1H NMR relaxation investigation of acetylcholinesterase inhibitors from huperzine A and derivative

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Abstract

The binding properties of huperzine A (1) with Torpediniforms Nacline acetylcholinesterase (TnAChE) were investigated by 1H NMR methods. The noselective, selective and double-selective spin-lattice relaxation rates were acquired in absent and present of TnAChE at a ratio [ligand]/[protein]=1:0.005. The selective relaxation rates shown protons of 1 had dipole–dipole interaction with protein active site protons. The motional correlation time of bound ligand was calculated by double-selective relaxation rate at 1 τ2,3=40.5 ns at 298 K, which showed 1 had high affinity with TnAChE. The experiments give a possible method to use TnAChE to locate the new huperzine A derivatives as AChE inhibitors.

The binding properties of huperzine A (1) with Torpediniforms Nacline acetylcholinesterase (TnAChE) were investigated by 1H NMR methods. The experiments give a possible method to use TnAChE to locate the new huperzine A derivatices as AChE inhibitors.

Introduction

The enzyme acetylcholinesterase (AChE) catalyzes the hydrolysis of the ester bound of acetylcholine (ACh) to terminate the impulse transmitted action of ACh through cholinegic synapses.1 The Torpedo Acetylcholinesterase contains 14 α-helices and 12 stranded mixed β-sheet. The mixed structure of AChE with its ligands showed it contained an ‘active-site gorge’.2 When ACh binds to this gorge, it is quickly hydrolyzed into acid and choline. Although the basic reason of Alzheimer's disease (AD) is not clear so far, AD is firmly associated with impairment in Cholinergic transmission by present study. A number of AChE inhibitors have been considered as candidates for the symptomatic treatment of AD as the most useful relieving strategy.3 (−)-huperzine A (1) (Fig. 1) is a natural compound first isolated from Chinese medicine Huperzia serrata (Thumb.) in 1986.4 Compound 1 is a potent, reversible and selective inhibitor of AChE with a rapid absorption and penetration into the brain in animal tests. It exhibits memory-enhancing activities in animal and clinical trials. Compared to tacrine and donepezil, 1 possesses a longer duration of action and higher therapeutic index, and the peripheral cholinergic side effects are minimal at therapeutic doses.5 NMR is one of the most suitable methods capable of determining the conformation of the bound ligands. The small molecule protons spin-relaxation rates have been proved as a very suitable parameter in the ligand–macromolecules complexes studies. Even if the macromolecules concentration is just 0.5% of the ligands, the protons spin-relaxation rates of ligands will sensitively change to the binding process.6, 7 The parameters have been used in evaluating the tacrine derivatives interacting with AChE.8 Here, we present NMR data of 1 and its analogue interacting with TnAChE to evaluated the possibility of using this method to procedure new AChE inhibitor of 1 derivatives.

1 was isolated from H. serrata (Thumb.) as previously reported.4 TnAChE was isolated from the electric organs of Torpediniforms Nacline timelei by affinity chromatography.9 Its activity was assayed using a colorimetric method.10 Solutions were prepared in deuterium oxide 100% (Sigma) buffered at 50 mM pH 7.0 (sodium phosphate buffer). All solutions were carefully deoxygenated by sealing off the sample after filling the nitrogen. All NMR experiments were carried out on Varian Inova 600 spectrometer at the controlled temperature of 298K.

Chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) through the water resonance calibrated at 298 K. Proton spin-lattice relaxation rates were measured with inversion recovery pulse sequences and calculated by exponential regression analysis of recovery curves of longitudinal magnetization components. Single- and double-selective proton spin-lattice relaxation rates were measured with inversion recovery pulse sequences implemented with DANTE or double-DANTE sequences.11, 12 All relaxation rates were calculated in the initial rate approximation.13

Section snippets

Chemical shifts

If there were self-stacking occurrence in a ligand, the molecules of the ligand would exist in the solution in mixture of dimer and monomer forms in different concentration. Then the chemical shifts of ligand protons would be affected by concentration of the ligand. In order to observe the self-aggregation state of the sample, the chemical shifts of protons in 1 were investigated under different concentrations 0.2–30.0 mM. The concentration dependence of the 1H NMR chemical shifts H-3 of 1 at

Proton spin-lattice relaxation rates

The relaxation rates of protons in 1 were recorded in non-selective (Rns) and selective (Rs) meaner in the absent (Rfree) and in the present of AchE (Robs) state. The non-selective (Rns) and selective (Rs) meaner were decided by the following equations:Rins=jiρij+jiσijiRis=jiρijiin the equations, ρij and σij are the direct- and cross-relaxation rates for any Hi–Hj interaction and sum is extended to all the dipolarly connected protons. ρ* is contributions of other relaxation

The molecular motion rate

In order to ascertain 1 binding to TnAChE tightly to induce strong intermolecular dipole–dipole interactions. The molecular motion rate τ was calculated by the cross-relaxation rate, which obtained by double-selective relaxation method. Comparing the , , the main difference of Rns and Rs was the absent of cross-relaxation rates:σij=Riij−Riswhere Riij is the double-selective relaxation rate measured for Hi upon selective excitation of Hi and Hj, Rsel is the single-selective relaxation rate

Controlled experiment

When the ligand was bound to the active site of protein, the relaxation rate was affected by the slowing down of molecular motions as well as by the occurrence of intermolecular 1H–1H dipole–dipole interaction of protons in the protein core. If a ligand just sticks to the surface of a protein, it will also slowing down of the ligand motion and give some contributes to the molecular dipole interacting change. In order to make it clear that 1 combined to the active core of TnAChE, another small

Conclusion

By enhancement of Rns/Rs value, the molecular motion rate and comparison with controlled compound, 1 combined to the TnAChE was observed undoubtedly. Although the addition concentration of TnAChE was just 5 μM, we still observed the spin-lattice relaxation change of protons in 1. The experiments give us a possible method to use TnAChE to locate the new huperzine A derivatives as AChE inhibitors.

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