Shape-based virtual screening,docking, and molecular dynamics simulations to identify Mtb-ASADH inhibitors

Aspartate β-semialdehyde dehydrogenase (ASADH) is a key enzyme for the biosynthesis of essential amino acids and several important metabolites in microbes. Inhibition of ASADH enzyme is a promising drug target strategy against Mycobacterium tuberculosis (Mtb). In this work, in silico approach was used to identify potent inhibitors of Mtb-ASADH. Aspartyl β-difluorophosphonate (β-AFP), a known lead compound, was used to understand the molecular recognition interactions (using molecular docking and molecular dynamics analysis). This analysis helped in validating the computational protocol and established the participation of Arg99, Glu224, Cys130, Arg249, and His256 amino acids as the key amino acids in stabilizing ligand–enzyme interactions for effective binding, an essential feature is H-bonding interactions with the two arginyl residues at the two ends of the ligand. Best binding conformation of β-AFP was selected as a template for shape-based virtual screening (ZINC and NCI databases) to identify compounds that competitively inhibit the Mtb-ASADH. The top rank hits were further subjected to ADME and toxicity filters. Final filter was based on molecular docking analysis. Each screened molecule carries the characteristics of the highly electronegative groups on both sides separated by an average distance of 6?Å. Finally, the best predicted 20 compounds exhibited minimum three H-bonding interactions with Arg99 and Arg249. These identified hits can be further used for designing the more potent inhibitors against ASADH family. MD simulations were also performed on two selected compounds (NSC4862 and ZINC02534243) for further validation. During the MD simulations, both compounds showed same H-bonding interactions and remained bound to key active residues of Mtb-ASADH.     

Pharmacoinformatics analysis to identify inhibitors of Mtb-ASADH

Aspartate-semialdehyde dehydrogenase (ASADH; EC 1.2.1.11) is a key enzyme in the biosynthesis of essential amino acids in prokaryotes and fungi, inhibition of ASADH leads to the development of novel antitubercular agents. In the present work, a combined structure and ligand-based pharmacophore modeling, molecular docking, and molecular dynamics (MD) approaches were employed to identify potent inhibitors of mycobacterium tuberculosis (Mtb)-ASADH. The structure-based pharmacophore hypothesis consists of three hydrogen bond acceptor (HBA), two negatively ionizable, and one positively ionizable center, while ligand-based pharmacophore consists of additional one HBA and one hydrogen bond donor features. The validated pharmacophore models were used to screen the chemical databases (ZINC and NCI). The screened hits were subjected to ADME and toxicity filters, and subsequently to the molecular docking analysis. Best-docked 25 compounds carry the characteristics of highly electronegative functional groups (–COOH and –NO₂) on both sides and exhibited the H-bonding interactions with highly conserved residues Arg99, Arg249, and His256. For further validation of docking results, MD simulation studies were carried out on two representative compounds NSC51108 and ZINC04203124. Both the compounds remain bound to the key active residues of Mtb-ASADH during the MD simulations. These identified hits can be further used for lead optimization and in the design more potent inhibitors against Mtb-ASADH.     

Identification of novel natural inhibitors of Opisthorchis felineus cytochrome P450 using structure-based screening and molecular dynamic simulation

Opisthorchis felineus is the etiological agent of opisthorchiasis in humans. O. felineus cytochrome P450 (OfCYP450) is an important enzyme in the parasite xenobiotic metabolism. To identify the potential anti-opisthorchid compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,65,869) against the OfCYP450. The ligands were screened against OfCYP450 in four sequential docking modes that resulted in 361 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 10 compounds were found to fit well with re-docking studies. After refinement by docking and drug-likeness analyses, four potential inhibitors (ZINC2358298, ZINC8790946, ZINC70707116, and ZINC85878789) were identified. These ligands with reference compounds (itraconazole and fluconazole) were further subjected to molecular dynamics simulation (MDS) and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the OfCYP450 and bound complexes. The binding energy analyses were also calculated. The results suggested that the compounds had a negative binding energy with ?259.41, ?110.09, ?188.25, ?163.30, ?202.10, and ?158.79 kJ mol^?1 for itraconazole, fluconazole, and compounds with IDs ZINC2358298, ZINC8790946, ZINC70707116, and ZINC85878789, respectively. These lead compounds displayed significant pharmacological and structural properties to be drug candidates. On the basis of MDS results and binding energy analyses, we concluded that ZINC8790946, ZINC70707116, and ZINC85878789 have excellent potential to inhibit OfCYP450.     

Identification and evaluation of bioactive natural products as potential inhibitors of human microtubule affinity-regulating kinase 4 (MARK4)

Microtubule affinity-regulating kinase 4 (MARK4) has recently been identified as a potential drug target for several complex diseases including cancer, diabetes and neurodegenerative disorders. Inhibition of MARK4 activity is an appealing therapeutic option to treat such diseases. Here, we have performed structure-based virtual high-throughput screening of 100,000 naturally occurring compounds from ZINC database against MARK4 to find its potential inhibitors. The resulted hits were selected, based on the binding affinities, docking scores and selectivity. Further, binding energy calculation, Lipinski filtration and ADMET prediction were carried out to find safe and better hits against MARK4. Best 10 compounds bearing high specificity and binding efficiency were selected, and their binding pattern to MARK4 was analyzed in detail. Finally, 100 ns molecular dynamics simulation was performed to evaluate; the dynamics stability of MARK4-compound complex. In conclusion, these selected natural compounds from ZINC database might be potential leads against MARK4, and can further be exploited in drug design and development for associated diseases.     

Docking based virtual screening and molecular dynamics study to identify potential monoacylglycerol lipase inhibitors

Monoacylglycerol lipase (MAGL) is one of the key enzymes of the endocannabinoid system (ECS). It hydrolyzes one of the major endocannabinoid, 2-arachidonoylglycerol (2-AG), an endogenous full agonist at G protein coupled cannabinoid receptors CB1 and CB2. Numerous studies showed that MGL inhibitors are potentially useful for the treatment of pain, inflammation, cancer and CNS disorders. These provocative findings suggested that pharmacological inhibition of MAGL function may confer significant therapeutic benefits. In this study, we presented hybrid ligand and structure-based approaches to obtain a novel set of virtual leads as MAGL inhibitors. The constraints used in this study, were Glide score, binding free energy estimates and ADME properties to screen the ZINC database, containing approximately 21 million compounds. A total of seven virtual hits were obtained, which showed significant binding affinity towards MAGL protein. Ligand, ZINC24092691 was employed in complex form with the protein MAGL, for molecular dynamics simulation study, because of its excellent glide score, binding free energy and ADME properties. The RMSD of ZINC24092691 was observed to stay at 0.1 nm (1 Å) in most of the trajectories, which further confirmed its ability to inhibit the protein MAGL. The hits were then evaluated for their ability to inhibit human MAGL. The compound ZINC24092691 displayed the noteworthy inhibitory activity reducing MAGL activity to 21.15% at 100 nM concentration, with an IC₅₀ value of 10 nM.     

Dynamic structure based pharmacophore modeling of the Acetylcholinesterase reveals several potential inhibitors

Acetylcholinesterase is a critical enzyme that regulates neurotransmission by catalyzing the breakdown of neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for therapeutic drugs that treat Alzheimer’s disease. Since, the degree of flexibility of the side chains of the residues in the active-site gorge of Acetylcholinesterase is diverse it results in different bound ligand conformations. The side-chain conformations of Ser293, Tyr341, Leu76, and Val73 are flexible, while the side-chain conformations of Tyr72, Tyr 124, Ser125, Phe295, and Arg296 appear to be fixed. In this study, multi-conformation dynamic pharmacophore models from the donepezyl-binding pocket based on highly populated structures chosen from molecular dynamics simulations were used for screening compounds that can properly bind acetylcholinesterase. Based on these structures, three pharmacophore models were generated. Consequently, 14 hits were retrieved as final candidates by utilizing virtual screening of ZINC database and molecular docking.     

Identification of potential inhibitors of Fasciola gigantica thioredoxin1: computational screening,molecular dynamics simulation,and binding free energy studies

Fasciola gigantica is the causative organism of fascioliasis and is responsible for major economic losses in livestock production globally. F. gigantica thioredoxin1 (FgTrx1) is an important redox-active enzyme involved in maintaining the redox homeostasis in the cell. To identify a potential anti-fasciolid compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,740) against the FgTrx1 structure. The ligands were docked against FgTrx1 and 309 ligands were found to have better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 30 compounds were found to fit well for re-docking studies. After refinement by molecular docking and drug-likeness analysis, three potential inhibitors (ZINC15970091, ZINC9312362, and ZINC9312661) were identified. These three ligands were further subjected to molecular dynamics simulation (MDS) to compare the dynamics and stability of the protein structure after binding of the ligands. The binding free energy analyses were calculated to determine the intermolecular interactions. The results suggested that the two compounds had a binding free energy of –82.237, and –109.52 kJ.mol^?1 for compounds with IDs ZINC9312362 and ZINC9312661, respectively. These predicted compounds displayed considerable pharmacological and structural properties to be drug candidates. We concluded that these two compounds could be potential drug candidates to fight against F. gigantica parasites.     

3D-QSAR pharmacophore-based virtual screening,molecular docking and molecular dynamics simulation toward identifying lead compounds for NS2B–NS3 protease inhibitors

NS2B–NS3 protease has been identified to serve as lead drug design target due to its significant role in West Nile viral (WNV) and dengue virus (DENV) reproduction and replication. There are currently no approved chemotherapeutic drugs and effective vaccines to inhibit DENV and WNV infections. In this work, 3D-QSAR pharmacophore model has been developed to discover potential inhibitory candidates. Validation through Fischer’s model and decoy test indicate that the developed 3D pharmacophore model is highly predictive for DENV inhibitors, which was then employed to screen ZINC chemical library to obtain reasonable hits. Following ADMET filtering, 15 hits were subjected to further filter through molecular docking and CoMFA modeling. Finally, top three hits were identified as lead compounds or potential inhibitory candidates with IC₅₀ values of ～0.4637?µM and fitness of ～57.73. It is implied from CoMFA modeling that substituents at the side site of benzotriazole such as a p-nitro group (e.g. biphenyl head) and a carbonyl (e.g. carboxylate function) at the side site of furan or amino group may improve bioactivity of ZINC85645245, respectively. Molecular dynamics simulations (MDS) were performed to discover new interactions and reinforce the binding modes from docking for the hits also. The QSAR and MDS results obtained from this work should be useful in determining structural requirements for inhibitor development as well as in designing more potential inhibitors for NS2B–NS3 protease.     

On scaffold hopping: Challenges in the discovery of sulfated small molecules as mimetics of glycosaminoglycans

The design of sulfated, small, nonsaccharide molecules as modulators of proteins is still in its infancy as standard drug discovery tools such as library of diverse sulfated molecules and in silico docking and scoring protocol have not been firmly established. Databases, such as ZINC, contain too few sulfate-containing nonsaccharide molecules, which severely limits the identification of new hits. Lack of a generally applicable protocol for scaffold hopping limits the development of sulfated small molecules as synthetic mimetics of the highly sulfated glycosaminoglycans. We explored a sequential ligand-based (LBVS) and structure-based virtual screening (SBVS) approach starting from our initial discovery of monosulfated benzofurans to discover alternative scaffolds as allosteric modulators of thrombin, a key coagulation enzyme. Screening the ZINC database containing nearly 1 million nonsulfated small molecules using a pharmacophore developed from the parent sulfated benzofurans followed by a genetic algorithm-based dual-filter docking and scoring screening identified a group of 10 promising hits, of which three top-scoring hits were synthesized. Each was found to selectively inhibit human alpha-thrombin suggesting the possibility of this approach for scaffold hopping. Michaelis–Menten kinetics showed allosteric inhibition mechanism for the best molecule and human plasma studies confirmed good anticoagulation potential as expected. Our simple sequential LBVS and SBVS approach is likely to be useful as a general strategy for identification of sulfated small molecules hits as modulators of glycosaminoglycan–protein interactions.     

Isolation,Synthesis and AChE Inhibitory Potential of Some Novel Cinnamyl Esters of Taraxerol,the Major Metabolite of the Mangrove Bruguiera cylindrica

Systematic chemical screening of the leaves of Bruguiera cylindrica, the tree mangrove of Rhizophoraceae family, afforded five single and pure compounds. The structures of the isolated compounds were established by their spectroscopic data as taraxerol ( 1 ), 3β‐(E)‐coumaroyltaraxerol ( 2 ), 3β‐(Z)‐coumaroyltaraxerol ( 3 ), β‐sitosterol ( 4 ), and eicosanol ( 5 ). In view of significant accumulation and interesting biological activities, taraxerol ( 1 ) was chemically transformed to synthesize a series of ten cinnamyl esters in very good to excellent yields. The synthesized analogues along with the parent compound were evaluated for their AChE inhibitory potential, BBB permeability and cytotoxicity against Neuro 2A cell line. Among the tested samples, compound 9 showed promising AChE inhibition with significantly low IC₅₀ values, low cytotoxicity and high BBB permeability. Hence, compound 9 can be considered as a lead molecule for further development as potent AChE inhibitor.     

In vitro inhibitory profile of NDGA against AChE and its in silico structural modifications based on ADME profile

Acetylcholinesterase (AChE) inhibitors are currently in focus for the pharmacotherapy of Alzheimer’s disease (AD). These inhibitors increase the level of acetylcholine in the brain and facilitate cholinergic neurotransmission. AChE inhibitors such as rivastigmine, galantamine, physostigmine and huperzine are obtained from plants, indicating that plants can serve as a potential source for novel AChE inhibitors. We have performed a virtual screening of diverse natural products with distinct chemical structure against AChE. NDGA was one among the top scored compounds and was selected for enzyme kinetic studies. The IC₅₀ of NDGA on AChE was 46.2 μM. However, NDGA showed very poor central nervous system (CNS) activity and blood–brain barrier (BBB) penetration. In silico structural modification on NDGA was carried out in order to obtain derivatives with better CNS activity as well as BBB penetration. The studies revealed that some of the designed compounds can be used as lead molecules for the development of drugs against AD

Design,Synthesis, Molecular Docking,and Cholinesterase Inhibitory Potential of Phthalimide‐Dithiocarbamate Hybrids as New Agents for Treatment of Alzheimer's Disease

A novel series of phthalimide‐dithiocarbamate hybrids was synthesized and evaluated for in vitro inhibitory potentials against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The anti‐cholinesterase results indicated that among the synthesized compounds, the compounds 7g and 7h showed the most potent anti‐AChE and anti‐BuChE activities, respectively. Molecular docking and dynamic studies of the compounds 7g and 7h , respectively, in the active site of AChE and BuChE revealed that these compounds as well interacted with studied cholinesterases. These compounds also possessed drug‐like properties and were able to cross the BBB.     

Virtual screening for novel COX-2 inhibitors using the ZINC database

Cyclooxygenase-2 (COX-2) enzyme binds to arachidonic acid and releases metabolites that are used to induce pain and inflammation. COX-2 selective inhibitors such as celecoxib, rofecoxib and valdecoxib are currently used to reduce inflammatory response. However, they lack anti-thrombotic activity and hence lead to cardiovascular and renal liabilities apart from gastrointestinal irritation. Therefore, there is still a need to develop more potent COX-2 inhibitors. In this paper, we report the screening of various compounds from the ZINC database (contains 4.6 million small molecule compounds) using the eHiTS (electronic High Throughput Screening) software tool against the COX-2 protein. The strategy employed can be conveniently split into two categories, viz. screening and docking, respectively. Screening was performed using molecular constraints tool to filter compounds with physico-chemical properties similar to the 6COX bound ligand SC-558. The analysis resulted in 1042 Lipinski compliant hits which are docked and scored to identify structurally novel ligands that make similar interactions to those of known ligands or may have different interactions with other parts of the binding site. Our screening approach identified two molecules ZINC00663976 (eHITS score of -7.135 kcal/mol) and ZINC02062094 (eHITS score of -7.242 kcal/mol) from the ZINC database. Their energy scores are better than the 6COX bound co-crystallized ligand SC-558 with an eHiTS score of -6.559 kcal/mol. Both the ligands were docked within the binding pocket forming interactions with Leu352, Phe518, Met522, Val523, Ala527 and Ser353. Visual inspection suggested similar orientation and binding mode for ZINC02062094 with SC-558 ligand. The NH group of the ligand formed hydrogen bond interactions with the backbone NH of Ala527.     

Identifying novel small molecule antagonists for mLST8 protein using computational approaches

Mammalian lethal with SEC13 protein 8 (mLST8), is an indispensable protein subunit of mammalian target of rapamycin (mTOR) signaling pathway that interacts with the kinase domain of mTOR protein, thereby stabilizing its active site. Experimental studies reported the over expression of mLST8 in human colon and prostate cancers by activation of both mTORC1/2 complexes and subsequent downstream substrates leading to tumor progression. Considering its role, targeting mLST8 protein would be a therapeutic approach against tumor progression in colon and prostate cancers. Hence, using in silico structure based drug design approach, the comparative binding patterns of 1,1′-binapthyl-2,2′diol (BINOL), 1-(2-carboxynaphth-1yl)-2-naphthoic acid (SCF-12) and their analogs in the cavity of mLST8 were explored. ADME and binding energy calculations led to the identification of five compounds with favorable Glide (G) scores and implicated the importance of Asn132 and Gln225 as key binding residues. Molecular dynamics (MD) simulations and free energy landscape (FEL) approaches helped in elucidating the binding mechanism and suggested the possibility of ligands 1–3 namely, ZINC01765622, ZINC62723702 and ZINC02576980 to be promising antagonists for mLST8. Thus, this study substantiates the prospect of targeting mLST8 protein using potent hits which could hinder tumor progression in colon and prostate cancers.     

Inhibition of Acetylcholinesterase by three New Pyridinium Compounds and their Effect on Phosphonylation of the Enzyme

Abstract

Three new mono-pyridinium compounds were prepared: 1-phenacyl-2-methylpyridinium chloride (1), 1-benzoylethylpyridinium chloride (2) and 1-benzoylethylpyridinium-4-aldoxime chloride (3) and assayed in vitro for their inhibitory effect on human blood acetylcholinesterase (EC 3.1.1.7, AChE). All the three compounds inhibited AChE reversibly; their binding affinity for the enzyme was compared with their protective effect (PI) on AChE phosphonylation by soman and VX. Compound 1 was found to bind to both the catalytic and the allosteric (substrate inhibition) sites of the enzyme with estimated dissociation constants of 6.9 μM (K_cat) and 27 μM (K_all), respectively. Compound 2 bound to the catalytic site with K_cat= 59 μM and compound 3 only to the allosteric site with K_all = 328 μM. PI was evaluated from phosphonylation measured in the absence and in presence of the compounds applied in a concentration corresponding to their K_cat or K_all value, and was also calculated from theoretical equations deduced from the reversible inhibition of the enzyme. Compounds 1 and 3 protected the enzyme from phosphonylation by soman and VX, whereas no protection was observed in the presence of compound 2 under the same conditions. Irrespective of the binding sites to AChE, PI for compounds 1 and 3 evaluated from phosphonylation agreed with PI calculated from reversible inhibition. Compound 3 was found to be a weak reactivator of methylphosphonylated AChE with k_r = 1.1 × 10²Lmol^-1 min^-1.     

Virtual screening using the ligand ZINC database for novel lipoxygenase-3 inhibitors

The leukotrienes constitute a group of arachidonic acid-derived compounds with biologic activities suggesting important roles in inflammation and immediate hypersensitivity. Epidermis-type lipoxygenase-3 (ALOXE3), a distinct subclass within the multigene family of mammalian lipoxygenases, is a novel isoenzyme involved in the metabolism of leukotrienes and plays a very important role in skin barrier functions. Lipoxygenase selective inhibitors such as azelastine and zileuton are currently used to reduce inflammatory response. Nausea, pharyngolaryngeal pain, headache, nasal burning and somnolence are the most frequently reported adverse effects of these drugs. Therefore, there is still a need to develop more potent lipoxygenase inhibitors. In this paper, we report the screening of various compounds from the ZINC database (contains over 21 million compounds) using the Molegro Virtual Docker software against the ALOXE3 protein. Screening was performed using molecular constraints tool to filter compounds with physico-chemical properties similar to the 1N8Q bound ligand protocatechuic acid. The analysis resulted in 4319 Lipinski compliant hits which are docked and scored to identify structurally novel ligands that make similar interactions to those of known ligands or may have different interactions with other parts of the binding site. Our screening approach identified four molecules ZINC84299674; ZINC76643455; ZINC84299122 & ZINC75626957 with MolDock score of -128.901, -120.22, -116.873 & - 102.116 kcal/mol, respectively. Their energy scores were better than the 1N8Q bound co-crystallized ligand protocatechuic acid (with MolDock score of -77.225 kcal/mol). All the ligands were docked within the binding pocket forming interactions with amino acid residues.     

In-silico analysis of Sirt2 from Schistosoma mansoni: structures,conformations, and interactions with inhibitors

Sirtuins are NAD+-dependent lysine deacetylases member of the class III HDAC family. These are demonstrated to be therapeutic targets in parasitic diseases like schistosomiasis. Observations suggested that sirtuin enzyme is necessary for the functionality of fe/male reproductive system, due to which SmSirt2 is treated as a potential therapeutic target. There are no structural and molecular features of SmSirt2 have been reported yet. In this study, homology modeling has been used to determine the three-dimensional features of the SmSITRT2. Further, structure validation has been performed by energy minimization and Ramachandran plot. Validated structures are further subjected to molecular docking and virtual screening to find the best lead molecules for downstream analysis. Ten lead molecules were selected while comparing virtual screening of hSirt2 and SmSirt2 both. These leads are further compared with AKG2 which is known inhibitor of hSirt2 (?8.8 kcal/mol). Out of selected 10 leads, four of them (ZINC23995485 (?9.5 kcal/mol), ZINC53298162 (?9.4 kcal/mol), ZINC70927268 (?10.0 kcal/mol), ZINC89878705 (?11.2 kcal/mol)) have shown better interaction with SmSirt2, in which ZINC89878705 (?11.2 kcal/mol) shows a more compact packing as compared to AKG2 and rest of ligands. These molecules could be further subject to in vitro study and model of SmSirt2 has been proposed for further structure-based drug design projects concerning sirtuins from Schistosoma mansoni.     

Design,synthesis and evaluation of quinolinone derivatives containing dithiocarbamate moiety as multifunctional AChE inhibitors for the treatment of Alzheimer’s disease

Abstract

A series of novel quinolinone derivatives bearing dithiocarbamate moiety were designed and synthesised as multifunctional AChE inhibitors for the treatment of AD. Most of these compounds exhibited strong and clearly selective inhibition to eeAChE. Among them, compound 4c was identified as the most potent inhibitor to both eeAChE and hAChE (IC₅₀ = 0.22?μM for eeAChE; IC₅₀ = 0.16?μM for hAChE), and it was also the best inhibitor to AChE-induced Aβ aggregation (29.02% at 100?μM) and an efficient inhibitor to self-induced Aβ aggregation (30.67% at 25?μM). Kinetic and molecular modelling studies indicated that compound 4c was a mixed-type inhibitor, which could interact simultaneously with the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 4c had good ability to cross the BBB, showed no toxicity on SH-SY5Y neuroblastoma cells and was well tolerated in mice at doses up to 2500?mg/kg (po).     

Synthesis, biological evaluation and molecular modelling of diversely functionalized heterocyclic derivatives as inhibitors of acetylcholinesterase/butyrylcholinesterase and modulators of Ca2+ channels and nicotinic receptors

The synthesis and the biological activity of compounds 5-40 as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca(2+) channels and nicotinic receptors, are described. These molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedl?nder reaction with selected cycloalkanones. These compounds are moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the 'oxazolo-tacrine' derivatives are devoid of any activity. All compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca(2+) channels, and three of them, 64, 19 and 67, the non-L type of Ca(2+) channels. Molecular modelling studies suggest that these compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both, the catalytic and peripheral binding sites of the enzyme.