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Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011 Sep; 155(3):219–230. DOI 10.5507/bp.2011.036
© M. Pohanka
CHOLINESTERASES, A TARGET OF PHARMACOLOGY AND TOXICOLOGY
Miroslav Pohanka
Faculty of Military Health Sciences, University of Defense, Trebesska 1575, Hradec Kralove, Czech Republic
University Hospital Hradec Kralove, Sokolska 581, Hradec Kralove
E-mail: miroslav.pohanka@gmail.com
Received: February 24, 2011; Accepted with revision: June 3, 2011
Key words: Acetylcholinesterase/Butyrylcholinesterase/Alzheimer’s disease/Myasthenia gravis/Huperzine/Donepezil/
Rivastigmine/Galantamine
Background. Cholinesterases are a group of serine hydrolases that split the neurotransmitter acetylcholine (ACh)
and terminate its action. Of the two types, butyrylcholinesterase and acetylcholinesterase (AChE), AChE plays the key
role in ending cholinergic neurotransmission. Cholinesterase inhibitors are substances, either natural or man-made that
interfere with the break-down of ACh and prolong its action. Hence their relevance to toxicology and pharmacology.
Methods and Results. The present review summarizes current knowledge of the cholinesterases and their inhibi-
tion. Particular attention is paid to the toxicology and pharmacology of cholinesterase-related inhibitors such as nerve
agents (e.g. sarin, soman, tabun, VX), pesticides (e.g. paraoxon, parathion, malathion, malaoxon, carbofuran), selected
plants and fungal secondary metabolites (e.g. aflatoxins), drugs for Alzheimer’s disease (e.g. huperzine, metrifonate,
tacrine, donepezil) and Myasthenia gravis (e.g. pyridostigmine) treatment and other compounds (propidium, ethidium,
decamethonium).
Conclusions. The crucial role of the cholinesterases in neural transmission makes them a primary target of a large
number of cholinesterase-inhibiting drugs and toxins. In pharmacology, this has relevance to the treatment of neuro-
degenerative disorders.
INTRODUCTION
The cholinergic system is based on the neurotransmit-
ter acetylcholine (ACh), firstly recognized by Loewi in
1920s (ref.1) and found widely distributed in both cen-
tral and peripheral nervous systems. The two basic types
of acetylcholine receptors in the nervous system and at
neuromuscular junctions are: muscarinic acetylcholine
receptors (mAChR) and nicotinic acetylcholine recep-
tors (nAChR). Acetylcholine receptors are also found
expressed in multiple cells including endothelial and im-
mune system cells2.
Cholinesterases are a family of enzymes that kata-
lyse the hydrolysis of ACh into choline and acetic acid,
an essential process allowing for the restoration of the
cholinergic neuron. Cholinesterases are divided into two:
acetylcholinesterase (AChE; EC 3.1.1.7.) and butyrylcho-
linesterase (BuChE; EC 3.1.1.8). AChE participates in
cholinergic neurotransmission by hydrolyzing acetylcho-
line. It is expressed in nerve and blood cells. Compared to
AChE, the importance of BuChE is not well understood.
BuChE was known as plasmatic cholinesterase or pseu-
docholinesterase. Similarly, AChE was called blood, also
erythrocytal cholinesterase as its activity remains in the
cell mass after blood centrifugation. The name AChE de-
rives from the natural substrate acetylcholine as opposed
to BuChE that has no natural substrate. An absence or
mutation of BuChE leads to a medical condition (see
below) that shows itself only in the presence of some
drugs (e.g. succinylcholine) and toxins (e.g. cocaine)
(ref.3), due to its ability to split artificial substrates. This
review’s main focus is on cholinesterases as targets of
toxins and drugs. The biochemistry of AChE and BuChE
is also discussed. Drugs and toxins are divided in chapters
according to target sites.
BUTYRYLCHOLINESTERASE
Though BuChE activity is prevalent in the human
body, its’ physiological function is not completely under-
stood. BuChE deficient individuals are generally healthy
with no manifest signs of disease4. The case is similar for
mice with a damaged BuChE gene5,6. BuChE deficient
individuals have increased sensitivity to muscle relaxants
such as succinylcholine, resulting in lasting breath insuf-
ficiency7. Peoples with the deficient, K type BuChE have
lower plasma activity as well as lower affinity for succi-
nylcholine8. The K allele is widely-spread especially in
the Caucasus area. In recent publications, a link between
K type BuChE and lower incidence of Alzheimer’s disease
has been described9 However, more research is needed to
examine this connection. Regular BuChE is sensitive to
inhibition caused by dibucaine (or cinchocaine in some
sources) whereas AChE and K type BuChE are relatively
resistant to dibucaine. Biochemical examination of K type
BuChE is based on serum/plasma BuChE assessment
with and without dibucaine. The output is called a dibu-
caine number (DN). This represents the percentage of
inhibited BuChE. People with regular BuChE have a high
dibucaine number (DN ≥ 75), heterozygotes have medial
220 M. Pohanka
inhibition of DN ~ 40–70, and K type homozygotes have
nearly non-inhibited BuChE (DN < 20) (ref.10).
In comparison with AChE, BuChE is not constituted
in situ but in different organs, mainly in the liver11. BuChE
reaches serum levels of 5 mg/ml with a half time of 12
days12. Assay of BuChE activity in plasma can also serve
as a liver function test. BuChE activity decreases until
complex liver necrosis occurs. However, the importance
of BuChE as a liver function marker is limited by low
sensitivity. Genetic aspects (see above) or intoxication
with some compounds such as organophosphate pesti-
cides and/or organophosphonate nerve agents (see below)
are sources of false positive findings. BuChE is capable of
detoxifying a large number of exogenous substances: pro-
caine13, succinylcholine14, cocaine15, heroin, acetylsalicylic
acid16, and it can also protect the body from the impact
of organophosphorus AChE inhibitors17. However the pri-
mary reason for the existence of BuChE is still unknown.
As mentioned, BuChE, is named according to its pref-
erence for the artificial substrate butyrylcholine. BuChE
can split butyrylcholine with higher turnover number than
AChE. BuChE is also able to hydrolyze much slower than
AChE, indole derivatives18, adipoylcholine19, benzoylcho-
line20, acetylcholine/acetylthiocholine21,22 , butyrylcholine/
butyrylthiocholine23,24 and propionylcholine/propionylthi-
ocholine25,26. On the other hand, BuChE is not able to split
acetyl-β-methyl-thiocholine oracetyl-β-methyl-choline27,28
whereas AChE can. A summary of substrates and reaction
products is depicted below (Table 1).
The fact that BuChE has wider substrate specificity
than AChE is structurally determined. BuChE is a te-
trameric glycoprotein composed of four subunits. Both
dimeric and monomeric forms are stable and ubiquitous
in the body29. All subunits are identical and composed of
574 amino acids with an overall molecular weight close to
85 kDa. As described by Lockridge et al.30 the structural
similarity to AChE is 54% and to bovine thyroglobuline
28%, which leaves ample differences: BuChE is not in-
hibited by substrate excess as is typical for AChE (ref.31),
the active site is wider for BuChE (ref.32), BuChE is sen-
sitive to inhibition by tetraisopropyl pyrophosphoramide
(iso-OMPA; see text about AChE), and the inhibition is
considered to be a fast proof whether a sample contains
BuChE or AChE.
ACETYLCHOLINESTERASE
AChE and BuChE are similar, resembling each other
by more than 50% but their significance and localiza-
tion in the body are very different. AChE is expressed in
cholinergic neurons. Relatively high AChE activity can
also be found in blood cells responsible for the degrada-
tion of plasma acetylcholine33. The primary function of
AChE is rapid splitting of acetylcholine and terminating
cholinergic neurotransmission. Individuals with inhibited
AChE or knock out AChE mice have over-stimulated ace-
tylcholine receptors34. Although, AChE deficient mice are
viable, they have reduced musculature with changed mor-
phology35 and levels of extracellular acetylcholine nearly
sixty times higher than normal. It seems that BuChE is
able to partially recover the AChE missing activity in the
deficient animals36.
The structure of AChE has been extensively investigat-
ed since the 1990s. The first experiments were conducted
on AChE in the electric eel (Torpedo californica) due to
its availability37. This was also considered an informal
model until the commercialization of human recombinant
AChEs. The AChE active site as well as the whole AChE
structure is evolutionary conservative and it contains
common regions similar to the other serine hydrolases.
Cholinesterases are a type α/β hydrolase folded with an
α helix bound with β sheet that contains a catalytic do-
main38 with catalytic triad Ser – His – Glu, the same as
in AChE, BuChE, and lipases. A similar structure can
be also found in carboxyesterases where glutamate is re-
Table 1. Selected substrates and products of AChE respectivelly BuChE catalyzed hydrolysis.
Substrate Product Enzyme Reference
Succinylcholine + water succinate + choline BuChE 14
Adipoylcholine + water adipoate + choline BuChE 19
Benzoylcholine + water benzoiate + choline BuChE 20
Acetylcholine + water acetate + choline AChE > BuChE 21
Acetylthiocholine + water acetate + thiocholine AChE > BuChE 22, 67
Butyrylcholine + water butyrate + choline BuChE > AChE 23
Butyrylthiocholine + water butyrate + thiocholine BuChE > AChE 24, 61, 67
Propionylcholine + water propionate + choline BuChE, AChE 25
Propionylthiocholine + water propionate + thiocholine BuChE, AChE 26, 67
Acetyl-β-methyl-thiocholine + water β-metyl-thiocholine + acetate AChE 27
Acetyl-β-methyl-choline + water β-methyl-choline + acetate AChE 28
221
Cholinesterases, a target of pharmacology and toxicology
placed by aspartate39. Serine is a part of a stable sequence
Gly-Glu(His)-Ser-Gly-Ala/Gly (ref.40).
The electric eel AChE’s active site lies on a bottom of
long and narrow cavity 20 Å deep. The active site con-
tains a catalytic triad within an esteratic site with amino
acid positions for the electric eel AChE: Ser 200, His
440 and Glu 327 (ref.41). The anionic site (also α-anionic
site) is another part of the active site and it is close to the
esteratic site. The anionic site is composed of the amino
acids Trp 86, Tyr 337 and Phe 338 for murine AChE
(ref.42) or Trp 84, Tyr 121 and Phe 330 for the electric eel
AChE (ref.43,44). While the esteratic site hydrolyzes the
ester bond, the anionic site interacts with the acetylcho-
line quaternary ammonium atom and is responsible for
its correct orientation. Entry into the active site through
the cavity composed of aromatic amino acids, i.e. aro-
matic gorge, enables higher selectivity for acetylcholine.
Substrate penetration is allowed by cation – π interactions
between acetylcholine quaternary ammonium atom and
π electrons of phenylalanine, tryptophan and tyrosine
aromatic cores45,46.
The peripheral anionic site (also β-anionic site) is lo-
calized on the AChE surface around the cavity entrance.
This site was recognized as a target for multiple AChE
activity modulators and the first experiments began in the
1960s (ref.47). The aromatic site contains loops and it has
good conformational flexibility. Tyr 70, Asp 72, Tyr 121,
Trp 279 and Tyr 334 amino acids residues are the most
significant residues in the peripheral anionic site48. As
described in the following chapters, the peripheral anionic
site is a target for a number of toxins and also promis-
ing drugs49,50. It probably plays an important role in the
development of Alzheimer’s disease. Amyloid β peptide
interacts with the peripheral anionic site resulting in the
formation of amyloid plaques and consequent damage to
cholinergic neurons51.
Both AChE and BuChE form mainly tetramer G4 but
they can also form dimmer G2 that can be secreted as
a water soluble molecule52. Monomeric AChE has mo-
lecular weight of 69 kDa (ref.53). The predominant part
of AChE localized in the central nervous system con-
tains botgh hydrophilic and hydrophobic regions i.e. it is
amphiphilic. There are differences between amphiphilic
and non-amphiphilic cholinesterases. The amphiphiphilic
cholinesterase contains G4 catalytic tetramer and one
non-catalytic subunit P (ref.54). The P subunit has a mo-
lecular weight 20 kDa and it is asymmetrically bound to
two G subunits. From a chemical point of view, it is glyco-
phosphatidylinositol (GPI) called GPI anchor (ref.55,56).
In the human AChE, the liphophilic part of the GPI an-
chor is palmitate57. The biological role of individual AChE
forms can be ascertained from an experiment done on
monkey brains58. 85% of AChE are tetramers with a sedi-
mentation constant 9.7 S, 10% is dimeric (5.7 S) and 5%
monomeric (3.2 S). In total, 83% AChE molecules are
amphiphilic and only 17% hydrophilic. Besides free and
membrane bound AChE, there is also collagen bound
AChE. From the symbols introduced by Massoulie and
Bon, bound AChE is abbreviated AChET and one collagen
oligomer is connected with one (A4), two (A8) or three
(A12) tetrameric AChE molecules59,60.
AChE and BuChE have different abilities to split sub-
strates. Compared to BuChE, AChE is not able to hydro-
lyze high molecular weight esters butAChE has higher
affinity for acetylcholine and BuChE for butyrylcholine.
The differences in affinity to substrate are probably caused
by changes in the aromatic gorge disposition. Substitution
of two phenylalanines to leucine and valine in the elec-
tric eel AChE aromatic gorge possessed butyrylcholine
turnover rate at a similar level to BuChE. Further, mu-
tated AChE was sensitive to inhibition by iso-OMPA and
was not inhibited by propidium, a peripheral anionic site
inhibitor61. Differences in AChE and BuChE structures
are revealed by huperzine A. This Alzheimer’s disease
drug is a strong AChE inhibitor binding to the peripheral
anionic site; however, BuChE intacts in the presence of
huperzine62. Another Alzheimer’s disease drug, tacrine,
binds into the α-anionic site. It inhibits AChE as well as
BuChE to a comparable degree63. A similar situation to
tacrin is common for other drugs containing quaternary
ammonium, nerve agents, and neurotoxic pesticides64-67.
The basic parameters for AChE and BuChE are summa-
rized below (Table 2).
Table 2. Basic parameters of AChE and BuChE.
AChE BuChE References
Subunit 69 kDa 85 kDa 29, 30, 53
Quaternary structure Tetramer predominate, mono-, di-
and trimeric forms can also occur 29, 54, 55, 66, 68, 69
Conversion of acetyl-β-methyl-(thio)choline High low 27, 28, 71, 72
Inhibition by excess of substrate Yes no 68, 69, 70
Inhibition by iso-OMPA* No yes 61, 75
Inhibition by nerve agents Yes yes 65, 66
Inhibition by huperzine Yes no 62
*Iso-OMPA: tetraisopropyl pyrophosphoramide
222 M. Pohanka
Apropos AChE enzymology, there are differences be-
tween AChE and BuChE. Inhibition by excess of substrate
is probably the most important fact68,69. The biological
significance of AChE inhibition by substrate excess has
not yet been proven. One theory however looks feasible70.
After acetylcholine vesicles are released into the neuro-
synaptic cleft, AChE is inhibited and the receptors are
stimulated. The termination signal, which has a square
plot in cholinergic nerves, is fast once the ACh level drops
below threshold concentration. The basic biochemical
test for estimating whether a sample contains AChE and
not BuChE is based on assessment of acetylthiocholine
and acetyl-β-methyl-thiocholine as fast conversion in low
concentrations of substrate and slow conversion in high
concentration71,72 Specific inhibitors introduced later are
also applicable. Non-enzymatic functions of AChE are
probable and the research is on-going. Interestingly, body
growth and cell adhesion are probably partially connected
with AChE (ref.73,74).
CHOLINESTERASE INHIBITORS
Cholinesterase inhibitors (e.g. drugs, natural toxins,
pesticides, chemical warfare agents) are a wide group of
chemical compounds with different physico-chemical
properties. AChE inhibitors play a significant role in
the biochemical processes of the human body due to
the physiological importance of AChE. Specific BuChE
inhibitors, such as iso-OMPA (Fig. 1), have mainly diag-
nostic importance75. Lower interest in BuChE inhibitors
can be explained by probable BuChE physiological redun-
dancy. For example, drugs suppressing the manifestation
of Alzheimer’s disease through impact on the cholinergic
system are predominantly selective inhibitors of AChE
(ref.76). Selective inhibitors of BuChE have also been in-
vestigated as potential drugs for Alzheimer’s disease77;
but to a lesser degree than AChE. Inhibition of AChE
also plays an important role in nerve agent toxicology.
Intact BuChE however can temporarily substitute inhib-
ited AChE and is able to slowly hydrolyze accumulated
acetylcholine78.
3. Compounds bound at the peripheral (β) anionic site
(e.g. huperzine, propidium).
Inhibitors binding at the esteratic part of active site
Inhibitors of the esteratic part found on the active
site are compounds with the chemical structure of or-
ganophosphorus or carbamate derivatives. Inhibitors of
esteratic subsite of the active centre are mainly toxins,
chemical warfare agents or pesticides. Of course some
are used as drugs. These compounds interact with ser-
ine in the catalytical triad of active site, providing sta-
ble esters. Organophosphorus compounds create stable,
covalently bound adducts with spontaneous dissociation
once covalently connected with serine hydroxyl. Some
drugs containing the oxime group are able to split or-
ganophoshorus moiety from the active site resulting in
liberation and enzyme reactivation. Obidoxime, trime-
doxime, pralidoxime (2-PAM) and asoxime (HI-6) can
be mentioned as commercially available drugs3. After
a specific time interval (from minutes up hours) for each
organophosphorus inhibitor, the bound inhibitor under-
goes dealkylation called “aging” (ref. 80). The aging has
no beneficial effect on the enzyme as it remains inactive.
In comparison with organosphosphorus inhibitors, the
carbamate moiety is spontaneously hydrolyzed and liber-
ated. AChE becomes active again. Carbamates are prob-
ably able to bind through non-covalent interactions81. The
mechanism of AChE inhibition by an organophosphonate
is depicted below (Fig. 2).
Fig. 1. Tetraisopropyl pyrophosphoramide (iso-OMPA).
Compounds inhibiting AChE can be divided into three
basic groups79:
1. Compounds binding at the active site interact with
either esteratic (e.g. nerve agents) or anionic site (e.g.
tacrine).
2. Compounds interacting with the aromatic gorge (e.g.
decamethonium).
Fig. 2. Inhibition of AChE by nerve agent sarin (reaction
1) and the consequent aging (reaction 2). Serine
hydroxyl is indicated in AChE molecule by an
abbreviation (Ser-OH).
Nerve agents are organophosphonate compounds
used in chemical warfare. The older group of nerve agents
called G series was discovered before World War II. Tabun
was the first known nerve agent first synthesized in 1936
by professor Gerhard Schrader. After World War II, the
most toxic nerve agents called the V series were exten-
sively investigated82. Tabun (abbreviated GA according to
NATO), sarin (GB), soman (GD) and cyclosarin (GF)
are representatives of the G series nerve agents. Among
VX nerve agents are the Russian VX (VR) and Chinese
(VC) variants83. The chemical structures of selected nerve
agents are depicted in (Fig. 3).
223
Cholinesterases, a target of pharmacology and toxicology
Fig. 3. Selected nerve agents.
Nerve agents are extremely hazardous due to superior
penetration ability into the human body by all routes and
their high toxicity. Nerve agents differ from pesticides in
their toxicity and rapid bodily dissemination The median
lethal dose (LD50) is different for individual nerve agents:
e.g. for subcutaneous administrations to rat, the LD50s
are 193 μg/kg for tabun, 103 μg/kg for sarin, 75 μg/kg for
soman, and 12 μg/kg for VX (ref.84-86). Median lethal con-
centration and time (LCt50) in rats for sarin is 150 mg/m3
for ten minutes lasting inhalation87. The toxicity of nerve
agents is much more apparent than standard pesticides:
e.g. the commercially available organophosphate pesticide
primiphos-methyl (e.g. preparation Actellic 50EC) has
a declared LD50 for rat males and per oral administration
≥ 1,500 mg/kg.
The G series of nerve agents penetrate the body by all
routes and spread quickly through the organism. In com-
parison with G series, the V series of nerve agents are able
to penetrate via the lungs and skin with ease; however,
V agents create sub-epithelial reservoirs and the agent is
slowly released from the reservoirs88. It should be empha-
sized that nerve agents as well as organophosphate and
carbamate pesticides are quite reactive. Apart from bind-
ing to AChE and BuChE, they can bind to multiple organs
and tissues in the body. Inhibition of AChE is the most
crucial from the toxicological point of view whereas from
the therapeutic point of view the most significant fact is
the inhibition of carboxylesterase 1. Carboxylesterase 1 is
able to recover its activity even after sarin inhibition. This
fact encourages scientists in the search for an effective
scavenger that would serve as a prophylactic against nerve
agent intoxication89. The other effective enzyme is serum
paraoxonase (PON). The PON is able to split organo-
phosphate and thereby detoxify the poisoned person. On
the other hand, PON activity is quite low and fluctuates
greatly in the general population90.
The less toxic variant of organophosphonate nerve
agents are organophosphate pesticides. Highly toxic
organophosphate pesticides are e.g. paraoxon ethyl,
paraoxon methyl, and malaoxon. These compounds are
approximately equally toxic to warm-blooded as well as
cold-blooded organisms. Due to the effort to enhance pes-
ticide specificity, numerous derivatives of highly toxic pes-
ticides have been prepared to reduce the toxicity towards
warm-blooded organisms and retain toxicity to insects.
Thioforms of organophosphates such as parathion ethyl,
parathion methyl and malathion are some relevant exam-
ples. The thioforms of organophosphate pesticides are
converted into the above mentioned oxoforms by mixed
function oxidases (MFO). The activation proceeds in
cold-blooded organisms but this is not common in warm-
blooded organisms where no metabolizing or dealkylation
into non toxic compound take place91.
Carbamates are the second group of pesticides inhib-
iting cholinesterases. From the chemical point of view,
they are N-alkyl and N,N-dialkyl carbamates. The natural
derivate of carbamate is physostigmine. It is produced as
a secondary metabolite in the African plant Physostigma
venenosum (Fabaceae). Physostigmine is a strong revers-
ible inhibitor of AChE. It has broad use in Myasthenia
gravis treatment as it increases acetylcholine levels in the
damaged neurosynaptic clefts and also as a prophylac-
tic to nerve agent exposure as it blocks the irreversible
binding of nerve agents92,93. Carbamates are pseudo-
irreversible inhibitors of cholinesterases; the carbamoyl
moiety can be split from cholinesterase by spontaneous
hydrolysis94. Carbamates cannot penetrate the blood
brain barrier in the healthy body; however, stress con-
ditions can enhance diffusion into the central nervous
system95. Organophosphate and carbamate compounds
are not only used in agriculture but for medical purposes
too. Rivastigmine is a drug available for the symptomatic
treatment of Parkinson’s as well as Alzheimer’s disease96.
Trichlorfon (metrifonate) has similar application in medi-
cine to rivastigmine though it was used as a pesticide in
the past97.
The majority of countries have strong regulations on
the application of pesticides; e.g. in the European Union it
is regulated by the directive 91/41/EHS. Individual prepa-
rations are approved for commercialization and the list is
regularly updated. Commonly used and relatively safe for
warm-blooded organisms, are mainly pesticides: organo-
phosphates – chlorpyrifos, fenitrothion, pirimiphos-me-
thyl, dimethoate, phosalone and carbamates – pirimicarb,
224 M. Pohanka
Fig. 4. Selected organophosphate and carbamate inhibitors of cholinesterases.
carbofuran, carbosulfan, methiocarb, fenoxycarb. The
structures of selected organophosphate and carbamate
compounds are shown below (Fig. 4).
Inhibitors of the α-anionic site
Cholinesterase inhibitors binding to the α-anionic
site are a group of chemical compounds containing cer-
tain common motives. Firstly, these compounds typi-
cally contain condensed aromatic cores. Secondly, there
should be quarternary ammonium or nitrogen included
as a heteroatom. Acrdines and tetrahydroacridines can be
mentioned as examples. Quinolines and isoquinolines are
other common structures interacting with the α-anionic
site of cholinesterases. In comparison with the esteratic
site inhibitors, compounds interacting with the α-anionic
site are reversible inhibitors. 9-amino-1,2,3,4,-tetrahy-
droacridine known as tacrine, which is also considered
one of the most important inhibitors of the α-anionic site
able to suppress Alzheimer’s disease manifestation. It is
marketed worldwide under the trade name Cognex. The
main disadvantage of tacrine is its relatively high hepato-
toxicity98. There is an effort underway to find less toxic
derivatives of tacrine99. In the past, 7-methoxytacrine
was extensively investigated as a promising substitute
to tacrine. This compound is less toxic than tacrine and
some in vitro as well as in vivo tests proved superior to
tacrine100,101. Protoberbrine alkaloids are strong natural
inhibitors of AChE. Berberine, palmatine, jatrorrhizine
and epiberberine are examples. These substances are con-
sidered promising drugs for Alzheimer’s disease sympto-
matic treatment102.
Galantamine (Nivalin) is another well known drug
interacting with the α-anionic site. It is an alkaloid
from the Caucasian snowdrop (Galanthus woronowii,
Amaryllidaceae). The properties of galantamine were
firstly recognized by Mashkovsky and Kruglikova-Lvova in
the 1950s (ref.103). Beside the α-anionic site, galantamine
also binds at another important part of the AChE active
site including aromatic gorge104,105. The formulas of these
compounds are shown in (Fig. 5).
Inhibitors binding into aromatic gorge
The aromatic gorge is not a typical target for
cholinesterase inhibitors. On the other hand, inhibitors
interacting with the α-anionic site will probably also in-
teract with the aromatic gorge. Galantamine can be men-
tioned as an example (see above). In silico methods and
structural analyses have shown that some bisquarternary
compounds such as the depolarizing muscle relaxant
decamethonium (Fig. 6) provide a stable complex with
225
Cholinesterases, a target of pharmacology and toxicology
Fig. 5. Selected inhibitors that bind onto the α-anionic site.
Fig. 6. Decamethonium (anions are not considered).
Fig. 7. Selected structures that bind to peripheral anionic site (anions are not
considered).
the aromatic gorge due to electrostatic interaction106. But
the main decamethonium effect is not on AChE (ref.107).
Inhibitors of peripheral (β) anionic site
The peripheral anionic site is the main target of many
pharmacologically important compounds rather than
toxins. Much attention is given to the peripheral anionic
site due to the link to Alzheimer’s disease. Lack of ace-
tylcholine was considered as a major factor in the cause
of Alzheimer’s disease (AD). The deposition of amyloid
plaque in AD may be accelerated or even triggered by
interaction of β-amyloid with the peripheral anionic
site. Inhibitors binding at the peripheral anionic site are
considered not only symptomatic drugs for Alzheimer’s
disease, but also probably causative ones108. It should be
emphasized though that the etiology of Alzheimer’s dis-
ease is not thoroughly understood and the actual function
of AChE is still being investigated.
Aflatoxins are natural hepatocarcinogens activated
by liver cytochrome P450. They probably interact with
the peripheral anionic site. Despite strong inhibition of
AChE, it seems that BuChE has no sensitivity to aflatoxin
as shown for aflatoxin B1 (ref.109-111). Inhibition of AChE
was confirmed after the onset of cholinergic symptoms
following aflatoxin exposure112. The mechanism of aflatox-
in interaction with AChE is not well-explained and more
supporting experiments are needed. Double-stranded
DNA fluorescence dyes are also inhibitors of AChE.
Propidium113 as well as ethydium114 are proven inhibitors,
binding at the peripheral anionic site. The structure of
alfatoxin B1, ethidium and propidium are depicted below
(Fig. 7).
The effects of certain ions on AChE remain unclear.
Oxidative state III+ aluminum ions have been investigated.
X–
X–X–
226 M. Pohanka
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