Acetylcholinesterase

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Acetylcholinesterase (AChE) is key enzyme in the nervous system of animals. By rapid hydrolysis of the neurotransmitter, acetylcholine (ACh), AChE terminates neurotransmission at cholinergic synapses. It is a very fast enzyme, especially for a serine hydrolase, functioning at a rate approaching that of a diffusion-controlled reaction. AChE inhibitors are among the key drugs approved by the FDA for management of Alzheimer's disease (AD). The powerful toxicity of organophosphorus (OP) poisons is attributed primarily to their potent AChE inhibitors.

1 Key Enzyme in the Nervous System
2 Treatment of Alzheimer's disease
3 Organophosphorus acid anhydride nerve agents
4 Selected 3D Structures of AChE
5 Additional Resources
6 External Links
7 References

Cholinergic Synapse

Key Enzyme in the Nervous System

Solution of the three-dimensional (3D) structure of Torpedo californica acetylcholinesterase (TcAChE) in 1991 opened up new horizons in research on an enzyme that had already been the subject of intensive investigation.^[1] The unanticipated structure of this extremely rapid enzyme, in which the active site was found to be buried at the bottom of a , lined by (colored dark magenta), led to a revision of the views then held concerning substrate traffic, recognition and hydrolysis.^[2] To understand how those aromatic residues behave with the enzyme, see Flexibility of aromatic residues in acetylcholinesterase. Solution of the 3D structure of acetylcholinesterase led to a series of theoretical and experimental studies, which took advantage of recent advances in theoretical techniques for treatment of proteins, such as molecular dynamics and electrostatics and to site-directed mutagenesis, utilizing suitable expression systems. Acetylcholinesterase hydrolysizes the neurotransmitter acetylcholine , producing group. directly binds (via its nucleophilic Oγ atom) within the catalytic triad (ACh/TcAChE structure 2ace). The residues are also important in the ligand recognition. See also: AChE inhibitors and substrates

Treatment of Alzheimer's disease

Alzheimer's disease (AD) is a disorder that attacks the central nervous system through progressive degeneration of its neurons. AD occurs in around 10% of the elderly and, as yet, there is no known cure. Patients with this disease develop dementia which becomes more severe as the disease progresses. It was suggested that symptoms of AD are caused by decrease of activity of cholinergic neocortical and hippocampal neurons. Treatment of AD by ACh precursors and cholinergic agonists was ineffective or caused severe side effects. ACh hydrolysis by AChE causes termination of cholinergic neurotransmission. Therefore, compounds which inhibit AChE might significantly increase the levels of ACh depleted in AD. Indeed, it was shown that AChE inhibitors improve the cognitive abilities of AD patients at early stages of the disease development.

1 The first generation of AD drugs - monovalent AChE inhibitors
2 The second generation of AD drugs - bivalent AChE inhibitors
- 2.1 Compound 3
- 2.2 Aricept (donepezil, E2020)

The first generation of AD drugs - monovalent AChE inhibitors

The first generation of AD drugs were AChE inhibitors: alcaloids like (-)-Huperzine A (HupA) and (-)-galanthamine (GAL, Reminyl); synthetic compounds tacrine (Cognex) and rivastigmine (Exelon).

HupA

HupA, discovered by Chinese scientists from 1980s, has been proved to be a powerful, highly specific, and reversible inhibitor of AChE. The crystal structure of the complex of TcAChE with HupA at 2.5 Å resolution (1vot) was determined in 1997 and it shows an unexpected orientation for the inhibitor with surprisingly few strong direct interactions with protein residues to explain its high affinity. HupA binds to TcAChE at the active site, and its in comparison to ACh. The principal interactions of are including: a direct (colored orange) through a water molecule as a linker at the bottom of the gorge; cation-π interactions between the amino group of (colored lime) with the distance between the nitrogen and the centroid of the aromatic rings of 4.8 and 4.7 Å, respectively; at the top of the gorge, hydrogen bonds through two water molecules as linkers formed between the amino group of (colored magenta). An unusually short (~3.0 Å) C-H→O HB has been seen between the ethylidene methyl group of (colored crimson).

Galanthamine

(GAL). (red) is an alkaloid from the flower snowdrop (Galanthus nivalis). The X-ray crystal structure of the TcAChE/GAL complex (1dx6) was determined at 2.3 Å resolution. The inhibitor binds at the base of the active site gorge of TcAChE, interacting with both the choline-binding site (Trp84) and the acyl-binding pocket (Phe288, Phe290). The tertiary amine appears to make a non-conventional hydrogen bond, via its N-methyl group, to Asp72. The hydroxyl group of the inhibitor makes a strong hydrogen bond (2.7 Å) with Glu199. ACh (gray) is shown for comparison. In the X-ray crystal structure of TcAChE/ complex which was determined at 2.8 Å resolution, the tacrine is seen (magenta) bound in the active site of TcAChE (1acj). ACh (gray) is shown for comparison.

Rivastigmine

is a carbamate inhibitor of AChE, and it is currenly used in therapy of Alzheimer's disease. Rivastigmine (colored yellow) interacts with TcAChE (colored lime) at the (1gqr). The carbamyl moiety of rivastigmine is to the active-site S200 Oγ. The second part of rivastigmine (the leaving group), NAP ((−)-S-3-[1-(dimethylamino)ethyl]phenol) is also held in the active-site gorge, but it is from the carbamyl moiety, hence, carbamylation took place. The of TcAChE/NAP (colored magenta) is known (1gqs). The TcAChE active-site residues which are interacting with NAP are colored violet. NAP is located in a similar region of TcAChE active site, but with different orientation than that of the NAP part (colored yellow) in the TcAChE/rivastigmine complex. Only H440 and F330 significantly change their side-chain conformations. of the TcAChE active sites in 4 different structures (TcAChE/rivastigmine (1gqr), TcAChE/NAP (1gqs), native TcAChE (2ace), and TcAChE/VX (1vxr, TcAChE colored white and VX black) reveals that the conformation of H440 in the TcAChE/NAP structure is very similar its conformation in the native TcAChE (2ace), but the distance between H440 Nδ and E327 Oε is significantly longer in the TcAChE/rivastigmine and the TcAChE/VX complexes. This structural change disrupts the catalytic triad consisting of S200, E327, H440. This could explain the very slow kinetics of AChE reactivation after its inhibition by rivastigmine.

The second generation of AD drugs - bivalent AChE inhibitors

The active site of consists of . First of them is the "catalytic anionic site" (CAS), which involves mentioned above catalytic triad (colored orange) and the conserved residues and also participating in ligands recognition. Another conserved residue (colored cyan) is situated at the second binding subsite, termed the "peripheral anionic site" (PAS), ~14 Å from CAS. Therefore, the ligands that will be able to interact with both these subsites, will be more potent AChE inhibitors in comparison to compounds interacting only with CAS (mentioned in the previous section "The first generation of AD drugs - monovalent AChE inhibitors"). One of the ways to produce such ligands is to introduce two active substances into one compound. If it is spatially necessary these subunits could be divided by alkyl linker with suitable length. For example, according to the strategy of the use of a bivalent ligand, the (RS)-(±)-tacrine-(10)-hupyridone ((R)-3 or (S)-3) was designed and synthesized. It consists of mentioned above (colored magenta), 10-carbon (yellow), and (red). The tacrine moiety of this inhibitor binds at the CAS, the linker spans the gorge, and the hupyridone moiety binds at the PAS. There are also only PAS-binding AChE inhibitors, (magenta) is a good example of them. of the crystal structure of the edrophonium/TcAChE (CAS-binding inhibitor) (2ack) on the thioflavin T/TcAChE complex structure (2j3q) shows that these ligands' positions do not overlap. Of note is that Phe330, which is part of the CAS, is the single residue interacting with thioflavin T. This residue is the only one which significantly to avoid clashes in comparison to other CAS residues of the edrophonium/TcAChE complex.

Compound 3

Described above, (abbreviated as ; colored red) is a CAS-binding inhibitor and it is currently used in therapy of the AD. Conjugate of GAL through the (8 carbons, yellow) with a (blueviolet) called compound 3 has a larger affinity for AChE than that of GAL alone. This is similar to previously described cases of bivalent ligands (e.g. (RS)-(±)-tacrine-(10)-hupyridone). A comparison between /TcAChE (1w4l) and structure (1dx6) shows an identical binding mode of the GAL-moiety (transparent red) of compound 3 to that of GAL alone (blue) at the CAS. A PEG molecule (gray) is located at the active site of the galanthamine/TcAChE structure. The alkyl linker spans the active-site gorge and the phthalimido moiety of compound 3 is situated near Trp279 at the PAS. Compound 3 has higher affinity to TcAChE than GAL. This can be explained by the higher number of interactions between compound 3 (which interacts not only with residues within CAS but also within PAS) and TcAChE relative to GAL.

Aricept (donepezil, E2020)

is one of the most interesting drugs that have been designed as AChE bivalent inhibitors. It was developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies prior to elucidation of the 3D structure of Torpedo californica AChE (TcAChE) (1ea5). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range. The X-ray structure of the E2020-TcAChE complex shows that E2020 has a along the active-site gorge, extending from the anionic subsite () of the active site, at the bottom, to the peripheral anionic site (), at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole' but only . The X-ray structure shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE, to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.

Organophosphorus acid anhydride nerve agents

Organophosphorus (OP) acid anhydride nerve agents are potent inhibitors which rapidly phosphonylate AChE and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. As was mentioned above, AChE hydrolysizes the neurotransmitter , producing group. directly binds catalytic (via its nucleophilic Oγ atom). , O-(1,2,2-trimethylpropyl) methylphosphonofluoridate (fluorine atom is colored violet and phosphorus atom is colored darkmagenta), is one of the most toxic OPs. Soman inhibits AChE by to catalytic Ser200, . This process implicates nucleophilic attack of the Ser200 nucleophilic Oγ atom on the phosphorus atom of soman, with concomitant departure of its fluoride atom. After that AChE catalyzes the of the soman or other OP. This causes irreversible inhibition of AChE, "aged" soman/AChE conjugate can not be reactivated. However, before “aging”, at the step of , AChE can be by nucleophiles, such as pralidoxime (2-PAM), resulting in of the phosphonyl adduct from Ser200 Oγ. At the (2wfz) the catalytic His440 forms hydrogen bonds with Ser200 Oγ and Glu327 Oε1 via its Nε2 and Nδ1 nitrogens, respectively. The O2 atom of soman is within hydrogen bonding distance of His440 Nε2. Soman O1 mimicks carbonyl oxygen of ACh. A water molecule 1001 interacting with soman O2 is represented as a red ball. The active site residues of the nonaged soman/TcAChE are colored yellow. The O2 atom of the (2wg0) forms a salt bridge with His440 Nε2. The active site residues of the aged soman/TcAChE are colored pink. of the structures of the nonaged (2wfz) and aged (2wg0) conjugates reveals a small, but important, change within the active site - the imidazole ring of His440 is tilted back to a native-like conformation after dealkylation. The water molecule 1001, which interacts with soman O2 in the nonaged crystal structure, is not within hydrogen bonding distance of O2 in the aged crystal structure. 2-PAM binds poorly to the nonaged phosphonylated enzyme (its electron density was not found) and binds in an after soman aging to TcAChE (2wg1).

Selected 3D Structures of AChE

Acetylcholinesterase - AChE native

3lii – hAChE - recombinant human
1ea5, 2ace – TcAChE – trigonal – Torpedo californica
2j3d – TcAChE – monoclinic
1w75 – TcAChE – orthorhombic
1eea – TcAChE – cubic
2vt6, 2vt7 – TcAChE – different dosage
1qid to 1qim - TcAChE synchrotron radiation damage
1j06, 1maa – mAChE - mouse
1qo9 – DmAChE - Drosophila
1c2o, 1c2b – electrophorus AChE – Electric eel

AChE inhibitors (In Different Languages)

1eve AChE-Aricept complex, 1eve (Arabic), 1eve (Chinese), 1eve (Italian), 1eve (Russian), 1eve (Spanish), 1eve (Turkish)
1vot AChE-Huperzine A complex, 1vot (Chinese)

AChE active site inhibitors conjugating at the bottom of the active site gorge

2w9i – TcAChE + methylene blue
2wls – MosAChE + AMTS13
2vq6 – TcAChE + 2-PAM
2j3q – TcAChE + Thioflavin T
2ha0 – mAChE + ketoamyltrimethylammonium
2h9y – mAChE + TMTFA
1gpk, 1gpn, 1vot – TcAChE + huperzine
1gqr – TcAChE + rivastigmine
1gqs – TcAChE + NAP
1e66 – TcAChE + huprine
1dx4, 1qon – DmAChE + tacrine derivative
1oce – TcAChE + MF268
1ax9, 1ack – TcAChE + edrophonium
1amn – TcAChE + TMTFA
1acj – TcAChE + tacrine

AChE peripheral site inhibitors conjugating at the surface of the protein

1ku6 - mAChE + fasciculin 2
1ku6, 1mah - mAChE + fasciculin 2
1j07 - mAChE + decidium
1n5m - mAChE + gallamine
1n5r - mAChE + propidium
1b41, 1f8u - hAChE + fasciculin 2
1fss - TcAChE + fasciculin 2

AChE bis inhibitors spanning the active site gorge

3i6m – TcAChE + N-piperidinopropyl galanthamine
3i6z - TcAChE + saccharinohexyl galanthamine
1zgb, 1zgc – TcAChE + tacrine (10) hupyridone
2w6c – TcAChE + bis-(-)-nor-meptazinol
2ckm, 2cmf – TcAChE + bis-tacrine
2cek – TcAChE + N-[8-(1,2,3,4-tetrahydroacridin-9-ylthio)octyl]-1,2,3,4-tetrahydroacridin-9-amine
1ut6 - TcAChE + N-9-(1,2,3,4-tetrahydroacridinyl)-1,8-diaminooctane
1odc - TcAChE + N-4-quinolyl-N-9-(1,2,3,4-tetrahydroacridinyl)-1,8-diaminooctane
1w4l, 1w6r, 1w76, 1dx6, 1qti - TcAChE + galanthamine and derivative
1q83, 1q84 - mAChE + TZ2PA6
1h22, 1h23 – TcAChE + bis-hupyridone
1hbj – TcAChE + quinoline derivativev
1e3q – TcAChE + bw284c51
1eve – TcAChE + e2020
1acl – TcAChE + decamethonium

AChE organophosphate inhibitors causing irreversible inhibition

2wu3 – mAChE + fenamiphos and HI-6
2wu4 – mAChE + fenamiphos and ortho-7
2jgf - mAChE + fenamiphos
2wfz, 2wg0, 1som - TcAChE + soman
2wg1 - TcAChE + soman + 2-PAM
2whp, 2whq, 2whr – mAChE + sarin and HI-6
2jgg - mAChE + sarin
2jgl - mAChE + VX and sarin
1cfj - TcAChE + sarin, GB
3dl4, 3dl7 – mAChE + tabun
2jey – mAChE + HLO-7
2c0p, 2c0q - mAChE + tabun
2jez - mAChE + tabun + HLO-7
2jf0 - mAChE + tabun + Ortho-7
2jgh - mAChE + VX
1vxo, 1vxr - TcAChE + VX
2jgi, 2jgm - mAChE + DFP
1dfp - TcAChE + DFP
2jgj, 2jgk, 2jge - mAChE + methamidophos
2gyu - mAChE + HI-6
2gyv - mAChE + Ortho-7
2gyw - mAChE + obidoxime

AChE substrate analogues mimicking the binding of the substrate acetylcholine

2ha4 – mAChE (mutant) + acetylcholine
2vja, 2vjb, 2vjc, 2vjd, 2cf5 – TcAChE + 4-oxo-N,N,N-trimethylpentanaminium
2v96, 2v97, 2v98, 2v99 – TcAChE + 1-(2-nitrophenyl)-2,2,2-trifluoroethyl-arsenocholine
2ha2 – mAChE + succinylcholine
2ha3 - mAChE + choline
2ha5 – mAChE (mutant) + acetylthiocholine
2ha6 – mAChE (mutant) + succinylthiocholine
2ha7 – mAChE (mutant) + butyrylthiocholine
2ch4, 2c58 – TcAChE + acetylthiocholine
2c5g – TcAChE + thiocholine

Others...

2j4f – TcAChE + Hg
1vzj – TcAChE tetramerization domain
1jjb – TcAChE + PEG

Additional Resources

For additional information, see:
Alzheimer's Disease
AChE inhibitors and substrates
AChE inhibitors and substrates (Part II)
AChE inhibitors and substrates (Part III)
AChE bivalent inhibitors
AChE bivalent inhibitors (Part II)

External Links

Acetylcholinesterase Tutorial by Karl Oberholser, Messiah College
PDB Molecule of the Month - Acetylcholinesterase
Movies: X-ray Damage in ACh & Nature's Vacuum Cleaner by R. Gillilan, Cornell Univ

References

↑ Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science. 1991 Aug 23;253(5022):872-9. PMID:1678899
↑ Botti SA, Felder CE, Lifson S, Sussman JL, Silman I. A modular treatment of molecular traffic through the active site of cholinesterase. Biophys J. 1999 Nov;77(5):2430-50. PMID:10545346

Proteopedia Page Contributors and Editors (what is this?)

Michal Harel, Joel L. Sussman, Alexander Berchansky, David Canner, Eran Hodis, Clifford Felder, Jaime Prilusky, Harry Greenblatt, Yechun Xu

Retrieved from "http://52.214.119.220/wiki/index.php/Acetylcholinesterase"

@@ Line 22: / Line 22: @@
 [http://en.wikipedia.org/wiki/Alzheimer's_disease Alzheimer's disease] (AD) is a disorder that attacks the [http://en.wikipedia.org/wiki/Central_nervous_system central nervous system] through progressive degeneration of its neurons. AD occurs in around 10% of the elderly and, as yet, there is no known cure. Patients with this disease develop [http://en.wikipedia.org/wiki/Dementia dementia] which becomes more severe as the disease progresses. It was suggested that symptoms of AD are caused by decrease of activity of [http://en.wikipedia.org/wiki/Cholinergic cholinergic] [http://en.wikipedia.org/wiki/Neocortex neocortical] and [http://en.wikipedia.org/wiki/Hippocampus hippocampal] neurons. Treatment of AD by ACh precursors and [http://en.wikipedia.org/wiki/Cholinergic cholinergic] [http://en.wikipedia.org/wiki/Agonist agonists] was ineffective or caused severe side effects. ACh hydrolysis by AChE causes termination of cholinergic neurotransmission. Therefore, compounds which inhibit AChE might significantly increase the levels of ACh depleted in AD. Indeed, it was shown that [http://en.wikipedia.org/wiki/Acetylcholinesterase_inhibitor AChE inhibitors] improve the cognitive abilities of AD patients at early stages of the disease development.
+<StructureSection load='1vot' size='600' side='right' scene='1vot/Com_view/1'>
 === The first generation of AD drugs - monovalent AChE inhibitors ===
 The first generation of AD drugs were AChE inhibitors: alcaloids like [http://en.wikipedia.org/wiki/Huperzine_A (-)-Huperzine A (HupA)] and [http://en.wikipedia.org/wiki/Galantamine (-)-galanthamine (GAL, Reminyl)]; [http://en.wikipedia.org/wiki/Chemical_synthesis synthetic] compounds [http://en.wikipedia.org/wiki/Tacrine tacrine (Cognex)] and [http://en.wikipedia.org/wiki/Rivastigmine rivastigmine (Exelon)].
-<applet load='1vot' size='500' frame='true' align='right'
+====HupA====
-scene='1vot/Com_view/1' />
 '''HupA''', discovered by Chinese scientists from 1980s, has been proved to be a powerful, highly specific, and [http://en.wikipedia.org/wiki/Enzyme_inhibitor#Reversible_inhibitors reversible inhibitor] of AChE. The [http://en.wikipedia.org/wiki/X-ray_crystallography crystal structure] of the complex of ''Tc''AChE with HupA at 2.5 Å resolution ([[1vot]]) was determined in 1997 and it shows an unexpected orientation for the inhibitor with surprisingly few strong direct interactions with protein residues to explain its high affinity. <font color='blueviolet'><b>HupA</b></font> binds to ''Tc''AChE at the active site, and its <scene name='1vot/Active_site/8'>observed orientation is almost orthogonal</scene> in comparison to <font color='gray'><b>ACh</b></font>. The principal interactions of <scene name='1vot/1vot_ache_interactions/2'>HupA with TcAChE</scene> are including: a direct <scene name='1vot/1vot_199_130_117/2'>hydrogen bond with Tyr130 and HBs with Glu199 and Gly117 </scene> <font color='orange'><b>(colored orange)</b></font> through a water molecule as a linker at the bottom of the gorge; [http://en.wikipedia.org/wiki/Cation-pi_interaction cation-π] interactions between the amino group of <scene name='1vot/1vot_84_330/2'>HupA and Trp84 and Phe330</scene> <font color='lime'><b>(colored lime)</b></font> with the distance between the nitrogen and the centroid of the aromatic rings of 4.8 and 4.7 Å, respectively; at the top of the gorge, [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] through two water molecules as linkers formed between the amino group of <scene name='1vot/1vot_70_72_81_85_121/3'>HupA and Tyr70, Asp72, Ser81, Asn85 and Tyr121</scene> <font color='magenta'><b>(colored magenta)</b></font>. An unusually short (~3.0 Å) C-H→O HB has been seen between the ethylidene methyl group of <scene name='1vot/1vot_440/2'>HupA and the main chain oxygen of His440</scene> <font color='crimson'><b>(colored crimson)</b></font>.
-{{Clear}}
+====Galanthamine====
-<applet load='ACHE2.pdb' size='500' frame='true' align='right'
-scene='AChE_inhibitors_and_substrates/Com_view_gal/1' />
-[http://en.wikipedia.org/wiki/Galantamine Galanthamine]. <scene name='AChE_inhibitors_and_substrates/Com_view_gal/2'>GAL</scene> <font color='red'><b>(red)</b></font> is an [http://en.wikipedia.org/wiki/Alkaloid alkaloid] from the flower  snowdrop ([http://en.wikipedia.org/wiki/Galanthus ''Galanthus nivalis'']). The [http://en.wikipedia.org/wiki/X-ray_crystallography X-ray crystal structure] of the ''Tc''AChE/GAL complex ([[1dx6]]) was determined at 2.3 Å resolution. The inhibitor binds at the base of the [http://en.wikipedia.org/wiki/Active_site active site] gorge of ''Tc''AChE, interacting with both the choline-binding site (Trp84) and the acyl-binding pocket (Phe288, Phe290). The [http://en.wikipedia.org/wiki/Amine tertiary amine] appears to make a non-conventional [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond], via its N-methyl group, to Asp72. The [http://en.wikipedia.org/wiki/Hydroxyl#Hydroxyl_group hydroxyl group] of the inhibitor makes a strong hydrogen bond (2.7 Å) with Glu199. <font color='gray'><b>ACh (gray)</b></font> is shown for comparison.
+<scene name='AChE_inhibitors_and_substrates/Com_view_gal/1'>Galanthamine</scene> (GAL). <scene name='AChE_inhibitors_and_substrates/Com_view_gal/2'>GAL</scene> <font color='red'><b>(red)</b></font> is an [http://en.wikipedia.org/wiki/Alkaloid alkaloid] from the flower  snowdrop ([http://en.wikipedia.org/wiki/Galanthus ''Galanthus nivalis'']). The [http://en.wikipedia.org/wiki/X-ray_crystallography X-ray crystal structure] of the ''Tc''AChE/GAL complex ([[1dx6]]) was determined at 2.3 Å resolution. The inhibitor binds at the base of the [http://en.wikipedia.org/wiki/Active_site active site] gorge of ''Tc''AChE, interacting with both the choline-binding site (Trp84) and the acyl-binding pocket (Phe288, Phe290). The [http://en.wikipedia.org/wiki/Amine tertiary amine] appears to make a non-conventional [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond], via its N-methyl group, to Asp72. The [http://en.wikipedia.org/wiki/Hydroxyl#Hydroxyl_group hydroxyl group] of the inhibitor makes a strong hydrogen bond (2.7 Å) with Glu199. <font color='gray'><b>ACh (gray)</b></font> is shown for comparison.
 In the X-ray crystal structure of ''Tc''AChE/<scene name='AChE_inhibitors_and_substrates/Com_view_tacrine/2'>tacrine</scene> complex which was determined at 2.8 Å resolution, the [http://en.wikipedia.org/wiki/Tacrine tacrine] is seen <font color='magenta'><b>(magenta)</b></font> bound in the active site of ''Tc''AChE ([[1acj]]). <font color='gray'><b>ACh (gray)</b></font> is shown for comparison.
-{{Clear}}
-<applet load='1gqr' size='500' frame='true' align='right'
-scene='1gqr/Com_view/1' />
-[http://en.wikipedia.org/wiki/Rivastigmine Rivastigmine (Exelon)] is a [http://en.wikipedia.org/wiki/Carbamate carbamate] inhibitor of AChE, and it is currenly used in therapy of [http://en.wikipedia.org/wiki/Alzheimer's_disease Alzheimer's disease]. Rivastigmine (colored yellow) interacts with ''Tc''AChE <font color='lime'><b>(colored lime)</b></font> at the <scene name='1gqr/Active_site/4'>active-site gorge</scene> ([[1gqr]]). The carbamyl moiety of rivastigmine is <scene name='1gqr/Active_site/9'>covalently bound</scene> to the active-site S200 Oγ. The second part of rivastigmine (the leaving group), NAP ((−)-S-3-[1-(dimethylamino)ethyl]phenol) is also held in the active-site gorge, but it is <scene name='1gqr/Active_site/6'>separated</scene> from the carbamyl moiety, hence, carbamylation took place. The <scene name='1gqr/Active_site/7'>crystal structure</scene> of ''Tc''AChE/<font color='magenta'><b>NAP (colored magenta)</b></font> is known ([[1gqs]]). The <font color='violet'><b>''Tc''AChE active-site residues</b></font> which are interacting with NAP are <font color='violet'><b>colored violet</b></font>. NAP is located in a similar region of ''Tc''AChE active site, but with different orientation than that of the NAP part (colored yellow) in the  ''Tc''AChE/rivastigmine complex. Only H440 and F330 significantly change their side-chain conformations. <scene name='1gqr/Active_site/8'>Overlap</scene> of the ''Tc''AChE active sites in 4 different structures (<font color='lime'><b>''Tc''AChE</b></font>/rivastigmine ([[1gqr]]), <font color='violet'><b>''Tc''AChE</b></font>/<font color='magenta'><b>NAP</b></font> ([[1gqs]]), <font color='cyan'><b>native ''Tc''AChE</b></font> ([[2ace]]), and ''Tc''AChE/'''VX''' ([[1vxr]], ''Tc''AChE colored white and VX black) reveals that the conformation of H440 in the ''Tc''AChE/NAP structure is very similar its conformation in the native ''Tc''AChE ([[2ace]]), but the distance between H440 Nδ and E327 Oε is significantly longer in the ''Tc''AChE/rivastigmine and the ''Tc''AChE/'''VX''' complexes. This structural change disrupts the [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] consisting of S200, E327, H440. This could explain the very slow kinetics of AChE reactivation after its inhibition by rivastigmine.
+====Rivastigmine====
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+<scene name='1gqr/Com_view/1'>Rivastigmine (Exelon)</scene> is a [http://en.wikipedia.org/wiki/Carbamate carbamate] inhibitor of AChE, and it is currenly used in therapy of [http://en.wikipedia.org/wiki/Alzheimer's_disease Alzheimer's disease]. Rivastigmine (colored yellow) interacts with ''Tc''AChE <font color='lime'><b>(colored lime)</b></font> at the <scene name='1gqr/Active_site/4'>active-site gorge</scene> ([[1gqr]]). The carbamyl moiety of rivastigmine is <scene name='1gqr/Active_site/9'>covalently bound</scene> to the active-site S200 Oγ. The second part of rivastigmine (the leaving group), NAP ((−)-S-3-[1-(dimethylamino)ethyl]phenol) is also held in the active-site gorge, but it is <scene name='1gqr/Active_site/6'>separated</scene> from the carbamyl moiety, hence, carbamylation took place. The <scene name='1gqr/Active_site/7'>crystal structure</scene> of ''Tc''AChE/<font color='magenta'><b>NAP (colored magenta)</b></font> is known ([[1gqs]]). The <font color='violet'><b>''Tc''AChE active-site residues</b></font> which are interacting with NAP are <font color='violet'><b>colored violet</b></font>. NAP is located in a similar region of ''Tc''AChE active site, but with different orientation than that of the NAP part (colored yellow) in the  ''Tc''AChE/rivastigmine complex. Only H440 and F330 significantly change their side-chain conformations. <scene name='1gqr/Active_site/8'>Overlap</scene> of the ''Tc''AChE active sites in 4 different structures (<font color='lime'><b>''Tc''AChE</b></font>/rivastigmine ([[1gqr]]), <font color='violet'><b>''Tc''AChE</b></font>/<font color='magenta'><b>NAP</b></font> ([[1gqs]]), <font color='cyan'><b>native ''Tc''AChE</b></font> ([[2ace]]), and ''Tc''AChE/'''VX''' ([[1vxr]], ''Tc''AChE colored white and VX black) reveals that the conformation of H440 in the ''Tc''AChE/NAP structure is very similar its conformation in the native ''Tc''AChE ([[2ace]]), but the distance between H440 Nδ and E327 Oε is significantly longer in the ''Tc''AChE/rivastigmine and the ''Tc''AChE/'''VX''' complexes. This structural change disrupts the [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] consisting of S200, E327, H440. This could explain the very slow kinetics of AChE reactivation after its inhibition by rivastigmine.
 === The second generation of AD drugs - bivalent AChE inhibitors ===
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+The active site of <scene name='1zgb/Com_view/1'>TcAChE</scene> consists of <scene name='1zgb/Act_site/3'>two binding subsites</scene>. First of them is the "catalytic anionic site" ('''CAS'''), which involves mentioned above [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] <scene name='1zgb/Act_site/8'>Ser200, His440, and Glu327</scene> <font color='orange'><b>(colored orange)</b></font> and the [http://en.wikipedia.org/wiki/Conserved_sequence#Conserved_protein_sequences_and_Structures conserved residues] <scene name='1zgb/Act_site/5'>Trp84</scene> and <scene name='1zgb/Act_site/10'>Phe330</scene> also participating in ligands recognition. Another conserved residue <scene name='1zgb/Act_site/11'>Trp279</scene> <font color='cyan'><b>(colored cyan)</b></font> is situated at the second binding subsite, termed the "peripheral anionic site" ('''PAS'''), ~14 Å from '''CAS'''. Therefore, the ligands that will be able to interact with both these subsites, will be more potent [http://en.wikipedia.org/wiki/Acetylcholinesterase_inhibitor AChE inhibitors] in comparison to compounds interacting only with CAS (mentioned in the previous section "The first generation of AD drugs - monovalent AChE inhibitors"). One of the ways to produce such ligands is to introduce two active substances into one compound. If it is spatially necessary these subunits could be divided by alkyl linker with suitable length. For example, according to the strategy of the use of a bivalent ligand, the <scene name='1zgb/Comp/7'>inhibitor</scene> '''(''RS'')-(±)-tacrine-(10)-hupyridone''' ((R)-3 or (S)-3) was designed and synthesized. It consists of mentioned above <scene name='1zgb/Comp/8'>tacrine</scene> <font color='magenta'><b>(colored magenta)</b></font>, 10-carbon <scene name='1zgb/Comp/9'>linker</scene> <font color='yellow'><b>(yellow)</b></font>, and <scene name='1zgb/Comp/10'>hupyridone</scene> <font color='red'><b>(red)</b></font>. The tacrine moiety of this inhibitor binds at the CAS, the linker spans the <scene name='1zgb/Act_site/12'>active-site</scene> gorge, and the hupyridone moiety binds at the PAS. There are also only PAS-binding AChE inhibitors, <scene name='2j3q/Active_site/6'>Thioflavin T</scene> <font color='magenta'><b>(magenta)</b></font> is a good example of them. <scene name='2j3q/Active_site/7'>Superposition</scene> of the crystal structure of the <font color='red'><b>edrophonium</b></font>/''Tc''AChE (CAS-binding inhibitor) ([[2ack]]) on the <font color='magenta'><b>thioflavin T</b></font>/''Tc''AChE complex structure ([[2j3q]]) shows that these ligands' positions do not overlap. Of note is that Phe330, which is part of the CAS, is the single residue interacting with <font color='magenta'><b>thioflavin T</b></font>.  This residue is the only one which significantly <scene name='2j3q/Active_site/9'>changes its conformation</scene> to avoid clashes in comparison to other CAS residues of the <font color='red'><b>edrophonium</b></font>/''Tc''AChE complex.
-The <scene name='1zgb/Act_site/3'>active site</scene> of ''Tc''AChE consists of two binding subsites. First of them is the "catalytic anionic site" ('''CAS'''), which involves mentioned above [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] <scene name='1zgb/Act_site/8'>Ser200, His440, and Glu327</scene> <font color='orange'><b>(colored orange)</b></font> and the [http://en.wikipedia.org/wiki/Conserved_sequence#Conserved_protein_sequences_and_Structures conserved residues] <scene name='1zgb/Act_site/5'>Trp84</scene> and <scene name='1zgb/Act_site/10'>Phe330</scene> also participating in ligands recognition. Another conserved residue <scene name='1zgb/Act_site/11'>Trp279</scene> <font color='cyan'><b>(colored cyan)</b></font> is situated at the second binding subsite, termed the "peripheral anionic site" ('''PAS'''), ~14 Å from '''CAS'''. Therefore, the ligands that will be able to interact with both these subsites, will be more potent [http://en.wikipedia.org/wiki/Acetylcholinesterase_inhibitor AChE inhibitors] in comparison to compounds interacting only with CAS (mentioned in the previous section "The first generation of AD drugs - monovalent AChE inhibitors"). One of the ways to produce such ligands is to introduce two active substances into one compound. If it is spatially necessary these subunits could be divided by alkyl linker with suitable length. For example, according to the strategy of the use of a bivalent ligand, the <scene name='1zgb/Comp/7'>inhibitor</scene> '''(''RS'')-(±)-tacrine-(10)-hupyridone''' ((R)-3 or (S)-3) was designed and synthesized. It consists of mentioned above <scene name='1zgb/Comp/8'>tacrine</scene> <font color='magenta'><b>(colored magenta)</b></font>, 10-carbon <scene name='1zgb/Comp/9'>linker</scene> <font color='yellow'><b>(yellow)</b></font>, and <scene name='1zgb/Comp/10'>hupyridone</scene> <font color='red'><b>(red)</b></font>. The tacrine moiety of this inhibitor binds at the CAS, the linker spans the <scene name='1zgb/Act_site/12'>active-site</scene> gorge, and the hupyridone moiety binds at the PAS. There are also only PAS-binding AChE inhibitors, <scene name='2j3q/Active_site/6'>Thioflavin T</scene> <font color='magenta'><b>(magenta)</b></font> is a good example of them. <scene name='2j3q/Active_site/7'>Superposition</scene> of the crystal structure of the <font color='red'><b>edrophonium</b></font>/''Tc''AChE (CAS-binding inhibitor) ([[2ack]]) on the <font color='magenta'><b>thioflavin T</b></font>/''Tc''AChE complex structure ([[2j3q]]) shows that these ligands' positions do not overlap. Of note is that Phe330, which is part of the CAS, is the single residue interacting with <font color='magenta'><b>thioflavin T</b></font>.  This residue is the only one which significantly <scene name='2j3q/Active_site/9'>changes its conformation</scene> to avoid clashes in comparison to other CAS residues of the <font color='red'><b>edrophonium</b></font>/''Tc''AChE complex.
+====Compound 3====
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+Described above, <scene name='1w4l/Al/1'>Galantamine</scene> (abbreviated as <scene name='1w4l/Al/2'>GAL</scene>; <font color='red'><b>colored red</b></font>) is a CAS-binding inhibitor and it is currently used in therapy of the AD. Conjugate of GAL through the <scene name='1w4l/Al/3'>alkyl linker</scene> (8 carbons, <font color='black'><b>yellow</b></font>) with a <scene name='1w4l/Al/4'>phthalimido moiety</scene> <font color='blueviolet'><b>(blueviolet)</b></font> called '''compound 3''' has a larger affinity for AChE than that of GAL alone. This is similar to previously described cases of bivalent ligands (''e.g.'' '''(''RS'')-(±)-tacrine-(10)-hupyridone'''). A comparison between <scene name='1w4l/Comparison/1'>compound 3</scene>/''Tc''AChE ([[1w4l]]) and <scene name='1w4l/Comparison/2'>galanthamine/TcAChE</scene> structure ([[1dx6]]) shows an identical binding mode of the <font color='red'><b>GAL-moiety (transparent red)</b></font> of '''compound 3''' to that of <font color='blue'><b>GAL alone (blue)</b></font> at the CAS. A <font color='gray'><b>PEG molecule (gray)</b></font> is located at the active site of the galanthamine/''Tc''AChE structure. The alkyl linker spans the active-site gorge and the phthalimido moiety of '''compound 3''' is situated near Trp279 at the PAS.  '''Compound 3''' has  higher affinity to ''Tc''AChE than GAL. This can be explained by the higher number of interactions between '''compound 3''' (which interacts not only with residues within CAS but also within PAS) and ''Tc''AChE relative to GAL.
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-Described above '''Galantamine''' (<scene name='1w4l/Al/2'>GAL</scene>; <font color='red'><b>colored red</b></font>) is a CAS-binding inhibitor and it is currently used in therapy of the AD. Conjugate of GAL through the <scene name='1w4l/Al/3'>alkyl linker</scene> (8 carbons, <font color='black'><b>yellow</b></font>) with a <scene name='1w4l/Al/4'>phthalimido moiety</scene> <font color='blueviolet'><b>(blueviolet)</b></font> called '''compound 3''' has a larger affinity for AChE than that of GAL alone. This is similar to previously described cases of bivalent ligands (''e.g.'' '''(''RS'')-(±)-tacrine-(10)-hupyridone'''). A comparison between <scene name='1w4l/Comparison/1'>compound 3</scene>/''Tc''AChE ([[1w4l]]) and <scene name='1w4l/Comparison/2'>galanthamine/TcAChE</scene> structure ([[1dx6]]) shows an identical binding mode of the <font color='red'><b>GAL-moiety (transparent red)</b></font> of '''compound 3''' to that of <font color='blue'><b>GAL alone (blue)</b></font> at the CAS. A <font color='gray'><b>PEG molecule (gray)</b></font> is located at the active site of the galanthamine/''Tc''AChE structure. The alkyl linker spans the active-site gorge and the phthalimido moiety of '''compound 3''' is situated near Trp279 at the PAS.  '''Compound 3''' has  higher affinity to ''Tc''AChE than GAL. This can be explained by the higher number of interactions between '''compound 3''' (which interacts not only with residues within CAS but also within PAS) and ''Tc''AChE relative to GAL.
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+====Aricept (donepezil, E2020)====
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+<scene name='Main_Page/E2020_in_ache_spinning/1'>Aricept (E2020)</scene> is one of the most interesting drugs that have been designed as AChE bivalent inhibitors. It was developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies prior to elucidation of the 3D structure of ''Torpedo californica'' AChE (''Tc''AChE) ([[1ea5]]). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range. The X-ray structure of the E2020-''Tc''AChE complex shows that E2020 has a <scene name='1eve/E2020_close_up_with_84_279/13'>unique orientation</scene> along the active-site gorge, extending from the anionic subsite (<scene name='1eve/E2020_close_up_with_84lbld/7'>W84</scene>) of the active site, at the bottom, to the peripheral anionic site (<scene name='1eve/E2020_close_up_with_84_279lbld/5'>near W279</scene>), at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole' but only <scene name='1eve/E20_interactionshown/8'>indirectly via solvent molecules</scene>. The X-ray structure shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE, to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.
-[http://en.wikipedia.org/wiki/Donepezil Aricept (E2020)] is one of the most interesting drugs that have been designed as AChE bivalent inhibitors. It was developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies prior to elucidation of the 3D structure of ''Torpedo californica'' AChE (''Tc''AChE) ([[1ea5]]). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range. The X-ray structure of the E2020-''Tc''AChE complex shows that E2020 has a <scene name='1eve/E2020_close_up_with_84_279/13'>unique orientation</scene> along the active-site gorge, extending from the anionic subsite (<scene name='1eve/E2020_close_up_with_84lbld/7'>W84</scene>) of the active site, at the bottom, to the peripheral anionic site (<scene name='1eve/E2020_close_up_with_84_279lbld/5'>near W279</scene>), at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole' but only <scene name='1eve/E20_interactionshown/8'>indirectly via solvent molecules</scene>. The X-ray structure shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE, to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.
+</StructureSection>
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 == Organophosphorus acid anhydride nerve agents ==