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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Figure 1. Acetylcholinesterase inhibitors approved by FDA for the treatment of AD
Cholinesterase inhibitors such as tacrine, rivastigmine, donepezil and galantamine, which block the
breakdown of ACh has been used for the treatment of mild to moderate AD (Mayeux & Sano, 1999;
Silmana & Sussman, 2008, Mirjana, 2013). All of these drugs shown in Figure 1 are reversible AChE
inhibitors (AChEIs), and can restore the level of ACh in the brain of AD patients, and which plays a key
role in memory and cognition (Sugimoto, 2000; Colombers, 2004).
Hence, it is not surprising that AChEIs have shown better benefits in the treatment of AD than other
therapeutic strategies. Recently, interference on amyloid beta (Aβ) aggregation by AChEIs directed to
the development of a novel class of AChEIs. It was also experimentally observed that AChEIs moderates
the rate of amyloid deposition (Inestrosa, Dinamarca, Alvarez, & Center, 2008). The bifunctional role,
anti-aggregating Aβ (characteristic pathological role in AD) and anti-cholinesterase activity is mainly
contributed by these AChEIs with interactions on this enzyme (De Ferrari, Canales, Shin, Weiner, Silman,
& Inestrosa, 2001). Therefore, identifying selective AChEIs are highly demanding for CNS penetration
and good pharmacokinetic properties for the treatment of AD.
Many studies have focused on synthesizing and evaluating new AChEIs for the improvement of
symptoms of AD. One of the major strategies has been the use of in silico techniques such as QSAR
modeling, molecular docking, pharmacophore mapping, virtual screening etc. which has proven their
usefulness in pharmaceutical research for the selection/identification and/or design/optimization of new
chemical entities. QSAR is one of the most important areas in Chemoinformatics, and its advances have
widened the scope of rational drug design and the search for the mechanism of drug action (Hansch,
Hoekman, Leo, Weininger, & Selassie, 2002; Garg, Gandhi & Mohan, 2008). It is a well-established fact
that the chemical and pharmacological effects of a compound are closely related to its physico-chemical
properties. This in turn been calculated by various methods from the molecular structure (Gupta, 2007).
In addition, the in silico methods can expand screening for compounds that do not exist physically in the
chemical collections, therefore compensating for some of the most important limitations of the heavy
experimental measurements which include high-throughput in vitro screening methods.
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Figure 2. Enzymatic hydrolysis of ACh by AChE
ACETYLCHOLINESTERASE ENZYME
Dale in 1914 postulated that the physiological role of ACh at cholinergic synapses is terminated by an
enzyme system. It was not until 1926 that Loewi and Navratil experimentally established AChE existence
and in 1932 Stedman arranged a crude extract of an “ACh-splitting” enzyme from horse serum, which
he called “cholinesterase”. The enzyme AChE (ACh hydrolyser, EC 3.1.1.7) is a molecule 32 KDa
molecular weight. The principal biological role of AChE is the termination of impulse transmission at
cholinergic synapses by rapid hydrolysis of the neurotransmitter ACh to produce choline and an acetate
group (Barnard 1974), and is presented in Figure 2.
AChE is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous
system, where its activity serves to terminate the synaptic transmission. AChE has a very high catalytic
activity, and each molecule of AChE degrades about 5000 molecules of ACh per second. The choline
produced by the action of AChE is recycled - it is transported through reuptake into nerve terminals,
where it is used to synthesize new ACh molecules (Quinn, 1987). AChE exists in multiple molecular
forms, which possess similar catalytic properties, but differ in their oligomeric assembly and mode of
attachment to the cell surface. Knowledge of the three dimensional structure of AChE is essential for
understanding its remarkable catalytic efficacy.
General Structure of AChE
Ever since the crystal structure of Torpedo californica acetylcholinesterase (TcAChE) was solved in
1991, it opened up new horizons in the understanding of catalytic mechanisms and mode of action of
inhibitors with this enzyme (Sussman et al. 1991). TcAChE belongs to the class of α/β proteins (Levitt,
& Chothia, 1976) and consists of 12-stranded central mixed β-sheet surrounded by 14 α-helices as shown
in Figure 3. The enzyme has a β-sheet platform that bears the catalytic machinery and is rather similar
in all members of the family. Indeed, the three members of the catalytic triad appeared in the same order
along the polypeptide chain in all α/β hydrolase enzymes (Ollis et al. 1992). The α-helices and loops
are then ascribed by the task of handling the specificity element, substrates of different members of the
family being very varied. When TcAChE structure was determined, it initially appeared to be an entirely
new fold. However, it folds soon turned out to be strikingly similar to other proteins whose structures
had been solved almost the same time.
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Figure 3. Schematic ribbon diagram of the 3D structure of TcAChE monomer; 11-standard β-sheets
(light gray) surrounded by 15 α-helices (dark black) and 3 β-sheets without any hydrogen bond to the
central sheet; ACh in the binding site has been rendered in ball and stick format.
The Active-Site Gorge and the Catalytic Triad
The most remarkable feature of the structure of TcAChE, is a deep and narrow gorge, about 20 Å long.
This region, which penetrates more than half-way into the enzyme, and lined with the rings of 14 conserved aromatic residues is presented in Figure 4. The existence of a catalytic triad in AChE had previously been the subject of controversy. As revealed by the crystallographic structure of AChE and its
inhibitor complexes, the AChE active site contains a catalytic triad (S200, H440, E327) located at the
bottom of a deep and narrow gorge as shown in Figure 5. Early kinetic studies indicated that the catalytic site (CS) of AChE contains two subsites, the ‘esteratic’ and ‘anionic’ subsites (AS) (Rosenberry,
1975). These two subsites correspond to the catalytic machinery and the choline-binding pocket. The
‘esteratic’ subsite was believed to resemble the catalytic subsites of other serine hydrolases (Froede, &
Wilson, 1971). The AS, located near the bottom of the cavity (Kryger, Silman, & Sussman, 1999) and
interacts with the charged quaternary moiety of choline group of ACh was believed to be the binding
site both for quaternary ligands, such as edrophonium and N-methylacridinium. They act as competitive
inhibitors, and for quaternary oximes, which serve as effective reactivators of organophosphate-inhibited
AChE (Mooser, & Sigman, 1974).
Both chemical modification and spectroscopic studies supported the presence of aromatic residues
in the active site of AChE (Fuchs, Gurari, & Silman, 1974). These studies implicated a histidine (H440)
residue in the active site (Goeldner, & Hirth, 1980). S200-Oγ, which can be seen looking down the
gorge from the surface of the enzyme, is about 4 Å above the base of the gorge. The rings of 14 aromatic
residues contributed a substantial portion (~40%) of the surface of the gorge as shown in Figure 4. These
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Figure 4. 3D structure of TcAChE displayed as a ribbon diagram; the 14 conserved aromatic residues
are shown as sticks and mesh. A model of the substrate, ACh, bound in the active site, is shown at the
bottom of the active-site gorge, in ball and stick format.
residues, and their flanking sequences, which are highly conserved in AChEIs from different species was
N66 and I444. They are synthesized on the first exon, which codes for residues 1–480. It should be noted
that the gorge contains only a few acidic residues, which include D285 and E273 at the very top, D72,
hydrogen-bonded to Y334, about half-way down, and E199, near the base. The presence of a tryptophan
in the active site of AChE was predicted by spectroscopic and chemical modification studies (Maulet,
Camp, Gibney, Rachinsky, Ekströ, & Taylor, 1990). The subsequent affinity labeling study of Weise et
al. indeed identified W84 as part of the putative ‘anionic’ (choline) binding site. The hydroxyl groups
of Y121 (half-way up) and Y130 (at the bottom) both points into the gorge (Weise, Kreienkamp, Raba,
Pedak, Aaviksaar, & Hucho, 1990). The high aromatic content of the walls and base of the active-site
gorge, together with its dimensions, may help explain why biochemical studies revealed a variety of
hydrophobic and ‘anionic’ binding sites distinct from, or overlapping with the active site.
Peripheral Anionic Site
Besides the catalytic site, the peripheral anionic site (PAS) of AChE is placed on the surface of the protein. W279 and Y70 were introduced as the residues of PAS and is presented in Figure 5. Furthermore,
two sets of peptide sequences, residues 251-264 and 270-278, respectively, contribute to the PAS in
TcAChE for ACh and other quaternary ligands. These two neighboring peptide sequences on the surface
of the protein are both close to the rim of the gorge. Thus, the ligand association with the peripheral
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Figure 5. Schematic view of the active-site gorge of TcAChE
(Gupta S, 2012).
site may prevent access of the substrate to the gorge by physical hindrance to restrict entry to the gorge
by an allosteric mechanism, in which the active center conformation is altered (Taylor, & Radic, 1994).
This blockade can have functional significance. In particular, during ACh hydrolysis, an incoming ACh
molecule bound at PAS blocks the exit of a choline molecule generated in the active site, giving rise
to the substrate inhibition. Some ligands may be too bulky to penetrate it, but can partially block its
entrance. Certain elongated bis-quaternary compounds, many of which serve as potent inhibitors, may
attach at one extremity to the PAS, and at other end to any one of the various aromatic residues lining
the walls of the gorge, in certain cases spanning the gorge completely (Harel et al. 1993). Recently,
evidence was presented that AChE accelerates assembly of Aβ peptides into amyloid fibrils with the
involvement of PAS.
Comparison of Human hAChE and TcAChE
Among the available crystal structures of the AChE enzyme, docking studies were performed on the
donepezil bound crystal structure of TcAChE (PDB ID: 1EVE) (Kryger, Silman, & Sussman, 1999). We
have selected this crystal structure by considering that the size and shape complementarity as well as the
dual binding site nature of the donepezil compound was similar to our training set lead compound CD1.
TcAChE has almost identical amino acid residues with the human AChE (hAChE) (PDB code: 1B41)
at both the CS and PAS, apart from the substitution of F330 (Tc) with Y337 (human) (Kryger, Harel, &
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Shafferman, 2000). The multiple sequence alignment (MSA) of AChE sequences from Homo sapiens,
Mus musculus, Electric eel, and Torpedo californica is shown in Figure 6. MSA shows that the active
site of AChE is very well conserved across species. hAChE and mAChE sequences exhibit 88% identity
and 97% homology, while with hAChE and TcAChE sequences exhibit 64% identity and 88% homology.
Mechanism of Action
AChE is the enzyme that catalyzes the breakdown of the cationic neurotransmitter, ACh: a neurotransmitter important in cognitive function, in the nerve synapses. The catalytic triad has been termed as “charge
relay system”. In TcAChE, the triad includes S200, the imidazole ring of H440 and the carboxylic group
of E327. It acts through an acylation and deacylation process (Wang, Jiang, Chen, Ji, & Ye, 1999). In
the active site S200 and H440 are involved in reaction with ACh. During binding of ACh to AChE, the
acyl-enzyme is produced after proton transformation from S200 to the imidazole group of H440, and
then oxygen of S200 attacks to the substrate (ACh), this part is acylation and then the acylated enzyme
complex is finally rapidly hydrolyzed with the waters of binding pocket, regenerating active enzyme
by releasing acetic acid. During the acylation process, the cation-π interaction takes place between the
positively charged nitrogen in ACh and W84.
Furthermore, the component of oxyanion holes (G118, G119 and A201) forms the hydrogen bond
to the carbonyl group of ACh. In the acylation process, both the cation interaction and H-bonds exist
during the reaction. However, these two kinds of interactions indirectly affect the proton transfer from
S200 to H440 and the nucleophilic attack of the oxygen atom of S200 to the carbonyl of ACh.
BACKGROUND
AChEIs can be classified into different groups, based on their mode of interaction with the AChE enzyme. Inhibition can be either reversible, by competitively preventing the substrate from reaching the
active site; pseudo-irreversible, by covalent reaction with the active site serine, inactivating the catalytic
ability of the enzyme or irreversible. Competitive inhibitors act by blocking substrate at the active site,
non-competitive inhibitors by binding to the PAS.
Synaptic Cholinergic Drugs
In tissues, the most abundant of cholinesterase types are AChE and BuChE. As it was described earlier,
AChE is the predominant enzyme in the brain. Therefore, inhibition of AChE causes more bioavailability of
ACh at the synaptic area and consequently improving neurotransmission process. Principally, this method
is most useful for the treatment of patients with undamaged presynaptic neurons. They are still active in
synthesizing and releasing ACh. It works in the early stages of AD and loses effectiveness after usage in a
period of time. The activation of M2 muscarinic receptors that leads to inhibition of presynaptic release of
ACh might decrease the efficacy of AChEIs, through the counteracting effect. Despite this AChEIs have
shown suitable therapeutic effects for patients having AD. The only drugs currently accepted to treat the
AD are AChEIs (i.e. tacrine, donepezil, rivastigmine and galanthamine). Based on the mechanism of action
in AChE, different sort of AChEIs have been designed and classified as pseudo-irreversible, irreversible,
transition state analogue and reversible inhibitors respectively (Camps, & Munoz-Torrero, 2002).
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Figure 6. The multiple sequence alignment of AChE sequences from Homo sapiens, Mus musculus,
Electric eel, and Torpedo californica, boxes showing CS and PAS residues
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Irreversible AChEIs (Organophosphorus Compounds)
Organophosphates compounds such as di-isopropyl fluorophosphates (DFP) are very potent inhibitors
of AChE and are used as agricultural insecticides or as nerve gases in chemical warfare. One of the
representatives of this group is metrifonate (Figure 7), which irreversibly phosphorylates the enzyme.
Although its efficacy was acceptable, its application as a drug was withdrawn due to muscle weakness
and respiratory problem. These compounds react with the active site serine, forming a very stable covalent phosphoryl-enzyme complex.
Pseudo-Irreversible AChEIs
Pseudo-irreversible AChEIs includes the compounds having carbamates functional group. They are
carbamylated by a catalytic triad of the AChE binding site. The rate of hydrolization of their carbamoylated complex with S200 is slower than the rate of hydrolization of ACh-AChE complex. Physostigmine
was the first AChEI of this class that was studied for the treatment of AD, but it was rejected due to the
lack of efficacy resulting from its short half-life and variable bioavailability. To improve its potency,
several analogues of that with more lipophilic side chains have been designed. Rivastigmine (Exelon®)
is a miotine derivative. It is a carbamic AChEI with a short half-life and duration of action of about 10
hours. This drug inhibits AChE by carbamoylating the serine residue of the catalytic triad in a pseudoirreversible manner. However, it shows a good combination in brain selectivity, long duration in vivo
activity and its good neuroprotective property, which caused it to be accepted as a drug for AD treatment. Eptastigmine, a more recent physostigmine analogue, is the heptyl carbamate of (-)-eseroline.
Compared to physostigmine, this compound has a long duration of action. The structures of various
pseudo-irreversible AChEIs are presented in Figure 8.
Transition State Analogue Inhibitors
Trifluoromethylketones are effective inhibitors of this group having a reversible covalent interaction with
S200 of the AChE active site forming a tetrahedral-hemiketal transition state. In fact, among AChEIs,
m-(N,N,N-trimethylamino) trifluoro acetophenone is a highly potent reversible inhibitor (Figure 9).
However, its ionic nature prevents its capability to cross the blood-brain-barrier (BBB). Therefore, a
more lipophilic and non-ionic derivative zifrosilone (MDL-73745) can function better that works as its
transition state analogue form.
Figure 7. Chemical structure of metrifonate
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Figure 8. Chemical structure of pseudo-irreversible inhibitors of AChE
Figure 9. Chemical structure of m-(N,N,N-trimethylamino) trifluoroacetophenone and zifrosilone (MDL-
73745) as transition state analogue of AChEIs
Reversible AChEIs
In contrast to the above described classes of AChEIs, reversible AChEIs binds to the active site of the
enzyme and inhibits the activity of the substrate. Aminoacridines, N-benzylpiperidines and alkaloids
belongs to reversible AChEIs.
• Aminoacridines: Tacrine (THA, Cognex®) is one of the members of this group, which was launched
in 1993 as the first AChEI drug for the symptomatic treatment of AD. THA (9-amino-1,2,3,4-tetrahydroacridine) is a centrally active, reversible inhibitor of AChE that shows a moderately long duration
of action. Interestingly, it is more potent towards BuChE than AChE. However, it has other features
including blocking sodium and potassium channels, and has a direct effect on muscarinic receptors
(Schneider, 2000). Some disadvantages of these inhibitors are short half-life and induction of hepatotoxicity. However, it was the lead compound for synthesizing new derivatives, such as velnacrine and
suronacrine (Figure 10), showing reduced toxicity (Ros, Aleu, Marsal, & Solsona, 2000).
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QSAR Models towards Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease
Figure 10. Chemical structure of tacrine, velnacrine, and suronacrine
Figure 11. Structure of donepezil (E2020), TAK-147 and N- benzylpiperidine morpholino-benzisoxazole
• N-Benzylpiperidines: Donepezil (E2020) is the second drug approved by FDA for the treatment
of mild to moderate AD. Donepezil, a highly-selective, reversible AChEI developed by Eisai
Company in Japan, is the marker leader due to its once-daily dosing (Heydorn, 1997). It is a
potent, long-acting and selective AChEI, than that of BuChE. The X-ray structure shows that the
elongated donepezil molecule spans the entire length of the active-site gorge of the enzyme. It thus
interacts with both the anionic subsite at the bottom of the gorge, and with the PAS, near its entrance, via aromatic stacking interactions with the conserved aromatic residues. TAK-147 (Figure
11) is another N-benzylpiperidine derivative, which has less potency than donepezil, but its effect
on animals showed fewer side effects and is undergoing clinical trials. Other N-benzylpiperidine
derivatives have been introduced, in which indanone moiety of donepezil has been replaced by
different heterocyclic systems (Martinez, Fernandez, Castro, Conde, Rodriguez-Franco, Baños, &
Badia, 2000) such as N-benzylpiperidine benzisoxazoles (Figure 11). One of the benzisoxazole
derivatives is morpholino substituted molecule, which showed higher potency and selectivity than
donepezil and was very effective in animal models (Villalobos, Blake, Biggers, Butler, Chapin,
Chen, Ives, Jones, & Liston, 1994).
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