Publications
2002
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(2002) Biophysical Chemistry. 100, 1-3, p. 293-305 Abstractα-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC50 of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.
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(2002) Journal of Synchrotron Radiation. 9, 6, p. 342-346 Abstract
Irradiation of proteins with intense X-ray radiation produced by third-generation synchrotron sources generates specific structural and chemical alterations, including breakage of disulfide bonds and decarboxylation. In this paper, disulfide bond lengths in irradiated crystals of the enzyme Torpedo californica acetylcholinesterase are examined based on quantum simulations and on experimental data published previously. The experimental data suggest that one disulfide bond elongates by ∼0.7 Å upon X-ray irradiation as seen in a series of nine data sets collected on a single crystal. Simulation of the same bond suggests elongation by a similar value if a disulfide-radical anion is formed by trapping an electron. The absorption spectrum of a crystal irradiated under similar conditions shows a peak at ∼400 nm, which in aqueous solution has been attributed to disulfide radicals. The results suggest that the formation of disulfide radicals in protein crystals owing to X-ray irradiation can be observed experimentally, both by structural means and by absorption spectroscopy.
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(2002) Acta Crystallographica Section D: Biological Crystallography. 58, 10 I, p. 1765-1771 Abstract
The X-ray crystal structure of Torpedo californica acetylcholinesterase (TcAChE) complexed with BW284C51 {CO[-CH2CH2-pC6H4-N(CH 3)2 (CH2-CH=CH2)]2} is described and compared with the complexes of two other active-site gorge-spanning inhibitors, decamethonium and E2020. The inhibitor was soaked into TcAChE crystals in the trigonal space group P3121, yielding a complex which diffracted to 2.85 Å resolution. The structure was refined to an R factor of 19.0% and an Rfree of 23.4%; the final model contains the protein, inhibitor, 132 water molecules and three carbohydrate moieties. BW284C51 binds similarly to decamethonium and E2020, with its two phenyl and quaternary amino end-groups complexed to Trp84 in the catalytic site and to Trp279 in the peripheral binding site, and its central carbonyl group hydrogen bonded very weakly to Tyr121. Possible reasons for decamethonium's weaker binding are considered. The relative strength of binding of bisquaternary inhibitors to acetylcholinesterase and the effect of several mutations of the enzyme are discussed in the context of the respective X-ray structures of their complexes with the enzyme.
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(2002) Biochemistry. 41, 35, p. 10810-10818 Abstract
Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 μM, respectively, compared to 0.18 μM for (-)-huperzine A. The x-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 Å resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through π-π stacking and through van der Waals or C-H···π interactions with Trp84 and Phe330. Since their α-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H···π interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.
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(2002) Biochemistry. 41, 9, p. 2970-2981 Abstract
Huprine X is a novel acetylcholinesterase (ACHE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 Å resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117.
[All authors] -
(2002) Biochemistry. 41, 11, p. 3555-3564 Abstract
Rivastigmine, a carbamate inhibitor of acetylcholinesterase, is already in use for treatment of Alzheimer's disease under the trade name of Exelon. Rivastigmine carbamylates Torpedo californica acetylcholinesterase very slowly (ki = 2.0 M-1 min-1), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is > 1600-fold higher (ki = 3300 M-1 min-1). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (ki = 9 × 104 and 5 × 105 M-1 min-1, respectively). Spontaneous reactivation of all four conjugates is very slow, with
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(2002) Journal of Molecular Biology. 320, 4, p. 721-725 Abstract
The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 Å resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH2 groups of the inhibitor, which are engaged in C-H···π interactions that structurally mimic the cation-π interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H···O and C-H···π types.
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(2002) Journal of Physical Chemistry A. 106, 1, p. 157-164 Abstract
The theoretical investigation of tetramethylammonium (TMA)-imidazole and TMA-furan complexes has been performed to justify the preferred structure of the cation-aromatic complexes predicated by the analysis of molecular electrostatic potential (MEP) maps of isolated aromatic systems. Such maps have been shown to be helpful in predicting the cation binding sites in cation-aromatic complexes. Our computational results show that a large part of the binding energies in the systems studied are contributed by the classical cation-π interaction. However, the optimized structure obtained in the MP2 method might be different from that obtained by the DFT method due to the influence of dispersion forces. Dispersion forces have been found to be important in the systems studied, increasing the binding energies by ∼7% and 20% for the TMA-imidazole and TMA-furan systems, respectively.
[All authors] -
(2002) Journal of Theorical and Computational Chemistry. 1, 1, p. 81-92 Abstract
Quantum chemical DFT-B3LYP/6-31G* method and IR spectrometry have been used to investigate the natural and binding structures of Huperzine B (HupB) in order to better understand the interaction nature between acetylcholinesterase (AChE) and its inhibitor, with the view of designing new AChE inhibitors. The predicted and experimental results reveal that both the natural state and binding form of HupB adopt the chair conformation. Furthermore, the B3LYP/6-31G* results suggest that structure S1 should be the dominant form of the two possible chair structures (S1 and S2, Fig. 2). The calculated results also show that the condensed ring structure composing of rings A, B and C is very rigid. Therefore, its flexibility does not need to be considered when we try to dock this structure to its target. Indeed, this supposition is confirmed by the excellent alignment of the binding structure produced from our recent/break X-ray crystallographic structure of the HupB-AChE complex with the B3LYP/6-31G* predicted geometry. Among all the 111 predicted vibrational bands, the mode 110, which is resulted from the stretching of the bond N2–H and having the second highest frequency, is essential for the geometrical identification. The difference between our predicted strongest absorption band and experimental IR spectrum suggests that a strong intermolecular interaction, which could be a hydrogen bond, exists in HupB crystal. The electrostatic potential surface of HupB derived from our B3LYP/6-31G* CHelpG atomic charge suggests a mechanism of how HupB would interact with its target. In addition, the good agreement between predicted vibrational bands (scaled by a factor of 0.96) and experimental result shows that B3LYP/6-31G* is a good tool for studying such kind of natural compound.