As a contribution to the development of new dual/multifunctional drugs, a novel therapeutical scaffold merging key structural features from memantine and M30D was designed, synthesized, and explored for its AChE/BuChE inhibitory activity and neuroprotective effects. All synthetized hybrids were not able to inhibit AChE, but most of them exhibit inhibition with high selectivity toward butyrylcholinesterase (BuChE). Notably, among the tested compounds, amantadine/M30D hybrids with six, seven, nine, and twelve methylene groups in the spacer (5d, 5e, 5f, and 5g) not only highlighted having the best potency and selective butyrylcholinesterase inhibition greater than 83% but also, particularly 5e and 5d, elicited considerable neuroprotection when evaluated in pretreatment conditions, by reducing injury effects caused by glutamate with maximum protection reached about 47.82 +/- 0.81% (5e) and 42 +/- 2.20% (5d) in comparison with memantine (37.27 +/- 2.69%). Likewise, we chose 5e as the hit compound, which in a glutamate excitotoxity coculture model prevented astroglia reactivity and neuronal death, as well as a 91% restoration of calcium levels and an increasing ATP level in both pre-/post-treatments of 61.48 +/- 4.60 and 45.16 +/- 10.55%, respectively. Regarding docking studies, a blockade of the NMDA channel pore by 5e would explain its neuroprotective response. Finally, the hit compound 5e exhibited in vitro blood-brain barrier (BBB) permeability and human plasma stability, as well as an optimal in silico neuropharmacokinetic profile. From a therapeutic perspective, merging key pharmacophoric features from memantine and M30D provides a new medicinal scaffold with dual-/multifunctional properties and human plasma stability for the future development of potential drugs for treating AD.
Zephyranthes carinata Herb., a specie of the Amaryllidoideae subfamily, has been reported to have inhibitory activity against acetylcholinesterase. However, scientific evidence related to their bioactive alkaloids has been lacking. Thus, this study describes the isolation of the alkaloids of this plant, and their inhibition of the enzymes acetylcholinesterase (eeAChE) and butyrylcholinesterase (eqBuChE), being galanthine the main component. Additionally, haemanthamine, hamayne, lycoramine, lycorine, tazettine, trisphaeridine and vittatine/crinine were also isolated. The results showed that galanthine has significant activity at low micromolar concentrations for eeAChE (IC(50) = 1.96 microg/mL). The in-silico study allowed to establish at a molecular level the high affinity and the way galanthine interacts with the active site of the TcAChE enzyme, information that corroborates the result of the experimental IC(50). However, according to molecular dynamics (MD) analysis, it is also suggested that galanthine presents a different inhibition mode that the one observed for galanthamine, by presenting interaction with peripheral anionic binding site of the enzyme, which prevents the entrance and exit of molecules from the active site. Thus, in vitro screening assays plus rapid computer development play an essential role in the search for new cholinesterase inhibitors by identifying unknown bio-interactions between bioactive compounds and biological targets.
Title: Alkaloids of Amaryllidaceae as Inhibitors of Cholinesterases (AChEs and BChEs): An Integrated Bioguided Study Cortes N, Sierra K, Alzate F, Osorio EH, Osorio E Ref: Phytochem Anal, 29:217, 2018 : PubMed
INTRODUCTION: Enzymatic inhibition of acetylcholinesterase (AChE) is an essential therapeutic target for the treatment of Alzheimer's disease (AD) and AChE inhibitors are the first-line drugs for it treatment. However, butyrylcholinesterase (BChE), contributes critically to cholinergic dysfunction associated with AD. Thus, the development of novel therapeutics may involve the inhibition of both cholinesterase enzymes. OBJECTIVE: To evaluate, in an integrated bioguided study, cholinesterases alkaloidal inhibitors of Amaryllidaceae species. METHODOLOGY: The proposed method combines high-performance thin-layer chromatography (HPTLC) with data analysis by densitometry, enzymatic bioautography with different AChEs and BChEs, the detection of bioactive molecules through gas chromatography mass spectrometry (GC-MS) analysis of spots of interest, and theoretical in silico studies. RESULTS: To evaluate the bioguided method, the AChE and BChE inhibitory activities of seven Amaryllidaceae plant extracts were evaluated. The alkaloid extracts of Eucharis bonplandii exhibited a high level of inhibitory activity (IC50 = 0.72 +/- 0.05 mug/mL) against human recombinant AChE (hAChE). Regarding human serum BChE (hBChE), the bulb and leaf extracts of Crinum jagus had the highest activity (IC50 = 8.51 +/- 0.56 mug/mL and 11.04 +/- 1.21 mug/mL, respectively). In the HPTLC spots with high inhibitory activity, several alkaloids were detected using GC-MS, and some of these alkaloids were identified. Galanthamine, galanthamine N-oxide and powelline should be the most prominent inhibitors of substrate accommodation in the active site of the Torpedo californica AChE (TcAChE), hAChE and hBChE enzymes. CONCLUSIONS: These results are evidence of the chemical relevance of the Colombian's Amaryllidaceae species for the inhibition of cholinesterases and as potent sources for the palliative treatment of AD. Copyright (c) 2017 John Wiley & Sons, Ltd.
AIMS: Amaryllidaceae alkaloids exhibit a wide range of physiological effects, of which the acetylcholinesterase (AChE) inhibitory activity is the most relevant. However, scientific evidence related to their neuroprotective effectiveness against glutamate-induced toxicity has been lacking. Thus, the purpose of this study was to conduct a comparative study of the neuroprotective activity and the AChE inhibitory activity of species of Amaryllidaceae. MAIN METHODS: The neuroprotective activity against glutamate-induced toxicity was measured in rat cortical neurons and the Ellman method was employed for the quantification of acetylcholinesterase inhibitory activity of alkaloidal extracts of five species of Amaryllidaceae (Crinum jagus, Crinum bulbispermum, Hippeastrum barbatum, Hippeastrum puniceum and Zephyranthes carinata). The alkaloid Amaryllidaceae patterns based on GC/MS analyses were also investigated. KEY FINDINGS: The results showed that the alkaloidal extract from C. jagus presented a high neuroprotective activity in both pre- and post-treatments against a glutamate excitotoxic stimulus. Furthermore, the alkaloid extracts from C. jagus and Z. carinata revealed an inhibitory activity of AChE from the electric eel with IC50 values of 18.28+/-0.29 and 17.96+/-1.22mug/mL, respectively. In addition, 46 alkaloids were detected by GC/MS, and 20 of them were identified based on their mass spectra and retention index. The results suggest that the neuroprotective effects might be associated with lycorine and crinine-type alkaloids, whereas the acetylcholinesterase enzyme inhibitory activity could be related to galanthamine and lycorine-type alkaloids, although not based on synergistic processes. SIGNIFICANCE: In summary, Amaryllidaceae species are sources of alkaloids with potential use for Alzheimer's disease.
