Cholinesterases and Their Inhibitors

Abstract

The main focus of this section is to review the available information on ChEs (ChEs) and their inhibitors. The ChE enzymes cause damage to the cholinergic system by hydrolyzing the neurotransmitter acetylcholine (ACh). ChE inhibitors, playing an important role in the cholinergic system, are used in the treatment of Alzheimer’s disease (AD) because of their effects on maintaining ACh levels in brain regions and preventing Aβ accumulation by inhibiting ChE. In this context, it is important to develop many synthetic and natural origin ChE inhibitors for the treatment of abnormalities in the cholinergic system and disorders with neuropsychiatric symptoms. In this section, firstly, general information about ACh and its synthesis in the cholinergic system is given, then ChEs and their catalytic properties, their roles in AD, and their molecular forms are explained. In the following section, the active site of Cantis was defined. The anti-ChE activity of the developed inhibitors was discussed, and then the mechanism of their binding to the ChE active site was explained by molecular docking. In the final section, many types of ChE inhibitors are described and discussed in detail in this section, and the properties and binding mechanism of these inhibitors are summarized.

1. Introduction

Acetylcholine (ACh), one of the most important neurotransmitters, is the primary substrate for cholinesterases (ChEs). First identified in autonomic ganglia, neuromuscular junctions, and many synapses in the central nervous system (CNS), the ACh is also the primary neurotransmitter in preganglionic sympathetic and parasympathetic neurons and in the adrenal medulla. In the CNS, ACh is mainly found in interneurons and long axon cholinergic pathways [1, 2]. The ACh is formed as a result of esterification of acetic acid with quaternary ammonium alcohol choline. This ACh found in cholinergic neurons and other cell types is biosynthesized by the transfer of acetyl group from acetyl-coenzyme A to choline catalyzed by choline acetyl transferase (ChAT, EC 2.3.1.6). The synthesis of ACh by ChAT is presented in the following reaction.

$$\text{Acetyl}-\mathrm{CoA}+\text{Choline}\stackrel{\text{ChAT}}{\to }\text{Acetylcholine}+\mathrm{CoA}-\mathrm{SH}$$E1

The synthesis of acetyl-CoA, which is the precursor for ACh synthesis from various metabolic pathways with carbohydrate, protein, and fat metabolism, has been reported by many researchers [3].

The cholinergic system is based on ACh, which was first recognized by Loewi in the 1920s and is widely found in both the peripheral and central nervous systems. There are two types of receptors in the nervous system and neuromuscular junctions: ACh receptors (muscarinic and nicotinic). The cholinergic receptors are also known to be expressed in many cells, including immune system and endothelial cells [4]. The receptors are identified based on their response to specific antagonists and agonists [5]. Although three types (M1–M3) of muscarinic receptors have been identified pharmacologically, five types have been reported based on molecular cloning experiments [6]. Muscarinic receptors are metabotropic and use G proteins for signal transduction, while nicotinic receptors are ionotropic and use ligand-gated ion channels for signal transduction [7]. The M1 receptor, one of the muscarinic receptor types and most common in the cerebral cortex, has its highest concentrations in the anterior olfactory nucleus, cerebral cortex, olfactory tubercle, dentate gyrus, hippocampus, and nucleus accumbens. The M2 receptor, a presynaptic autoreceptor that directs cholinergic release, is located in brain regions with abundant cholinergic neurons [8]. M3 and M4 receptors are located in the mainly in the olfactory tubercle, diencephalic, striatum, and brainstem regions [9]. Two types of the nicotinic receptor have also been identified in the CNS. The receptors are commonly found in the thalamus, substantia nigra, and periaqueductal gray [6, 10].

2. Cholinesterases

The existence of ChEs, one of the serine hydrolase class enzymes, was first suggested by Henry Dale in 1914, and research on the subject has been continuing since the 1930s. Research on the determination of the sequence of amino acids in the active site of ChEs has been started since the early 1959 [11]. The ChEs are divided into two groups due to their substrate selectivity to ACh and butyrylcholine. AcetylChE (AChE, acetylcholine acetyl hydrolase, EC 3.1.1.7) is known as true ChE, while butyrylChE (BChE, acylcholine acyl hydrolase, EC 3.1.1.8) is known as pseudoChE, serum ChE, or nonspecific ChE. While AChE inhibits at high substrate concentrations, BChE is activated. These enzymes also differ from each other in terms of selective inhibition of enzymes. The enzymes, which show 65% similarity in their amino acid sequence contents, are encoded in the chromosomes (7 and 3) [12, 13, 14].

The AChE is found in erythrocyte membranes and many tissues such as central and peripheral tissues, nerve and muscles, motor and sensory fibers, and noncholinergic and cholinergic fibers [1]. Another enzyme, BChE, is mainly synthesized in the liver and released into the plasma and is known to show high activity in many tissues [12, 13]. AChE, one of the fastest known enzymes with catalytic activity, is responsible for the hydrolysis of ACh in cholinergic synapses [11, 14]. Although the basic physiological function of BChE is not yet fully known, it is important both pharmacologically and toxicologically due to its ability to break down ester drugs such as carbamates, aspirin, succinylcholine, cocaine, antidepressant drugs such as sertraline, amitriptyline, pesticides, organophosphate, and chemical warfare agents. It has been reported that the expression of AChE and BChE is increased in many pathological conditions such as glioma, lung cancers, meningioma, leukemia, and ovarian tumors [12, 15].

The ChEs differ in their catalytic activities according to the artificial substrate preference. Since ACh is the more selective substrate for AChE, it is more hydrolyzed by AChE than by BChE. As expressed in Table 1, the catalytic activity of the ChEs may vary depending on the type of substrates [16]. AChE and BChE are responsible for the hydrolysis of different types of choline esters.

Enzyme	Substrate	Product
AChE	Acetyl-β-methyl-thiocholine	β-metyl-thiocholine + acetate
AChE	Acetyl-β-methyl-choline	β-methyl-choline + acetate
AChE > BChE	Acetylcholine	acetate + choline
AChE > BChE	Acetylthiocholine	acetate + thiocholine
BChE > AChE	Butyrylcholine	butyrate + choline
BChE > AChE	Butyrylthiocholine	butyrate + thiocholine
BChE	Succinylcholine	succinate + choline
BChE	Benzoylcholine	benzoiate + choline

Table 1.

Selected substrates and products of AChE and BChE hydrolysis [16].

AChE, playing an important role in impulse transmission by hydrolyzing ACh in the central and peripheral nervous system, catalyzes the hydrolysis of the neurotransmitter ACh to choline and acetate. BChE is responsible for the hydrolysis of its more specific substrate (butyrylcholine) to butyrate and choline. Hydrolysis of ACh/butyrylcholine by the ChEs is presented in the following reaction.

E2

E3

The ChE activity method is based on the formation of 5-thio-2-nitrobenzoic acid, a yellow compound, with 5,5-ditiyobis(2-nitrobenzoik) asit (DTNB) of thiocholine, which is formed by the hydrolysis of ACh and BCh as substrate. The ChE activities were determined using the Ellman method. Reaction mixture contains acetylthiocholine iodide (10 mM) for AChE and butyrylthiocholine iodide (0,5 mM) for BChE as substrate, 5,5-ditiyobis(2-nitrobenzoik) asit (DTNB, 10 mM), and Tris–HCl buffer (pH 8, 1 M) [17, 18, 19].

2.1 The role of cholinesterases in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common type of dementia seen in the elderly population. Its etiology is not known exactly. This disease has been expressed as a neurodegenerative disease characterized by irreversible loss of nerve cells, difficulties in cholinergic nerve conduction, memory and mental functions, thinking and interpretation, and personality and behavioral disorders. It has been reported that amyloid β-peptide (Aβ) aggregation, neurofibrillary network formation originating from hyperphosphorylated tau proteins, oxidative stress, and low ACh levels are effective in the pathology of AD [20, 21, 22]. Some hypotheses have been proposed to explain the pathogenesis of AD.

Cholinergic hypothesis: It is one of the first hypotheses. In this hypothesis, it is thought that AD occurs due to the reduction of acetylcholine, an important neurotransmitter. Degeneration of cholinergic neurons in the basal forebrain of AD patients, as well as a significant decrease in cholinergic receptors and choline acetyltransferase (ChAT) levels in the cerebral cortex, was detected. Although many of the previous treatment approaches are based on this hypothesis, clinical studies have revealed that treatment strategies to increase ACh levels provide only symptomatic relief but are not effective in the development and treatment of the disease [23, 24]. However, there is evidence that the use of ChE inhibitors in new approaches based on this hypothesis may affect Aβ aggregation [25].

Amyloid cascade hypothesis: The hypothesis proposed in 1991 reports that Aβ deposits are the primary factor in AD. In AD, there is a series of neurodegenerative events triggered by the production, aggregation, storage, and toxicity of derivatives that occur after the production of amyloid precursor protein (APP) in the brain [26], and this study has been supported by molecular genetic, neuropathological, and biochemical studies. In this hypothesis, it is suggested that Aβ proteins, which accumulate in certain parts of the brain and form insoluble fibrils, later damage nerve cells by forming senile plaques; these cells break their connections with each other and reduce the amount of neurotransmitters [27].

Dysfunction of cholinergic neurotransmission in the brain causes an increase in AD symptoms. In the brain, choline-ester-based neurotransmitters are catalyzed by ChEs such as AChE and BChE. Therefore, ACh level decreases in the perisynaptic region of AD. Inhibition of AChE to prevent this reduction in ACh level is considered an important therapeutic target for the disease. The role of BChE in the progression of AD has been stated, and inhibition of BChE gains importance for treatment purposes. In addition, the presence of two types of the ChEs was detected between neuritic plaques and neurofibrillary tangles in AD brain. Thus, it has been suggested that both AChE and BChE may be involved in the formation of aggregates of the Aβ peptide in the AD brain [28]. Toxicity in AD brains varies depending on the amount of AChE and BChE, which have the potential to form complexes with Aβ aggregates [29, 30]. Depending on the disease, changes are seen in the amounts of AChE forms. The tetrameric form of AChE (G4), which predominates in the healthy brain, decreased at the onset of AD, while its monomeric and dimeric forms (G1 and G2) remained unchanged. In addition, studies have reported that the level of G1 and G2 forms is elevated in the plasma of AD patients [28, 31, 32].

2.2 Molecular forms of cholinesterases

The ChEs exist as amphiphilic or soluble molecular forms in tissues and body fluids [1, 12]. Monomeric, dimeric, and tetrameric molecular forms of ChE arise from the posttranslational modification of the expressed protein. The soluble monomeric and tetrameric membrane-bound forms are the predominant enzyme species in humans [10, 33] The forms are summarized below:

Amphiphilic dimers (Type 1): The dimeric form of AChE covalently bound to the membrane by a glycophosphatidylinositol extension, soluble only by detergents and aggregates in the absence of detergent. This form is found in erythrocytes, muscle, and lymphocytes of mammals.

Amphiphilic monomers and dimers (Type 2): Unlike Type 1, the form does not contain glycolipids anchor, does not aggregate in the absence of detergent, and can only be dissolved in salt solutions. The forms of the ChEs are frequently encountered in the muscle, brain, and intestine.

Hydrophobic-tailed tetramers (Type 3): This form of AChE, hydrophobically attached to the plasma membrane by a 20 kDa polypeptide anchor, is widely found in the CNS of mammals.

Collagen-like tailed or asymmetrical forms (Type 4): These forms can be identified by the presence of a collagen-like tail that allows them to attach to the basal lamina. This tail consists of collagen 3 helical subunits, each associated with a ChE tetramer. It finds it more common for AChE rather than BChE in neuromuscular junctions.

Soluble tetrameric form (G4): This form consists of four identical monomers and is stabilized by the interactions of hydrophobic amino acids at the C-terminus of the monomers. These forms are common in mammalian body fluids and soluble fractions of tissue homogenates. The form of ChE in the mammalian brain, mammalian body fluids, and soluble fractions of tissue homogenates is the tetrameric form (G4) [1].

2.3 Active site of cholinesterases

Homo sapiens AChE belonging to the serine hydrolases is known to have a very high catalytic activity, with each molecule of AChE degrading about 25,000 molecules of ACh per second [34, 35]. The enzyme’s active site contains the esteratic subsite and anionic subsite in catalytic center. There is a glutamate residue in the anionic region and a serine residue with a functional -OH group in the esteratic region (ES) and an imidazole ring. In addition to the ES, there is an acyl region and a choline-binding subsite at the catalytic active site (CAS) [34, 36]. The enzyme also contains the peripheral anionic site (PAS). The sites serve as sites for binding AChE and other quaternary ligands and involved in substrate-based inhibition [37]. The anionic site contains many amino acid residues such as Phe 330, Trp 84, and Tyr 121 for the electric eel AChE or Phe 338, Trp 86, and Tyr 337 for murine AChE. The PAS, localized on the AChE surface around the cavity entrance, was recognized as a target for multiple AChE activity modulators. Asp70 and Tyr332 residues in the PAS are involved in the binding of positively charged substrate/ligands and in the activity control of the enzyme. The site contains the most significant amino acids residues such as Asp 72, Tyr 121, Tyr 70, Trp 279, and Tyr 334 [16].

How the substrate binds to the active site of AChE (PDB code: 4EY5) via different types of amino acid residues is predicted by molecular docking. The quaternary nitrogen in the ACh interacts with the anionic site formed by the amino acid tryptophan (Trp), and through the aspartic acid and tyrosine located at the entrance of the active center, the substrates are directed to interact with the active center [38]. The molecular docking results show that ACh has Pi-cation interaction with Tyr337 and Trp86 in the active site of human AChE. In addition, nitrogen in the ACh formed a salt bridge with Glu202 (Figure 1). The AChE is composed of a hydrophobic cloud with Ala203, Hıs447, Gly120-Gly122, Gly448,Tyr133, Tyr124, and Phe297.

Figure 1.

2D and 3D interaction diagrams of ACh in the active site of human AChE (PDB ID: 4EY5).

3. Molecular docking

Molecular docking has gained importance with the increase in studies on drug development and design. Such studies have gained momentum with the creation of protein data banks. Molecular docking is a computer-generated tool for finding the optimal configuration and energy between the two if the protein and ligand structures are known. The basis of the method is to identify possible conformational states between the protein-ligand complex and to choose the complex with the lowest free energy. There are many applications such as Schrodinger, AutoDock, DOCK, and MolDock developed to simulate interactions (hydrogen bonding, hydrophobic interactions, Van der Waals) between two or more molecules [39]. Computational docking of test ligands to predict binding sites to the ChE active site becomes one approach to predict potential inhibitors against the target [40]. The docking studies were carried out using the GLIDE software [41].

Previous studies reported interactions with the AChE amino acid residues with various antipsychotic drugs. Recent studies reported interactions with critical residues such as Leu288, Ser292, Thr237, Val238, Gln368, Pi-cation interaction with Arg295, Pi-alkyl and alkyl interactions with Pro289, Val299, Pro367, 234 of AChE [42, 43] Donepezil and Pimozide ligands showed the lowest energy and the best favorable interactions. The Galanthamine (−7.3 kcal/mol) and Risvagtimine (−7.95 kcal/mol) showed higher binding energies than Donepezil (−8.5 kcal/mol) ligands [44].

In addition, in another study, binding free energy analysis was performed to understand that some drugs are potential candidates. It is a more suitable drug candidate with lower binding energies. Brexpiprazole and pimavanserin have binding free energies of −212.690 kcal/mol and − 108.626 kcal/mol, respectively, whereas Donepezil has binding energy of −180.517. The values, providing the highest contribution in terms of energy, are obtained from Van Der Waals forces. With significantly lower binding energy, these results indicate that Brexipyrazole with lower binding energy binds to the active site and other binding pockets in the protein with higher affinity than Donepezil and Pimavanserin [13]. In the study, it was stated that reference tacrine has higher binding affinity to AChE than to the natural substrate ACh [40]. In the present study, the tacrine was predicted to interact with AChE-binding site residues Trp84, Phe330, Tyr334, Ile 439, Trp432, Tyr442, and Gly441.The molecular docking results show that tacrine has Pi-stacking interaction with Phe 330 and Trp84 in the active site of human AChE. In addition, the AChE is composed of a hydrophobic cloud with Tyr334, Ile 439, Trp432, Tyr442, and Gly441 (Figure 2).

Figure 2.

2D and 3D interaction diagrams of tacrine in the active site of human AChE (PDB ID: 4EY5, Schrodinger 2017).

4. Cholinesterase inhibitors

ChE inhibitors constitute a wide group of compounds with different physicochemical properties, including drugs used in the treatment of many diseases, natural toxins, pesticides, and chemical warfare agents. The ChEs, which play an important role in the hydrolysis of pesticides, are also of great importance in food and agriculture research. Specific AChE inhibitors play an important role in the regulation of ACh metabolism due to the physiological importance of AChE, especially in the brain and blood. Since ACh is a more selective substrate for AChE, it takes a more active role in this metabolism than BChE. Specific BChE inhibitors such as tetraisopropyl pyrophosphoramide (iso-OMPA) are mainly of diagnostic importance [45, 46]. AChE inhibitors, one of the ChE inhibitors used in the treatment of many diseases, are more widely known than BChE. Drugs that suppress the development of AD through action on the cholinergic system are predominantly selective inhibitors of AChE. It is stated that these inhibitors also suppress Aβ aggregate formation and oxidative stress. Selective inhibitors of BChE for this disease have also been investigated as potential drugs for AD, but to a much lesser extent than AChE [47, 48, 49, 50]. Inhibition of AChE also plays an important role in nerve agent toxicology. However, BChE can sometimes replace AChE temporarily inhibited by these agents and slowly hydrolyze accumulated ACh [51].

Compounds with AChE inhibitory potential can be divided into three main groups as follows [52]:

1. Compounds binding at the active site interact with either anionic site (e.g., tacrine) or esteratic (e.g., nerve agents).

2. Compounds interacting with the aromatic gorge (e.g., decamethonium).

3. Compounds bound at the PAS (e.g., propidium, huperzine).

In vitro inhibition studies: The inhibition effects of compounds against AChE are determined with at least five different inhibitor concentrations. The IC₅₀ values of the compounds are calculated from activity (%)-[compound] graphs for each compound. The inhibition types and K_I constants are found by Lineweaver and Burk’s curves according to previous works [53, 54, 55].

4.1 ChE inhibitors with therapeutic potential

Many compounds such as donepezil, galantamine, metrifonate, physostigmine, rivastigmine and tacrine, and memantine have been developed as ChE inhibitors. ChE inhibitors, having a regulatory effect on ACh metabolism, have been developed for the treatment of many diseases. For example, donepezil, galantamine, rivastigmine, tacrine, and memantine are drugs used in the treatment of AD [56]. However, the efficacy of these drugs is limited, and they are known to have various dose-related side effects, especially at higher doses. Galantamine and donepezil have AChE inhibitory potential [57, 58], whereas rivastigmine has a reversible inhibitory effect for both AChE and BChE. In particular, donepezil is a highly selective inhibitor for AChE rather than BChE. In a study, the inhibitory potentials (IC₅₀ values) of donepezil, physostigmine, tacrine, and rivastigmine for AChE were found to be 6.7, 0.67, 77, and 4.3 nM, respectively [58].

Donepezil: Donepezil is a drug approved for the mild to moderate treatment of AD (Figure 3A). However, donepezil has many side effects such as muscle weakness loss of appetite, diarrhea, muscle cramps, nausea, and insomnia. In addition, when high doses of the drug are given to patients, many symptoms such as muscle weakness, low blood pressure, severe nausea, severe vomiting, and respiratory problems occur [59]. In addition to being a selective inhibitor of ChE, donepezil may have multiple mechanisms of action. They act at molecular and cellular levels in many pathways, including inhibition of various aspects of glutamate-induced excitotoxicity, stimulation of the neuroprotective isoform of AChE and reduction of oxidative-stress-dependent effects, and in the pathogenesis of AD [60]. The donepezil has a molecular structure that causes simultaneous inhibition by binding to the active and PAS of AChE. However, it has been stated that it does not interact directly with the catalytic triad or the oxyanion hole [61].

Figure 3.

The molecular structures of ChE inhibitors such as donepezil (a), galantamine (B), metrifonate (C), physostigmine (D), rivastigmine (E), and tacrine (F).

Galantamine: Galantamine, one of the alkaloid groups, is found in many plants and has been used as a medicine for decades in Russia and Eastern European countries for many purposes such as the treatment of myasthenia, myopathy, and CNS-related sensory and motor defects. The galantamine has the appropriate molecular structure to bind to nicotinic receptors in the cholinergic system (Figure 3B). As its effectiveness against ChE was revealed in the 1950s, it started to be used in the treatment of various neurological diseases [56]. It was approved for use after it was found to be effective in the treatment of cognitive and many symptoms related to AD. However, it also has many side effects such as severe nausea, convulsions, vomiting, stomach cramps, irregular breathing, and muscle weakness.

Metrifonate: Metrophonate, a prodrug of dichlorvos (2,2-dichlorovinyl dimethyl phosphate), is an irreversible AChE inhibitor with biphasic action (Figure 3C). Initially, it interacts competitively with the enzyme and then turns into a noncompetitive type of inhibition by phosphorylation of the enzyme esteratic site. In other words, metrifonate from the organophosphate group interacts with the esteratic site of the enzyme (Table 2). Having close inhibition effects on both AChE and BChE, metrifonate can be defined as a pseudo-irreversible ChE inhibitor [62].

Inhibitors	Class	Enzyme	Enzymatic active site
Donepezil	Piperidine	BChE > AChE	Anionic
Galantamine	Phenanthrene; alkaloid	AChE > BChE	—
Metrifonate	Organophosphate	BChE > AChE	Esteratic
Physostigmine	Carbamate	BChE > AChE	Esteratic
Rivastigmine	Carbamate	AChE > BChE	Esteratic
Tacrine	Acridine	BChE > AChE	Anionic

Table 2.

Pharmacological properties of some selective ChE inhibitors [10].

Physostigmine: Physostigmine (also known as Eserine) with AChE inhibitory effect is one of the compounds first isolated from Calabar bean in 1864. Although this compound is able to cross the blood–brain barrier (BBB), its use is limited due to its short half-life and many side effects such as stomach cramps, diarrhea, increased saliva production, and excessive sweating. Therefore, physostigmine is not approved for the treatment of AD. Physostigmine from the organophosphate group has a molecular structure to interact with the ES of the enzyme (Figure 3D). Based on this structure, many of its derivatives have been designed and synthesized. Among the derivatives, eseroline, tolserine, and phenserine have ChE inhibitory potential [56, 63].

Rivastigmine: Rivastigmine has been used for the treatment of moderate AD and dementia associated with Parkinson’s disease [64]. Although it is stated that rivastigmine may exert its pharmacological effect through the cholinergic system, its mechanism of action has still not been clarified. Rivastigmine binds reversibly to both AChE and BChE and can cause inhibition. Also, rivastigmine is a carbamate class compound that is converted to various phenolic derivatives that are rapidly excreted from the body. The compound has the potential to bind with high affinity to the ES of AChE during ACh hydrolysis [56, 65], and has the appropriate molecular structure to bind to ChE in the cholinergic system (Figure 3E). The rivastigmine can cause many side effects such as weight loss, diarrhea, stomach pain, and loss of appetite, and in overdose, it can cause numerous symptoms such as irregular heartbeat and chest pain [66].

Tacrine: Tacrine, synthesized in the 1930s, was first used as a muscle relaxant antagonist and respiratory stimulant, moreover, due to its therapeutic effect on the cholinergic system in AD patients, and was approved by the FDA in 1993. The amine group of tacrine, whose molecular structure is presented in Figure 3F, interacts with amino acid residues Phe330 and Trp84 located in the “anionic region” of AChE (Table 2). As a result of the interaction, it has been stated that it is an effective inhibitor developed for the ChEs [67]. However, the use of tacrine has been limited due to liver toxicity, short half-life, and many side effects such as loss of appetite, nausea, vomiting, and diarrhea [56, 68].

4.2 Naturally derived inhibitors

ChE inhibitors used in the treatment of Alzheimer’s, Parkinson’s, and many diseases have short half-lives and many side effects. For this reason, there has been an increased interest in studies on the determination of natural origin inhibitors. Although these types of compounds have less ChE inhibitory activity than synthetics, they have much less side effects due to their natural origin.

Phenolic compounds: A number of flavonoid compounds, which have free radical scavenging properties and important roles in the prevention of oxidative stress, are natural ChE inhibitors in vitro. Galangin, a flavonol group from Alpiniae officinarum, showed a strong inhibitory effect against AChE [69]. Many studies have reported that phenolic compounds, reducing oxidative stress due to antioxidant properties, have inhibitory effects on AChE. The inhibitory effect on the AChE of phenolic compounds strongly depends on the structure of a particular compound, especially the position and/or number of the C=O and OH groups [70]. Chlorogenic acid has an inhibition effect on AChE in the hippocampus and frontal cortex (IC₅₀: 98.17 μg/ml). In vitro, caffeic acid has an activation effect in the cerebral cortex, cerebellum, hypothalamus, whole blood, and lymphocytes, while it has an inhibition effect at the concentrations of 0.5, 1.0, 1.5, and 2 mM in the muscles [71]. Hydroquinone, chlorogenic acid, and 4-hydroxybenzoic acid have inhibitory potential against the AChE with IC₅₀ and K_I values in the range of 0.26 ± 0.01–36.34 ± 2.72 mM and 0.72 ± 0.00–29.23 ± 2.62 mM, respectively. The effectiveness of the compounds has been associated with its structure [49]. Consequently, as phenolic compounds have both AChE inhibitory effect and antioxidant properties, they can be considered as alternative drugs in the treatment of AD.

Cardanol: In 2009, various non-isoprenoid phenolic lipids obtained from Anacardium occidentale were investigated for their inhibitory activity against AChE. In particular, cardanol, a phenolic lipid, has shown promising results [56]. In one study, a novel series of cardanol derivatives was designed as AChE inhibitors and tested for their inhibitory effects against ChEs. The derivatives showed the highest inhibitory activity against AChE, with IC₅₀ of 6.6 μM [72].

Huperzine (Hup): Hup, obtained from the Huperzia serrata, is available in two types, Hup-A and Hup-B (Figure 4A and B, respectively). Hup-A is used in the treatment of AD and age-related memory loss and in improving cognitive functions by regulating ACh level. It is more effective than tacrine, galantamine, and rivastigmine and is a highly selective and potent inhibitor of AChE. However, it is less active against BChE compared with AChE. Many hybrids of Hup-A also have an AChE inhibitory effect [56, 73].

Figure 4.

The molecular structures of Hup-A and Hup-B.

4.3 Synthetic analogs

In order to prevent or reduce the side effects and toxicity of ChE inhibitors, which are known to be used as drugs, their synthetic analogs have been developed as ChE inhibitors [74]. However, the main problem of synthetic analogs is their inability to penetrate the blood–brain barrier (BBB), and their inhibitory effect may be lower compared with reference inhibitors [75]. The derivatives of tacrine have shown improved AChE-inhibitory activities compared with the tacrine used as drug [76]. Various analog compounds were synthesized and tested by Ali et al. in 2009. Most of them showed moderate AChE-inhibitory effects. Ali et al. suggested that the presence of methoxy groups on the phenyl ring significantly improved the inhibition of AChE [75]. Sulfonamide compounds, which have a wide range of biological applications such as antimicrobial, antiviral, diuretic, and anticancer agents, are found as active ingredients in many drugs. In recent years, many studies have been carried out on the design and synthesis of sulfonamide-derived compounds with ChE inhibitory potential. Many of the compounds have been reported to be selective inhibitors for AChE and BChE [19, 77, 78, 79, 80].

5. Conclusions

In conclusion, many synthetic and natural ChE inhibitors have been discovered recently. Due to the side effects of ChE inhibitors used in the treatment of many neurodegenerative diseases such as AD, studies conducted within the scope of the discovery and design of alternative inhibitors have gained importance. Although the side effects of natural origin inhibitors are much less than those of synthetic compounds, their effect is low. In some studies, derivatives of known inhibitors have been synthesized to increase the effectiveness of inhibitors and reduce their toxicity. The binding affinities of these derivatives to the enzyme active site vary depending on the structure. Therefore, the design of targeted inhibitors suitable for the enzyme active site by molecular docking method sheds light on the development of alternative drugs for treatment. The design of compounds with the ChE inhibitor potential by molecular docking method provides a significant advantage in terms of financial and workload since ineffective syntheses are not made.

Acknowledgments

I would like to thank Dr. Cüneyt Türkeş for his kindly helpful suggestions during the preparation of the book chapter.

Conflict of interest

The authors declare no conflict of interest.

Appendices and nomenclature

AD	Alzheimer disease
ACh	acetylcholine
AChE	acetylcholinesterase
Aβ	beta‐amyloid
BBB	blood-brain-barrier
BChE	butyrylcholinesterase
CAS	catalytic active site
CNS	central nervous system
ChE	cholinesterases
ChAT	choline acetyl transferase
DTNB	5,5-ditiyobis(2-nitrobenzoik) asit
ES	esteratic region
iso-OMPA	tetraisopropyl pyrophosphoramide
PAS	peripheral anionic site