AChE inhibitors and substrates

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Revision as of 09:13, 1 January 2009

AChE substrate

Solution of the three-dimensional (3D) structure of Torpedo californica acetylcholinesterase (TcAChE) in 1991 (Sussman et al. & Silman (1991)) opened up new horizons in research on an enzyme that had already been the subject of intensive investigation. 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 darkmagenta), led to a revision of the views then held concerning substrate traffic, recognition, and hydrolysis (Botti et al. Sussman & Silman (1999)). This 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 of site-directed mutagenesis, utilizing suitable expression systems. Acetylcholinesterase hydrolysizes the neurotransmitter (ACh), producing group. directly binds (via its nucleophilic Oγ atom) within the (ACh/TcAChE structure 2ace). The residues are also important in the ligand recognition (Ref 1).

AChE monovalent inhibitors

Alzheimer's disease (AD) is a disorder that attacks the central nervous system through progressive degeneration of its neurons. Patients with this disease develop dementia which becomes more severe as the disease progresses. It was suggested that symptoms of AD caused by decrease of activity of cholinergic neocortical and hippocampal neurons. Treatment for AD by acetylcholine (ACh) precursors and cholinergic agonists was ineffective or caused side effects. ACh hydrolysize by AChE causes termination of cholinergic neurotransmission. Therefore, compounds which inhibit AChE might significantly increase the levels of ACh reduced by Alzheimer's disease. Indeed, it was shown that AChE inhibitors improve the cognitive abilities of AD patients at early stages disease development. The first generation of AD drugs were AChE inhibitors: alcaloids (-)-Huperzine A (HupA) and (-)-galanthamine (GAL, Reminyl); synthetic compounds tacrine (Cognex) and rivastigmine (Exelon).

1) is found in an extract from a club moss that has been used in Chinese folk medicine. Its action has been associated to its ability to strongly inhibit AChE (it has high binding affinity to this enzyme). The X-ray structure of the complex of TcAChE with optically pure HupA (colored blueviolet) at 2.5 Å resolution (1vot) reveals that this inhibitor also binds to TcAChE active site mentioned above (Ser200, His440, Glu327, Trp84, and Phe330; colored orange), but its observed orientation is almost orthogonal in comparison to ACh (transparent gray; 2ace) (Ref 1). Ser200 (colored transparent yellow) is from ACh/TcAChE complex 2ace).

2) The X-ray crystal structure of (magenta) bound in the active site of TcAChE (1acj) was determined at 2.8 Å resolution. ACh (gray) is shown for comparison.

3) (red) is an alkaloid from the flower common snowdrop (Galanthus nivalis). The X-ray crystal structure of GAL bound in the active site of TcAChE (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.

4) The X-ray structure of Torpedo californica AChE (TcAChE) in complex with galanthamine iminium derivative (compound 5) was determined at 2.05 Å. The binding mode of this (cyan) with TcAChE is virtually identical to that of galanthamine (red) itself (1dx6). The TcAChE residues of the galanthamine/TcAChE are colored pink, while those of compound 5/TcAChE are in lime. The main structural change is side-chain movement of Phe330 in the compound 5/TcAChE complex, in comparison to that of galanthamine. The compound 5 differs by presence of (N⁺; blue) instead N of galanthamine. This chemical difference causes the structural change and slightly decreases affinity of compound 5 to TcAChE in comparison to galanthamine itself.

AChE bivalent inhibitors

The of TcAChE consists of two binding subsites. 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. 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. 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 in the section 'AChE monovalent inhibitors' (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.

The comparison of the (R)-3/TcAChE and tacrine/TcAChE complexes at the . A of the trigonal (R)-3/TcAChE structure (R)-3 colored cyan (TcAChE residues interacting with (R)-3 are colored sea-green) with the crystal structure of tacrine/TcAChE (1acj, (tacrine colored magenta; residues interacting with tacrine are colored pink) reveals a similar binding mode for the tacrine moiety. In both structures the tacrine ring is situated at the CAS, between the aromatic residues Trp84 and Phe330. Steric clash with the 10-carbon linker could explain the tilt observed for the Phe330 (yellow and transparent in the tacrine/TcAChE). Water molecules are shown as red spheres. The tacrine unit of (R)-3 N forms with His440 O (3.0 Å) similar to that of tacrine alone. Similarly to the tacrine/TcAChE structure the system of three water molecules at the CAS ((R)-3/TcAChE) binds the tacrine-linker N via hydrogen bonds to Ser81 O, Ser122 Oγ, and Asn85 Oδ1 (2.6-3.5 Å) .

The comparison of the (R)-3/TcAChE and bis-hupyridone/TcAChE complexes (1h22 and 1h23) at the . of the (R)-tacrine-(10)-hupyridone ((R)-3, cyan) and (S,S)-(-)-Bis(12)-hupyridone ((S,S)-(-)-4b, orange, i.e. 12-carbon-tether-linked hupyridone dimer) and (S,S)-(-)-Bis(10)-hupyridone ((S,S)-(-)-4a, plum) complexes demonstrates the binding mode of the hupyridone moiety. TcAChE residues of symmetry-related molecule are shown in magenta. X-ray structures of TcAChE complexed with these 10- and 12-carbon-tether-linked 2 (S,S)-(-)-4a and (S,S)-(-)-4b show one subunit bound at the , the linker spanning the gorge, and the other subunit bound at the . There are two connecting the hupyridone O to Lys11 Nζ and hupyridone N to Gln185 Oε1 of a symmetry-related molecule at (R)-3/TcAChE complex. Water molecules are shown as red spheres. Another hydrogen bond connects the hupyridone O to a water molecule, which is bound to Ser286 N. Similarly, the hupyridone-PAS unit of both (-)-4a and (-)-4b forms direct and an indirect hydrogen bonds with the protein backbone in the PAS region.

The of (R)-3 (cyan) and (S)-3 (orange) bound to the TcAChE active site in the orthorhombic forms is shown. The residues important for inhibitor binding are in green. In contrast to the trigonal form (1zgb), the residues Gln*185 and Lys*11 (yellow) of an other symmetry-related monomer do not form hydrogen bonds with the ligands. of (S,S)-(-)-4a (magenta) and (S)-3 (orange, orthorhombic TcAChE) demonstrates the similar mode of binding of the hupyridone unit at the PAS. The residues Trp279 (top) and Trp84 (bottom) represent the PAS and the CAS, respectively.

Described in the section 'AChE monovalent inhibitors' (GAL; colored red) is an AChE inhibitor and it is currently used in therapy of the AD. Conjugate of GAL through (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.

A comparison between the in complex with TcAChE and the 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. The PEG molecule (gray) is located at the active site of galanthamine/TcAChE structure. The alkyl linker spans the active-site gorge and phthalimido moiety of the compound 3 is situated near the Trp279 at the PAS. The compound 3 has larger affinity to TcAChE than GAL. It could be explained by increased number of interactions between compound 3 (which interacts not only with residues within CAS but also within PAS) and TcAChE in comparison to GAL.

Among the most interesting drugs that have been designed to inhibit acetylcholinesterase are those that have two binding sites that bind both the peripheral and catatylic sites simultaneously. Such drugs bind highly specificly and strongly. A good example is .

It appears that the principal interaction between the aceylcholine and the enzyme is relatively newly discovered cation-pi interactions between the cationic moiety of the substrate and the many aromatic residues lining the catalytic gorge. Unlike most interatomic interactions in chemistry, cation-pi interactions are unusual in that their energy hardly changes as the cationic and aromatic ring centers vary between 4 and 7 Angstroms apart, and for a wide variety of relative orientations of the aromatic rings. This gives the substrate an energetically smooth ride down the gorge with few bumps or barriers to impede passage down the gorge.

Most acetylcholinesterases have a net negative charge and a large patch of negative potential around the entrance to the active site gorge, which may be useful to attract the positively charged acetycholine substrate to the site. As one travels down the gorge, this potential becomes increasingly more and more negative, reaching a peak at the active site at the base. Because of this potential, the peripherial site is thought to act like a substrate trap, that forces practically molecule of substrate that reaches the peripheral site to travel down the gorge to the active site, that probably contributes greatly to the extremely rapid rate of degrading the substrate. This whole enzyme therefore acts like a brilliantly designed natural vacuum cleaner that clears the neurotransmitter out of the synapse extremely quickly. Yet to be solved, however, is how the products clear the active site rapidly, whether back through the gorge, or out a back door on the other side of the protein that quickly opens each catalytic cycle (Try 84 is actually near the surface of the 'underside' of the protein.)

Selected 3D Structures of AChE

• 2ace This is the original solved structure for Torpedo Californica
• 1ea5 This is one of the highest quality representative X-ray structures in the PDB.
• 1eve The E2020 (Aricept) complex.
• 1ax9 Endrophonium complex.
• 1vot Complex with Huperzine, a Chinese folk medicine.
• 1fss Complex with snake venum toxin Fasciculin-II.
• 1vzj Structure of the tetramerization domain of acetylcholinesterase.

More structures can be obtained by searching for AChE

References

Structure of acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A., Raves ML, Harel M, Pang YP, Silman I, Kozikowski AP, Sussman JL, Nat Struct Biol. 1997 Jan;4(1):57-63. PMID:8989325

Crystal packing mediates enantioselective ligand recognition at the peripheral site of acetylcholinesterase., Haviv H, Wong DM, Greenblatt HM, Carlier PR, Pang YP, Silman I, Sussman JL, J Am Chem Soc. 2005 Aug 10;127(31):11029-36. PMID:16076210

Potent, easily synthesized huperzine A-tacrine hybrid acetylcholinesterase inhibitors., Carlier PR, Du DM, Han Y, Liu J, Pang YP, Bioorg Med Chem Lett. 1999 Aug 16;9(16):2335-8. PMID:10476864

Acetylcholinesterase complexed with bivalent ligands related to huperzine a: experimental evidence for species-dependent protein-ligand complementarity., Wong DM, Greenblatt HM, Dvir H, Carlier PR, Han YF, Pang YP, Silman I, Sussman JL, J Am Chem Soc. 2003 Jan 15;125(2):363-73. PMID:12517147

Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein., Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I, Science. 1991 Aug 23;253(5022):872-9. PMID:1678899