Free energy landscape for the binding process of Huperzine A to acetylcholinesterase.
ABSTRACT: Drug-target residence time (t = 1/k(off), where k(off) is the dissociation rate constant) has become an important index in discovering better- or best-in-class drugs. However, little effort has been dedicated to developing computational methods that can accurately predict this kinetic parameter or related parameters, k(off) and activation free energy of dissociation (?G(off)?). In this paper, energy landscape theory that has been developed to understand protein folding and function is extended to develop a generally applicable computational framework that is able to construct a complete ligand-target binding free energy landscape. This enables both the binding affinity and the binding kinetics to be accurately estimated. We applied this method to simulate the binding event of the anti-Alzheimer's disease drug (-)-Huperzine A to its target acetylcholinesterase (AChE). The computational results are in excellent agreement with our concurrent experimental measurements. All of the predicted values of binding free energy and activation free energies of association and dissociation deviate from the experimental data only by less than 1 kcal/mol. The method also provides atomic resolution information for the (-)-Huperzine A binding pathway, which may be useful in designing more potent AChE inhibitors. We expect this methodology to be widely applicable to drug discovery and development.
Project description:<b>Aim:</b> The present study aimed to investigate huperzine A as an anti-Alzheimer agent based on the principle that a single compound can regulate multiple proteins and associated pathways, using system biology tools. <b>Methodology:</b> The simplified molecular-input line-entry system of huperzine A was retrieved from the PubChem database, and its targets were predicted using SwissTargetPrediction. These targets were matched with the proteins deposited in DisGeNET for Alzheimer disease and enriched in STRING to identify the probably regulated pathways, cellular components, biological processes, and molecular function. Furthermore, huperzine A was docked against acetylcholinesterase using AutoDock Vina, and simulations were performed with the Gromacs package to take into account the dynamics of the system and its effect on the stability and function of the ligands. <b>Results:</b> A total of 100 targets were predicted to be targeted by huperzine A, of which 42 were regulated at a minimum probability of 0.05. Similarly, 101 Kyoto Encyclopedia of Genes and Genomes pathways were triggered, in which neuroactive ligand-receptor interactions scored the least false discovery rate. Also, huperzine A was predicted to modulate 54 cellular components, 120 molecular functions, and 873 biological processes. Furthermore, huperzine A possessed a binding affinity of -8.7 kcal/mol with AChE and interacted within the active site of AChE <i>via</i> H-bonds and hydrophobic interactions.
Project description:Huperzine A (<b>1</b>, Hup A), a lycodine-type <i>Lycopodium</i> alkaloid isolated from Thai clubmosses <i>Huperzia squarrosa</i> (G. Forst.) Trevis., <i>H. carinata</i> (Desv. ex. Poir.) Trevis., <i>H. phlegmaria</i> (L.), and <i>Phlegmariurus nummulariifolius</i> (Blume) Chambers (Lycopodiaceae), exerts inhibitory activity on acetylcholinesterase, a known target for Alzheimer's disease therapy. This study investigated the structure-activity relationship of C(2)-functionalized and <i>O</i>- or <i>N</i>-methyl-substituted huperzine A derivatives. <i>In silico</i>-guided screening was performed to search for potential active compounds. Molecular docking analysis suggested that substitution at the C(2) position of Hup A with small functional groups could enhance binding affinity with AChE. Consequently, 12 C(2)-functionalized and four <i>O</i>- or <i>N</i>-methyl-substituted compounds were semi-synthesized and evaluated for their <i>ee</i>AChE and <i>eq</i>BChE inhibitory activities. The result showed that 2-methoxyhuperzine A (<b>10</b>) displayed moderate to high <i>ee</i>AChE inhibitory potency (IC<sub>50</sub> = 0.16 μM) with the best selectivity over <i>eq</i>BChE (selectivity index = 3633). Notably, this work showed a case of which computational analysis could be utilized as a tool to rationally screen and design promising drug molecules, getting rid of impotent molecules before going more deeply on labor-intensive and time-consuming drug discovery and development processes.
Project description:The first total synthesis of (−)-huperzine W (1) has been achieved. Key element of the synthesis is a highly convergent assemblage for the two rings system of target molecule utilizing an efficient Suzuki-Miyaura coupling reaction between chiral iodide 2 and 2-allylpyrrolidinone 4. Evaluation of the AchE inhibition of synthetic huperzine W was also carried out. <h4>Electronic Supplementary Material</h4> Supplementary material is available for this article at 10.1007/s13659-012-0084-2 and is accessible for authorized users.
Project description:AIM: To design novel bifunctional derivatives of huperzine B (HupB) based on the concept of dual binding site of acetylcholinesterase (AChE) and evaluate their pharmacological activities for seeking new drug candidates against Alzheimer's disease (AD). METHODS: Novel 16-substituted bifunctional derivatives of HupB were synthesized through chemical reactions. The inhibitory activities of the derivatives toward AChE and butyrylcholinesterase (BuChE) were determined in vitro by modified Ellman's method. Cell viability was quantified by the reduction of MTT. RESULTS: A new preparative method was developed for the generation of 16-substituted derivatives of HupB, and pharmacological trials indicated that the derivatives were multifunctional cholinesterase inhibitors targeting both AChE and BuChE. Among the derivatives tested, 9c, 9e, 9f, and 9i were 480 to 1360 times more potent as AChE inhibitors and 370 to 1560 times more potent as BuChE inhibitors than the parent HupB. Further preliminary pharmacological trials of derivatives 9c and 9i were performed, including examining the mechanism of AChE inhibition, the substrate kinetics of the enzyme inhibition, and protection against hydrogen peroxide (H2O2)-induced cytotoxicity in PC12 cells. CONCLUSION: Preliminary pharmacological evaluation indicated that 16-substituted derivatives of HupB, particularly 9c and 9i, would be potentially valuable new drug candidates for AD therapy, and further exploration is needed to evaluate their pharmacological and clinical efficacies.
Project description:The present study was designed to probe the effects of Huperzine A (HupA) on diabetes-associated cognitive decline (DACD) using a streptozotocin (STZ)-injected rat model. Diabetic rats were treated with HupA (0.05 and 0.1 mg/kg) for seven weeks. Memory functions were evaluated by the water maze test. Nissl staining was selected for detecting neuronal loss. Protein and mRNA levels of brain-derived neurotrophic factor (BDNF) were analyzed by ELISA and real-time PCR, respectively. The activities of choline acetylase (ChAT), Acetylcholinesterase (AChE), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), NF-?B p65 unit, TNF-?, IL-1?, IL-6 and caspase-3 were measured using corresponding kits. After seven weeks, diabetic rats exhibited remarkable reductions in: body weight, percentage of time spent in target quadrant, number of times crossing the platform, ChAT and BDNF levels, SOD, GSH-Px and CAT accompanied with increases in neuronal damage, plasma glucose levels, escape latency, mean path length, AChE, MDA level as well as CAT, NF-?B p65 unit, TNF-?, IL-1?, IL-6 and caspase-3 in cerebral cortex and hippocampus. Supplementation with HupA significantly and dose-dependently reversed the corresponding values in diabetes. It is concluded that HupA ameliorates DACD via modulating BDNF, oxidative stress, inflammation and apoptosis.
Project description:Nootropic agents or cognitive enhancers are purported to improve mental functions such as cognition, memory, or attention. The aim of our study was to determine the effects of two possible cognitive enhancers, huperzine A and IDRA 21, in normal young adult monkeys performing a visual memory task of varying degrees of difficulty. Huperzine A is a reversible acetylcholinesterase (AChE) inhibitor, its administration results in regionally specific increases in acetylcholine levels in the brain. In human clinical trials, Huperzine A resulted in cognitive improvement in patients with mild to moderate form of Alzheimer's disease (AD) showing its potential as a palliative agent in the treatment of AD. IDRA 21 is a positive allosteric modulator of glutamate AMPA receptors. It increases excitatory synaptic strength by attenuating rapid desensitization of AMPA receptors and may thus have beneficial therapeutic effects to ameliorate memory deficits in patients with cognitive impairments, including AD. The present study evaluated the effects of the two drugs in normal, intact, young adult monkeys to determine whether they can result in cognitive enhancement in a system that is presumably functioning optimally. Six young pigtail macaques (Macaca nemestrina) were trained on delayed non-matching-to-sample task, a measure of visual recognition memory, up to criterion of 90% correct responses on each of the four delays (10s, 30s, 60s, and 90s). They were then tested on two versions of the task: Task 1 included the four delays intermixed within a session and the monkeys performed it with the accuracy of 90%. Task 2 included, in each of 24 trials, a list of six objects presented in succession. Two objects from the list were then presented for choice paired with novel objects and following two of the four delays intermixed within a session. This task with a higher mnemonic demand yielded an average performance of 64% correct. Oral administration of huperzine A did not significantly affect the monkeys' performance on either task. However, a significant negative correlation was found between the baseline performance on each delay and the change in performance under huperzine A, suggesting that under conditions in which the subjects were performing poorly (55-69%), the drug resulted in improved performance, whereas no improvement was obtained when the baseline was close to 90%. In fact, when the subjects were performing very well, huperzine A tended to reduce the performance accuracy, indicating that in a system that functions optimally, the increased availability of acetylcholine does not improve performance or memory, especially when the animals are close to the maximum performance. In contrast, oral administration of IDRA 21 significantly improved performance on Task 2, especially on the longest delay. This finding supports the potential use of this drug in treatment of cognitive and memory disorders. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.
Project description:<i>Huperzia serrata</i> (Thunb.) Trevis is widely used in traditional asiatic medicine to treat many central disorders including, schizophrenia, cognitive dysfunction, and dementia. The major alkaloid, Huperzine A (HA), of <i>H. serrata</i> is a well-known competitive reversible inhibitor of acetylcholinesterase (AChE) with neuroprotective effects. Inspired by the tradition, we developed a green one-step method using microwave assisted extraction to generate an extract of <i>H. serrata</i>, called NSP01. This green extract conserves original neuropharmacological activity and chemical profile of traditional extract. The neuroprotective activity of NSP01 is based on a precise combination of three major constituents: HA and two phenolic acids, caffeic acid (CA) and ferulic acid (FA). We show that CA and FA potentiate HA-mediated neuroprotective activity. Importantly, the combination of HA with CA and FA does not potentiate the AChE inhibitory property of HA which is responsible for its adverse side effects. Collectively, these experimental findings demonstrated that NSP01, is a very promising plant extract for the prevention of Alzheimer's disease and memory deficits.
Project description:A catalytic mechanism for the butyrylcholinesterase (BChE)-catalyzed hydrolysis of acetylcholine (ACh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy calculations on both acylation and deacylation of BChE. It has been shown that the acylation with ACh includes two reaction steps, including nucleophilic attack on the carbonyl carbon of ACh and dissociation of choline ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of the substrate and dissociation between the carboxyl carbon of the substrate and the hydroxyl oxygen of the Ser198 side chain. Notably, despite the fact that acetylcholinesterase (AChE) and BChE are very similar enzymes, the acylation of BChE with ACh is rate-determining, which is remarkably different from the AChE-catalyzed hydrolysis of ACh, in which the deacylation is rate-determining. The computational prediction is consistent with available experimental kinetic data. The overall free energy barrier calculated for BChE-catalyzed hydrolysis of ACh is 13.8 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 13.3 kcal/mol.
Project description:Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes: (i) pretreatment with reversible blockers of AChE, (ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABA(A) receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life approximately 5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for alpha(5)-containing GABA(A) receptors. Coadministration of HUP (50 microg/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18-24 h. In HUP-pretreated mice, the ED(50) of IMI to block DFP-induced mortality is approximately 10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.
Project description:Tacrine (THA), the first clinically effective acetylcholinesterase (AChE) inhibitor and the first approved drug for the treatment of Alzheimer's disease (AD), was withdrawn from the market due to its side effects, particularly its hepatotoxicity. Nowadays, THA serves as a valuable scaffold for the design of novel agents potentially applicable for AD treatment. One such compound, namely 7-methoxytacrine (7-MEOTA), exhibits an intriguing profile, having suppressed hepatotoxicity and concomitantly retaining AChE inhibition properties. Another interesting class of AChE inhibitors represents Huprines, designed by merging two fragments of the known AChE inhibitors-THA and (-)-huperzine A. Several members of this compound family are more potent human AChE inhibitors than the parent compounds. The most promising are so-called huprines X and Y. Here, we report the design, synthesis, biological evaluation, and in silico studies of 2-methoxyhuprine that amalgamates structural features of 7-MEOTA and huprine Y in one molecule.