Differential senolytic inhibition of normal versus Aβ-associated cholinesterases: implications in aging and Alzheimer’s disease

Introduction

The concept of cellular senescence was first used to describe diploid cells that ceased to proliferate [1]. Under normal conditions, cellular senescence has been suggested to be involved in maintaining tissue homeostasis during embryonic development, wound healing and repair, and suppression of tumor proliferation [2]. However, chronic accumulation of these cells can lead to deleterious effects. Cellular senescence is described as a hallmark of aging and age-related conditions such as Alzheimer’s disease (AD) [2, 3].

Cellular senescence is identified by permanent cell cycle arrest and resistance to apoptosis in proliferative cells such as astrocytes, microglia, oligodendrocytes, and endothelial cells [2]. It is independently regulated by the tumor suppressor p53/p21 and p16/pRB pathways [3] that result in preventing G₁ to S phase transition in the cell cycle [4, 5]. Cellular senescence is triggered by activation of harmful genes and sustained endogenous and exogenous stresses, including DNA and oxidative damage, telomere shortening, mitogenic and oncogenic signaling, mitochondrial dysfunction, and chromatin and lysosomal alterations [2].

Senescent cells are characterized by increased accumulation of the lysosomal enzyme senescence-associated-β-galactosidase [6] and lipofuscin [7], as well as the robust secretion of senescence-associated secretory phenotype (SASP) factors [8] including activated inflammatory cytokines (e.g. interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNFα), chemokines, metalloproteinases, extracellular matrix components, and growth factors and regulators [2, 9]. Non-proliferative post-mitotic cells, like neurons, can also exhibit an increase in senescence-associated-β-galactosidase and lipofuscin. They also secrete SASP inflammatory factors [10], the hallmarks of senescence.

For more than 30 years, there has been an interest in the association between cognitive decline and cholinergic neurotransmission in aging, particularly in AD [11–13]. Regarding brain senescence, many facets of the cholinergic system, such as the neurotransmitter acetylcholine, synthesizing enzyme choline acetyltransferase (ChAT) and nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR, respectively), have received great attention [14–18]. However, there have been few studies exploring the relationship between senescence and cholinesterases (ChEs) [18–23].

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are related serine hydrolases that co-regulate the cholinergic system through hydrolysis of acetylcholine [24, 25]. In the normal brain, BChE is primarily expressed in white matter, glia and distinct subcortical populations of neurons important for cognition and behavior while AChE is found in neurons and neuropil throughout the brain, with very little in white matter [24, 26, 27]. Although both ChEs share structural similarities [28] and functional homology [29], both AChE and BChE have distinct functions in health and disease beyond neurotransmission [25, 30].

In normal aging, there is a gradual decline in cholinergic function resulting in reduced levels of acetylcholine, ChAT, nAChRs, mAChRs, and AChE [31]. On the other hand, BChE activity has been shown to increase with age in the normal brain [32]. These changes have been correlated with age-related cognitive decline [11, 33]. In AD, pronounced cholinergic dysfunction is attributed to widespread loss of cholinergic neurons and significantly reduced acetylcholine levels that contribute to the salient cognitive and behavioural deficits characteristic of the disease [34, 35]. In turn, AChE levels are significantly reduced [36], while BChE levels remain the same [37] or increase [36, 38]. AChE and BChE are associated with the pathological hallmarks of AD, amyloid-β (Aβ) plaques and tau neurofibrillary tangles [39, 40], however, these enzymes are not associated with other dementia-related neuropathologies [41–43]. A role for BChE in the maturation of Aβ plaques, and consequently, AD progression [39] has been suggested due to its distinct association with the “malignant” fibrillar plaques of AD brains but not the “benign” non-fibrillar plaques typically found in cognitively normal, aged brains [40, 41]. Plaque-bound ChEs appear to undergo a conformational change that alters their biochemical properties and differentiates these enzymes from those associated with normal neural elements [37, 44–46]. These biochemical changes lead to altered binding affinities [44, 47] and subsequent inhibitor sensitivities between ChEs associated with AD pathology and with normal neural elements [37, 44, 45]. Such alterations have significant implications in the design and development of potential next-generation therapeutic approaches targeting AChE and BChE for treatment of AD.

In addition to termination of cholinergic signaling, BChE is also involved in hydrolysis of the growth hormone secretagogue ghrelin, drug hydrolysis, lipid metabolism, detoxification of xenobiotics, and protein interaction and modification [25, 48, 49]. Moreover, both ChEs are also involved in cell proliferation and neural development [50, 51]. The non-cholinergic functions of AChE and BChE have been attributed to sharing similar sequence homologies of cell adhesion and ChE-like cell adhesion molecules [52, 53]. However, under pathological conditions, cell proliferation and apoptotic functions of ChEs appear altered. For instance, high levels of BChE activity and BCHE expression have been implicated in the rapid undifferentiated cellular proliferation of several brain tumor types [54, 55]. However, this rate of proliferation was reduced through the abolition of BCHE and BChE enzyme [55]. The BCHE gene has been shown to be upregulated in cell immortality in tumor development, whereby unchecked cell proliferation and evasion of cell death prevail, however, BChE suppression was shown to increase the rate of apoptotic processes [56].

Many approved and experimental cancer therapies are pro-apoptotic agents that exert their effects by targeting several intrinsic and extrinsic signaling pathways, including tumor suppressor pathways [57]. Furthermore, a number of these anti-cancer agents have been repurposed as senolytic agents as they also exhibit suppression of cellular senescence [58]. However, only a few senolytics have been identified as potential ChE inhibitors (ChEIs) [59, 60]. ChEIs that target AChE, BChE, or both [61], are a class of drugs that have been widely used for the treatment of several conditions, most notably the cognitive and behavioral symptoms associated with AD [62]. Several ChEIs, such as the AChE-selective donepezil and galantamine and the BChE-selective rivastigmine have shown to modulate apoptotic pathways and exert potent anti-inflammatory effects, in addition to their primary mode of action of increasing acetylcholine levels [63–65].

Cellular senescence is a significant contributor to low levels of chronic inflammation in normal aging, with notably increased levels of inflammatory cytokines in AD [2]. There is an established relationship between cholinergic function and immunomodulation [66–69]. Immune cells are equipped with the necessary cholinergic components, including ChAT, nAChRs and mAChRs, as well as AChE and BChE, required for modulation of cell activity through the cholinergic anti-inflammatory pathway (CAIP). ChEIs stimulate CAIP [70, 71], leading to decreases in macrophage and microglial secretion of pro-inflammatory cytokines such TNFα, IL-1β, and IL-6 [66, 67, 72].

Senolytic agents such as dasatinib, nintedanib, fisetin, and quercetin and the nootropic, anti-aging agent meclofenoxate hydrochloride (also referred to as centrophenoxine) (Figure 1) have undergone or are currently undergoing human clinical trials in patients with age-related diseases [73–75]. They, along with the senolytic GW2580, have also had promising results in ameliorating inflammation, decreasing pathological load, and improving cognition in preclinical models of aging and AD [76–84]. However, the mechanism(s) by which senescent cells and their pro-inflammatory SASP factors contribute to aging and neurodegeneration, including neuropathology and dysfunction of the cholinergic system and CAIP is unknown. It has been suggested that increasing the bioavailability of acetylcholine for CAIP activation, via ChE inhibition, could be an attractive avenue for decreasing neuroinflammation and modulating apoptotic activity in aging and AD [72, 85].

Figure 1. Chemical structures of approved and investigational senolytic (1-5) and nootropic (6) agents. Structures were drawn using ChemDoodle: 2D Chemical Drawing, Publishing and Informatics (Version 11.10, iChemLabs, LLC, Somerset, NJ, USA).

Because of the importance of ChEs in cell communication, the cell cycle, CAIP and AD neuropathology, we evaluated the enzyme kinetic properties of several senolytic and nootropic agents towards AChE and BChE to determine if they were inhibitors of these enzymes. In addition, since biochemical properties of ChEs associated with AD pathology are different from those associated with normal neural elements, using histochemical methods, we also evaluated whether these compounds inhibited ChEs that are associated with Aβ plaques in AD. We show that the selected senolytics and nootropic inhibit ChEs associated with plaques but not the enzymes associated with normal neural elements. Together, these findings suggest that these senolytic agents may exert their beneficial effects, in part, by inhibiting ChE-associated with Aβ pathology and providing new avenues for the development of the next generation of ChE inhibitors targeting AD pathology-associated ChEs.

Results

Enzyme kinetic studies

To determine experimental inhibition constants (K_i values) and mode of inhibition for senolytic and nootropic compounds 1-6 with ChEs, Lineweaver-Burk plots were generated (Figures 2, 3), as described previously [86]. Calculated experimental K_i values (μM) are shown in Table 1. Where ChE inhibition constants for compounds were identified previously in the literature [60, 87], K_i values observed herein were also similar (Table 1). All compounds showed some degree of ChE inhibition, except for 2 and 5 which did not inhibit BChE. Nintedanib (2) showed activity that was too low to conduct a full inhibition study.

Figure 2. Lineweaver-Burk (LB) plots showing inhibition of acetylcholinesterase (AChE) upon treatment with varying concentrations of senolytic and nootropic agents 1-6 (A–F, respectively). Each compound was assessed at a range of concentrations (0-83 μM), with 0 M (▲), middle concentration (●), and highest concentration (■). Slopes from LB plot trendlines were plotted against compound concentrations to generate K_i values as the x-intercept (insert graphs). Enzyme kinetic experiments were performed in triplicate and kinetic parameter values were averaged.

Figure 3. Lineweaver-Burk (LB) plots showing inhibition of butyrylcholinesterase (BChE) upon treatment with varying concentrations of senolytic and nootropic agents 1, 3, 4 and 6 (A–D, respectively). Compounds 2 and 5 showed too little inhibition to complete LB kinetics. Each compound was assessed at a range of concentrations (0-140 μM), with 0 M (▲), middle concentration (●), and highest concentration (■). Slopes from LB plot trendlines were plotted against compound concentrations to generate K_i values as the x-intercept (insert graphs). Enzyme kinetic experiments were performed in triplicate and kinetic parameter values were averaged.

Table 1. Experimental inhibition constants (K_i values) and mode of inhibition for compounds 1-6 with acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).

Compound	Experimental AChE Ki (μM) (Mode of inhibition)Literature AChE Ki values	Experimental BChE Ki (μM) (Mode of inhibition) Literature BChE Ki values
Dasatinib (1)	28.22	3.36
	(Mixed Non-competitive)	(Mixed Non-competitive)
Nintedanib (2)	7.10	No Inhibition
	(Mixed Non-competitive)
Fisetin (3)	109.81	94.35
	(Competitive)	(Uncompetitive)
	100.2a	94.2a
Quercetin (4)	62.19	51.49
	(Mixed Non-competitive)	(Mixed Non-competitive)
	38.3a, 76.2b	68.0a, 46.8b
GW2580 (5)	70.97	No Inhibition
	(Mixed Non-competitive)
Meclofenoxate Hydrochloride (6)	63.20	76.30
	(Mixed Non-competitive)	(Mixed Non-competitive)
aKi values taken from Katalinić et al. [60].
bKi values taken from Orhan et al. [87].

Modes of inhibition were determined from Lineweaver-Burk plots, with most compounds showing mixed non-competitive inhibition, often indicative of allosteric binding – double reciprocal plot trendlines crossing between x- and y-axes (Figures 2, 3). Fisetin (3) was the exception, showing robust competitive inhibition for AChE – trendlines crossing at the y-axis (Figure 2C), and clear uncompetitive inhibition with BChE – trendlines are parallel (Figure 3B). These results show that 3 outcompetes acetylthiocholine iodide (ATChI) for the active site of AChE; while 3 only targets the enzyme-butyrylthiocholine iodide (BTChI) complex at which point it could bind to the active or allosteric sites to render inhibition of BChE. All other senolytic and nootropic agents are likely binding to only allosteric sites or a combination of active and allosteric sites, to produce the observed mixed non-competitive inhibitory responses.

Histochemical studies

Of the AD brains selected for this study (Table 2), three cases were previously described to have robust Aβ-, AChE-, and BChE-associated plaque loads in the cerebral cortex [42, 46, 88]. The fourth case, BB11-010, also showed severe AD and ChE-associated pathology, consistent with the other selected cases.

Table 2. Demographic information for Alzheimer’s disease (AD) brain tissues used.

Case #	Neuropathological diagnosis	Sex	Age (y)	Brain weight (g)	Braak stage	CERADa plaque score
BB11-010	AD	M	93	1254	IV	Frequent
BB11-026	AD	M	98	1066	V	Frequent
BB11-039	AD	F	92	1009	IV	Frequent
BB12-035	AD	F	84	1120	VI	Frequent
aCERAD, Consortium to Establish a Registry for Alzheimer’s Disease

Histochemical staining was done at pH 6.8 to allow for the visualization of ChE activity associated with plaques, while pH 8.0 was used to visualize ChE activity associated with normal neural elements, as shown previously [37, 88]. The staining quality of ChEs in tissues selected for this study were examined using the standard Karnovsky-Roots (KR) substrate staining method and found to be appropriate prior to treatment with compounds 1-6.

The use of senolytic and nootropic compounds 1-6 in KR histochemical staining resulted in varying degrees of inhibition of AChE and BChE activity associated with normal neural elements (pH 8.0) and plaques (pH 6.8) in human AD brain tissues, ranging from no inhibition (-) to strong inhibition (xxx). Qualitative analysis of each of the four AD cases selected is described as the average rankings for each compound in Table 3. Representative photomicrographs showing AChE and BChE histochemical staining of AD brain tissues with or without treatment with compounds 1-6 are shown in Figures 4–9.

Table 3. Qualitative analysis of senolytic and nootropic compound inhibition of Karnovsky-Roots staining for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) associated with β-amyloid plaques (pH 6.8) and normal neural elements (pH 8.0) in human orbitofrontal cortex and thalamus Alzheimer’s disease brain tissues.

Senolytic compound	Staining results
	AChE pH 6.8	BChE pH 6.8	AChE pH 8.0	BChE pH 8.0
Dasatinib (1)	xx/xa	xxx	-	-
Nintedanib (2)	xxx	xxx	-	-
Fisetin (3)	See belowb	See belowb	See belowb	See belowb
Quercetin (4)	See belowb	See belowb	See belowb	See belowb
GW2580 (5)	-	x/xx	-	-
Meclofenoxate Hydrochloride (6)	xx/x	x	-	-
aThe inhibition in AChE or BChE staining resulting from senolytic and nootropic compound competition was categorized as: – (no inhibition), x (slight inhibition), xx (moderate inhibition), or xxx (strong inhibition).
bThe presence of these compounds in the reaction mixture led to the chelation and precipitation of metal ions necessary for the Karnovsky-Roots method. Thus, histochemical staining could not be done.

Figure 4. Effect of dasatinib (1) on histochemical staining of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Representative photomicrographs of histochemical staining of AChE (A–D) and BChE (E–H). Staining at pH 6.8 demonstrates AChE- (A) and BChE (E)-associated plaques in the Alzheimer’s disease (AD) orbitofrontal cortex (arrows). Staining at pH 8.0 demonstrates AChE (C) and BChE (G) associated with normal neural structures in the AD thalamus (arrowheads showing neurons). Dasatinib (1) inhibits AChE (B) and BChE (F) associated with AD plaques but not AChE (D) and BChE (H) associated with normal neural elements. Note, for ease of reference, identical images of the positive control staining of AChE and BChE at pH 6.8 and 8.0 (A, C, E, G) were used herein and in Figures 5-9 (A, C, E, G) to help compare directly the effects of each senolytic or nootropic agent on the standard Karnovsky-Roots (KR) histochemical staining method. Abbreviations: OFC, orbitofrontal cortex; VA, ventroanterior thalamic nucleus; WM, white matter. Scale bar = 500 μm.

Figure 5. Effect of nintedanib (2) on histochemical staining of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Representative photomicrographs of histochemical staining of AChE (A–D) and BChE (E–H). Staining at pH 6.8 demonstrates AChE- (A) and BChE (E)-associated plaques in the Alzheimer’s disease (AD) orbitofrontal cortex (arrows). Staining at pH 8.0 demonstrates AChE (C) and BChE (G) associated with normal neural structures in the AD thalamus (arrowheads showing neurons). Nintedanib (2) inhibits AChE (B) and BChE (F) associated with AD plaques but not AChE (D) and BChE (H) associated with normal neural elements. Note, for ease of reference, identical images of the positive control staining of AChE and BChE at pH 6.8 and 8.0 (A, C, E, G) were used herein and in Figures 4, 6–9 (A, C, E, G) to help compare directly the effects of each senolytic or nootropic agent on the standard Karnovsky-Roots (KR) histochemical staining method. Abbreviations: OFC, orbitofrontal cortex; VA, ventroanterior thalamic nucleus; WM, white matter. Scale bar = 500 μm.

Figure 6. Effect of fisetin (3) on histochemical staining of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Representative photomicrographs of histochemical staining of AChE (A–D) and BChE (E–H). Staining at pH 6.8 demonstrates AChE- (A) and BChE (E)-associated plaques in the Alzheimer’s disease (AD) orbitofrontal cortex (arrows). Staining at pH 8.0 demonstrates AChE (C) and BChE (G) associated with normal neural structures in the AD thalamus (arrowheads showing neurons). Staining reactions for AChE and BChE at pH 6.8 (B and F) or pH 8.0 (D and H) in the presence of fisetin (3) could not be done due to compound 3 precipitating out of solution, most likely due to chelating with metal ions required for the Karnovsky-Roots (KR) staining method. Note, for ease of reference, identical images of the positive control staining of AChE and BChE at pH 6.8 and 8.0 (A, C, E, G) were used herein and in Figures 4, 5, 7–9 (A, C, E, G) to help compare directly the effects of each senolytic or nootropic agent on the standard KR histochemical staining method. Abbreviations: OFC, orbitofrontal cortex; VA, ventroanterior thalamic nucleus; WM, white matter. Scale bar = 500 μm.

Figure 7. Effect of quercetin (4) on histochemical staining of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Representative photomicrographs of histochemical staining of AChE (A–D) and BChE (E–H). Staining at pH 6.8 demonstrates AChE- (A) and BChE (E)-associated plaques in the Alzheimer’s disease (AD) orbitofrontal cortex (arrows). Staining at pH 8.0 demonstrates AChE (C) and BChE (G) associated with normal neural structures in the AD thalamus (arrowheads showing neurons). Staining reactions for AChE and BChE at pH 6.8 (B and F) or pH 8.0 (D and H) in the presence of quercetin (4) could not be done due to compound 4 precipitating out of solution, most likely due to chelating with metal ions required for the Karnovsky-Roots (KR) staining method. Note, for ease of reference, identical images of the positive control staining of AChE and BChE at pH 6.8 and 8.0 (A, C, E, G) were used herein and in Figures 4–6, 8, 9 (A, C, E, G) to help compare directly the effects of each senolytic or nootropic agent on the standard BChE KR histochemical staining method. Abbreviations: OFC, orbitofrontal cortex; VA, ventroanterior thalamic nucleus; WM, white matter. Scale bar = 500 μm.

Figure 8. Effect of GW2580 (5) on histochemical staining of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Representative photomicrographs of histochemical staining of AChE (A–D) and BChE (E–H). Staining at pH 6.8 demonstrates AChE- (A) and BChE (E)-associated plaques in the Alzheimer’s disease (AD) orbitofrontal cortex (arrows). Staining at pH 8.0 demonstrates AChE (C) and BChE (G) associated with normal neural structures in the AD thalamus (arrowheads showing neurons). GW2580 (5) inhibits BChE (F) but not AChE (B) associated with AD plaques but not AChE (D) and BChE (H) associated with normal neural elements. Note, for ease of reference, identical images of the positive control staining of AChE and BChE at pH 6.8 and 8.0 (A, C, E, G) were used herein and in Figures 4–7, 9 (A, C, E, G) to help compare directly the effects of each senolytic or nootropic agent on the standard Karnovsky-Roots (KR) histochemical staining method. Abbreviations: OFC, orbitofrontal cortex; VA, ventroanterior thalamic nucleus; WM, white matter. Scale bar = 500 μm.

Figure 9. Effect of meclofenoxate hydrochloride (6) on histochemical staining of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Representative photomicrographs of histochemical staining of AChE (A–D) and BChE (E–H). Staining at pH 6.8 demonstrates AChE- (A) and BChE (E)-associated plaques in the Alzheimer’s disease (AD) orbitofrontal cortex (arrows). Staining at pH 8.0 demonstrates AChE (C) and BChE (G) associated with normal neural structures in the AD thalamus (arrowheads showing neurons). Meclofenoxate hydrochloride (6) inhibits AChE (B) and, to a certain extent, BChE (F) associated with AD plaques but not AChE (D) and BChE (H) associated with normal neural elements. Note, for ease of reference, identical images of the positive control staining of AChE and BChE at pH 6.8 and 8.0 (A, C, E, G) were used herein and in Figures 4-8 (A, C, E, G) to help compare directly the effects of each senolytic or nootropic agent on the standard Karnovsky-Roots (KR) histochemical staining method. Abbreviations: OFC, orbitofrontal cortex; VA, ventroanterior thalamic nucleus; WM, white matter. Scale bar = 500 μm.

In the presence of compound 1, AChE staining associated with plaques was reduced by varying degrees when compared with control staining at pH 6.8 (Table 3 and Figure 4A, 4B). While two of the AD cases showed complete inhibition of AChE-associated plaques, the remaining cases only showed moderate reduction in staining. Compound 1 showed complete inhibition of BChE activity associated with plaques in all cases, although white matter staining remained when compared to control staining at pH 6.8 (Table 3 and Figure 4E, 4F). There was no inhibition of AChE or BChE staining associated with normal neural elements (pH 8.0) in any cases, relative to controls, when treated with compound 1 (Figure 4C, 4D, 4G, 4H).

Similarly, complete inhibition of both AChE and BChE activity associated with plaques, compared to controls, was observed in the presence of compound 2 (Figure 5A, 5B, 5E, 5F). There was no inhibition of ChE staining associated with normal neural elements (Table 3 and Figure 5C, 5D, 5G, 5H).

The two flavonoids, compounds 3 and 4, produced identical results under KR histochemical conditions (Table 3 and Figures 6, 7). When the aqueous stock solutions of 3 and 4 were added to their KR staining solutions, there were noticeable color and solubility changes from the original stocks. The stock solution of compound 3 was a cloudy, peach color, which turned umber and precipitated upon addition to the KR solution. The stock solution of compound 4 was a cloudy, neon yellow color, which turned brown and likewise precipitated when added to the KR solution. These observations indicated that the lack of ChE staining at pH 6.8 and 8.0 for tissues tested with compounds 3 and 4 (Figures 6, 7) were likely due to both flavonoids forming complexes with metal ions of the KR solution, iron and/or copper [89, 90], necessary for the staining reaction to occur. Thus, producing the false impression of inhibitory activity.

Compound 5 did not inhibit staining of AChE associated with plaques; however, it did inhibit staining of BChE associated with plaques when compared to control staining (Table 3 and Figure 8A, 8B, 8E, 8F). Furthermore, compound 5 did not inhibit either ChE associated with normal neural elements compared to controls (Table 3 and Figure 8C, 8D, 8G, 8H).

Lastly, compound 6 showed moderate to strong inhibition of AChE-associated plaque staining and slight inhibition of BChE-associated plaque staining when compared to control staining for AChE and BChE, respectively (Figure 9A, 9B, 9E, 9F). Although reduced, the appearance of AChE inhibition associated with plaques varied when comparing all AD cases. Cases with a relatively higher plaque burden appeared to have less reduction in ChE staining than cases with a relatively lower plaque burden. When comparing appropriate control sections to those stained in the presence of compound 6, the degree of inhibition remained the same in amongst all cases (not shown). Compound 6 did not inhibit AChE or BChE staining associated with normal neural elements (Table 3 and Figure 9C, 9D, 9G, 9H).

Molecular docking studies

Cholinesterase inhibitors are known to bind to varying sites on ChEs to yield effects on catalytic activity. In silico molecular docking studies provide a means of assessing potential enzyme binding sites and amino acid residue interactions that could contribute to the observed activity of compounds. As such, understanding the structural characteristics and features of AChE and BChE is essential to appropriately investigate the potential mechanisms of action for active compounds. Structural features of ChEs used to analyze molecular docking studies were based on the published x-ray crystal structures of these enzymes [91, 92].

AChE and BChE possess many of the same structural components and similar amino acid residues that are essential for catalytic activity. Though the volume of the active site gorge and the diameter of the gorge opening are larger in BChE than in AChE (~500 ų vs. ~300 ų, ~16 Å vs. ~11 Å) [93], they both have a similar depth of ~20 Å and contain the same five main active site regions with key residues and structural features (acyl loop, Ω-loop, ε-helix) shown in Figure 10. These five regions include: 1) the catalytic active site (CAS) at the base of the gorge, comprised of the catalytic triad, serine (S203 for AChE, S198 for BChE), histidine (H447, H438), and glutamate (E334, E325); 2) the acyl binding pocket (ABP) at the mouth of the gorge; 3) the peripheral anionic site (PAS) at the mouth of the gorge; 4) the π-cationic site (PCS) midway down the gorge; and 5) the oxyanion hole (OAH) next to the catalytic serine at the bottom of the gorge (Supplementary Figure 1).

Figure 10. Structural components and amino acid residues that are essential for the catalytic activity of acetylcholinesterase (AChE) and butyrylcholinesterse (BChE). (A) 2D depiction of the entry/exit routes (“doors”) to the active site gorge of AChE. Amino acids are labelled and arranged according to their structural placement around the gorge, with those located on the opposite side to the viewing plane shown through the transparent green gorge wall. (B) AChE (PDB: 4M0E, 2.00 Å) [91] and BChE (BChE; PDB: 4TPK, 2.70 Å) [92] enzyme ribbon structures are overlayed to show structural conservation between the two enzymes. The catalytic triad residues are shown in yellow. (C, D) The five main compound binding site pockets (I-V) of AChE (C) and BChE (D) are identified as the enzyme active site gorge (lime green, I), pocket behind ChE acyl loop (pink, II), pocket behind ChE catalytic glutamate (orange, III), pocket behind ChE active site tryptophan (Back Door, blue, IV), and other binding pocket (teal, V). Structural features of AChE and BChE including the acyl loop (pink), Ω-loop (blue), and ε-helix (orange) are shown throughout each panel. Figures were generated using Microsoft PowerPoint for Microsoft 365 MSO (version 2409 build 18025.20104; Microsoft Corporation, Redmond, WA, USA) and Molecular Operating Environment 2022.02 (Chemical Computing Group ULC, Montreal, Quebec, Canada).

In addition to the main active site gorge, there are several proposed sites that are entry/exit routes for substrates and hydrolysis products that surround and share some residues of the active site gorge, the acyl and Ω-loops, and the ε-helix. These sites are denoted as the acyl, side, and back doors (Figure 10A) [94–97]. The discovery of the back door was originally hypothesized as an alternate exit route for choline from the active site of AChE following the hydrolysis of acetylcholine, and offered a possible explanation for its high turnover rate despite the restricted opening of the active site gorge [97]. The side and acyl doors were later proposed as additional entry/exit points to the gorge (Figure 10A) [94, 96, 98]. While these “doors” have not been described in detail in the literature for BChE, their key residues and structural components are likewise present in the BChE structure (Figure 10B), with many of the residues conserved between the two enzymes.

Utilizing a blind docking approach [99], with five docking poses generated in each docking experiment and repeated in triplicate, compounds 1-6 localized to the same five main ChE sites (Figure 10C, 10D) identified using the Molecular Operating Environment (MOE) Site Finder feature. These five sites are denoted as follows: (I) enzyme active site gorge, (II) pocket behind ChE acyl loop, (III) pocket behind ChE catalytic glutamate and Ɛ-helix, (IV) pocket behind key ChE active site tryptophan (“Back Door”), and (V) other site located on the opposite side of the enzyme, away from the active site gorge. While blind docking experiments with compounds 1-6 did not show all five of these sites in each experiment, compound blind docking poses were distributed between these five ChE enzyme locations (Supplementary Table 1). These five binding pockets are displayed for each ChE crystal structure in Figure 10C, 10D.

Senolytics 1 and 2 did not find the active site of either ChE enzyme in blind docking experiments (Supplementary Table 1). Compounds 3-6 localized to the active site of AChE, while compounds 3, 4, and 6 showed affinities for the active site of BChE (Supplementary Table 1). Compound 5 did not find the active site of BChE. Though compounds 3, 4, and 6 found the active site of both enzymes, only quercetin (4) with BChE and fisetin (3) with AChE showed predominant affinity for the active site gorge in blind docking poses (Supplementary Table 1). Compounds that did not find the active site (1, 2 for AChE; 1, 2, 5 for BChE) – or those that did not localize to the active site in the majority of blind docking poses (4, 5 for AChE; 3 for BChE) – showed affinity most frequently to one major site on each enzyme, site II for AChE and site III for BChE (Supplementary Table 1). Meclofenoxate hydrochloride (6) with both ChEs, and nintedanib (2) with AChE showed equal affinity (6/15 poses) for two sites. For compound 2, these sites were II and III of AChE (Supplementary Table 1), while for 6 these were sites I and III with BChE, and I and IV with AChE.

While it remains uncertain if the frequency of localization of blind docking poses to specific enzyme sites is indicative of true compound preference for that site, or simply a result of varying factors related to the blind modeling process [99–101], it is generally accepted that the failure of a compound to find the active site of an enzyme during blind docking experiments is predictive of the compound not interacting with that active site experimentally [99, 102–104]. Localization to sites beyond the active site gorge of an enzyme may indicate compound affinity for allosteric binding sites; thus, all compounds were assessed in site-directed docking experiments at all identified ChE sites. This resulted in some compounds having multiple calculated inhibition constants (K_i values) for different binding sites, which are summarized in the Supplementary Materials (Supplementary Table 2).

Predicted K_i values were generally within the same order of magnitude as experimentally determined K_i values but were highly variable depending on the enzyme site used for site-directed docking. For example, dasatinib (1) docked with BChE at site II had a sub-micromolar predicted K_i, while the dock directed to site III showed a predicted K_i that was almost identical to the experimental K_i value (Supplementary Table 2). Similarly, fisetin (3) and quercetin (4) found the active sites of both ChEs in blind docks but predicted K_i values at these sites were much lower than experimental values. However, the predicted K_i values at alternate sites for compounds 3 and 4 were closer to their experimental values (Table 1 and Supplementary Table 2). For AChE, site II consistently showed K_i values closest to those determined experimentally (Table 1 and Supplementary Table 2). For BChE, there was more variability in alternate sites, but site III produced more accurate K_i values for several compounds (Table 1 and Supplementary Table 2).

Key amino acid interactions between compounds 1-6 and residues of each ChE were analyzed for the top site-directed docking pose at each identified site (I-V; Supplementary Table 3). Their interaction type, bond lengths, and residues’ location on the enzyme were identified, with most being hydrogen bonding interactions. Overall, similar amino acid residue interactions were observed for compounds docked to the same site (I-V). Likewise, many of these residues were components of the acyl and Ω-loops, the Ɛ-helix, or were residues of the five main regions of the ChE active site gorge (CAS, ABP, PAS, PCS, and OAH).

Abbreviations

Aβ: amyloid-β; ABP: acyl binding pocket; AChE: acetylcholinesterase; AD: Alzheimer’s disease; ATChI: acetylthiocholine iodide; BChE: butyrylcholinesterase; Bibfl120: nintedanib; BTChI: butyrylthiocholine iodide; CAIP: cholinergic anti-inflammatory pathway; CAS: catalytic active site; ChAT: choline acetyltransferase; ChE: cholinesterase; ChEI: cholinesterase inhibitor; DAB: 3,3’-diaminobenzidine tetrahydrochloride; dH₂O: distilled water; DTNB: 5,5-dithio-bis-(2-nitrobenzoic acid); K_i: inhibition constant; H₂O₂: hydrogen peroxide; IL-1β: interleukin 1β; IL-6: interleukin 6; KR: Karnovsky-Roots; mAChR: muscarinic acetylcholine receptor; MOE: Molecular Operating Environment; nAChR: nicotinic acetylcholine receptor; OAH: oxyanion hole; PAS: peripheral anionic site; PB: phosphate buffer; PCS: π-cationic site; PDB: Protein Databank; SAMP8: senescence-accelerated prone 8 mouse; SASP: senescence-associated secretory phenotype; TNB: 5-thio-2-nitrobenzoic acid; TNFα: tumor necrosis factor α.

Author Contributions

Sultan Darvesh: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing; Meghan K. Cash: Writing – original draft, Writing – review and editing; Katrina Forrestall: Data curation, Formal analysis, Investigation, Validation, Visualization, Writing - original draft, Writing – review and editing; Hillary Maillet: Data curation, Formal analysis, Investigation, Validation, Visualization, Writing - original draft, Writing – review and editing; Dane Sands: Formal analysis, Investigation, Visualization, Writing – review and editing.

Acknowledgments

We would like to thank Andrew Davis for their technical assistance.

Conflicts of Interest

Sultan Darvesh is a scientific co-founder and stockholder in the Treventis Corporation, a biotech company focused on the development of diagnostic and therapeutic agents for Alzheimer’s disease. Sultan Darvesh is listed as an inventor on patents assigned to Treventis Corporation. Also, Sultan Darvesh is an Associate Editor of Current Alzheimer Research. All other authors do not have financial disclosures or conflicts of interest to declare.

Ethical Statement and Consent

All research experiments performed using post-mortem human brain tissues were approved by the Nova Scotia Health Research Ethics Board (REB FILE #: 1020654). The consent is not required because the brain tissues were obtained from the Maritime Brain Tissue Bank, Faculty of Medicine, Dalhousie University (Halifax, Nova Scotia, Canada).

Funding

This work was supported in part by the Canadian Institutes of Health Research (PJT – 153319), the Dalhousie Medical Research Foundation (DMRF Chemists, DMRF Clare Durland Fund in Alzheimer’s Disease Research; DMRF Research Grant – Robert and Barbara Pickett; DMRF Gillian’s Hope for MS Research Fund), and the Dalhousie University Endowed Irene MacDonald Sobey Endowed Chair in Curative Approaches to Alzheimer’s Disease.

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