indicating 69% AChEI activity. The latter possessed the highest in situ activity. All compounds, including donepezil, increased transendothelial electrical resistance (TEER) over 48 h exposure, suggesting decreased BBB-permeability of the bEnd.5 monolayer. Furthermore, all compounds were detected in both the apical and basolateral chambers of the in vitro BBB model. This suggests in vitro BBB permeation of the compounds and further supporting the in silico BBB permeability predictions. Factors such as efficacy, BBB permeability, and/or toxicity contribute to the high attrition rate noted for central nervous system targeting drugs. In this study 1,453 synthetic compounds were assessed for these aspects in search for potential treatment for AD. Among the 12 compounds that displayed AChEI activity, compound A51 had the greatest AChEI activity. However, A51 was predicted in silico to be impermeable to the BBB and would most likely not be further developed as it may not cross the BBB. Compound A136 was cytotoxic as monotherapy and in combination with donepezil, indicating its capability to kill neuronal cells. Due to the low in situ AChEI activity displayed by compounds A8, A73 and C53, these compounds might not inhibit AChE efficiently in vivo. Therefore, compound C53 alone (at the IC50 concentration) and combined with donepezil indicated the potential for further investigation as AChEIs given their promising acellular and cellular AChEI activity, in silico BBB-permeability, absence of cytotoxicity, and ability to penetrate the in vitro BBB monolayer.

Keywords: acetylcholinesterase, acetylcholinesterase inhibitors, Alzheimer’s disease, blood-brain barrier, cytotoxicity.