Geissoschizoline, a promising alkaloid for Alzheimer’s disease: Inhibition of human cholinesterases, anti-inflammatory effects and molecular docking

Highlights

• Geissoschizoline is a potent inhibitor of both humans AChE and BChE.

• Geissoschizoline plays a promising anti-inflammatory role.

• It reduces the microglial release of NO• and TNF-α.

• It shows mixed-type inhibition mechanism; interacts with AS and PAS sites of enzyme.

• Geissoschizoline behaves as dual-site inhibitor.

Abstract

Due to the lack of effective pharmacotherapy options to treats Alzheimer’s disease, new strategies have been approached in the search for multi-target molecules as therapeutic options. In this work, four indole alkaloids, geissoschizoline, geissoschizone, geissospermine, and 3′,4′,5′,6′-tetradehydrogeissospermine were isolated from Geissospermum vellosii (Pao pereira) and evaluated for their anticholinesterase activities. While geissospermine inhibited only butyrylcholinesterase (BChE), the other alkaloids behaved as non-selective inhibitors of acetylcholinesterase (AChE) and BChE. In cell viability tests, only geissoschizoline was not cytotoxic. Therefore, geissoschizoline actions were also evaluated in human cholinesterases, where it was twice as potent inhibitor of hBChE (IC₅₀ = 10.21 ± 0.01 µM) than hAChE (IC₅₀ = 20.40 ± 0.93 µM). On enzyme kinetic studies, geissoschizoline presented a mixed-type inhibition mechanism for both enzymes. Molecular docking studies pointed interactions of geissoschizoline with active site and peripheral anionic site of hAChE and hBChE, indicating a dual site inhibitor profile. Moreover, geissoschizoline also played a promising anti-inflammatory role, reducing microglial release of NO and TNF-α at a concentration (1 μM) ten and twenty times lower than the IC₅₀ values of hBChE and hAChE inhibition, respectively. These actions give geissoschizoline a strong neuroprotective character. In addition, the ability to inhibit hAChE and hBChE, with approximate inhibitory potencies, accredits this alkaloid for therapeutic use in the moderate to severe phase of AD. Thus, geissoschizoline emerges as a possible multi-target prototype that can be very useful in preventing neurodegeneration and restore neurotransmission.

Introduction

Alzheimer's disease (AD) remains a major scientific challenge. This disease is the most common form of dementia in elderly people, accounting for 60–80% of total cases of dementia worldwide, currently estimated at more than 45 million [1] and expected to triple by 2050 as a result of increased life expectancy [2]. AD is characterized by marked atrophy of the cerebral cortex and loss of cholinergic neurons from the basal forebrain [3]. Its major pathological features are related to neuronal degeneration and include extracellular deposition of amyloid beta (Aβ) plaques, intracellular formation of neurofibrillary tangles and neuroinflammation [4], [5].

Increasing evidences [6], [7], [8], [9] highlight the important role of neuroinflammation in the degenerative process of AD. The neuroinflammatory process is associated with increased populations of activated microglia and astrocytes by diversified stimuli, such as infection and tissue injury [10], [11]. In AD, these cells are attracted and activated by Aβ plaques [8], [12], and release a series of pro-inflammatory mediators, resulting in inflammatory responses that may further damage neuronal cells, stimulate Aβ synthesis and increase microglial activation through a positive feedback loop [6], [7]. Tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1β) are the main neurotoxic agents released by microglia stimulated by Aβ [12], [13], [14].

So far the therapeutic treatment remains the same as twenty years ago and is based on three anticholinesterase drugs (galantamine, rivastigmine and donepezil), which restore synaptic transmission but cannot contribute to the reduction of brain damage caused by the neuroinflammatory process already installed [15], [16]. To date, there is a general perception that drugs acting only on a specific target are not suitable for the treatment of AD. Therefore, approaches based on multi-target ligands are being increasingly applied in AD, in an attempt to obtain a drug capable of preventing the neurodegenerative process and re-establish neurotransmission [17], [18].

We have been working with Geissospermum vellosii Fr. All (Pao pereira), a Brazilian tree whose stembarks are rich in indole alkaloids. In previous works , we demonstrated the intense anticholinesterase activity, in vitro, of a fraction (PP) from stembarks of Pao pereira and, on memory tests in mice, PP reversed the scopolamine-induced amnesia in passive avoidance and in Morris water maze tests, with no noticeable peripheral or central cholinergic side effects [17]. Furthermore, in classic murine models of inflammation and pain, PP presented anti-inflammatory and antinociceptive activities [19].

In this work we isolated and purified four indole alkaloids from Pao pereira: geissoschizoline (1), geissoschizone (2), geissospermine (3), and 3′,4′,5′,6′-tetradehydrogeissospermine (4), an anhydronium base of geissospermine [20] (Fig. 1), and evaluated their cytotoxicity and anticholinesterase activities. For geissoschizoline (1), the most promising alkaloid, we also evaluated its ability to inhibit both humans acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE), performed the kinetics of inhibition studies, molecular docking, and investigated the neuroprotective and antineuroinflammatory activities in LPS-stimulated microglial cells. As far as we know, this is the first report showing the anticholinesterase activity of these alkaloids (1, 2, 3, 4) and the multi-target actions of geissoschizoline (1), highlighting this alkaloid as a possible therapeutic prototype to treats AD.