Structure-Based Discovery of Dual-Target Hits for Acetylcholinesterase and the α7 Nicotinic Acetylcholine Receptors: In Silico Studies and In Vitro Confirmation.

Despite extensive efforts in the development of drugs for complex neurodegenerative diseases, treatment often remains challenging or ineffective, and hence new treatment strategies are necessary. One approach is the design of multi-target drugs, which can potentially address the complex nature of disorders such as Alzheimer's disease. We report a method for high throughput virtual screening aimed at identifying new dual target hit molecules. One of the identified hits, N,N-dimethyl-1-(4-(3-methyl-[1,2,4]triazolo[4,3-a]pyrimidin-6-yl)phenyl)ethan-1-amine (Ý;mir-2), has dual-activity as an acetylcholinesterase (AChE) inhibitor and as an α7 nicotinic acetylcholine receptor (α7 nAChR) agonist. Using computational chemistry methods, parallel and independent screening of a virtual compound library consisting of 3,848,234 drug-like and commercially available molecules from the ZINC15 database, resulted in an intersecting set of 57 compounds, that potentially possess activity at both of the two protein targets. Based on ligand efficiency as well as scaffold and molecular diversity, 16 of these compounds were purchased for in vitro validation by Ellman's method and two-electrode voltage-clamp electrophysiology. Ý;mir-2 was shown to exhibit the desired activity profile (AChE IC50 = 2.58 ± 0.96 µM; α7 nAChR activation = 7.0 ± 0.9% at 200 µM) making it the first reported compound with this particular profile and providing further evidence of the feasibility of in silico methods for the identification of novel multi-target hit molecules.

Novel Approach for the Search for Chemical Scaffolds with Dual Activity with Acetylcholinesterase and the α7 Nicotinic Acetylcholine Receptor-A Perspective for the Treatment of Neurodegenerative Disorders.

Neurodegenerative disorders, including Alzheimer's disease, belong to the group of the most difficult and challenging conditions with very limited treatment options. Attempts to find new drugs in most cases fail at the clinical stage. New tactics to develop better drug candidates to manage these diseases are urgently needed. It is evident that better understanding of the neurodegeneration process is required and targeting multiple receptors may be essential. Herein, we present a novel approach, searching for dual active compounds interacting with acetylcholinesterase (AChE) and the α7 nicotinic acetylcholine receptor (nAChR) using computational chemistry methods including homology modelling and high throughput virtual screening. Activities of identified hits were evaluated at the two targets using the colorimetric method of Ellman and two-electrode voltage-clamp electrophysiology, respectively. Out of 87,250 compounds from a ZINC database of natural products and their derivatives, we identified two compounds, 8 and 9, with dual activity and balanced IC50 values of 10 and 5 µM at AChE, and 34 and 14 µM at α7 nAChR, respectively. This is the first report presenting successful use of virtual screening in finding compounds with dual mode of action inhibiting both the AChE enzyme and the α7 nAChR and shows that computational methods can be a valuable tool in the early lead discovery process.

G protein- and agonist-bound serotonin 5-HT2A receptor model activated by steered molecular dynamics simulations.

A 5-HT(2A) receptor model was constructed by homology modeling based on the ß(2)-adrenergic receptor and the G protein-bound opsin crystal structures. The 5-HT(2A) receptor model was transferred into an active conformation by an agonist ligand and a G(αq) peptide in four subsequent steered molecular dynamics (MD) simulations. The driving force for the transformation was the addition of several known intermolecular and receptor interhelical hydrogen bonds enforcing the necessary helical and rotameric movements. Subsquent MD simulations without constraints confirmed the stability of the activated receptor model as well as revealed new information about stabilizing residues and bonds. The active 5-HT(2A) receptor model was further validated by retrospective ligand screening of more than 9400 compounds, whereof 182 were known ligands. The results show that the model can be used in drug discovery for virtual screening and structure-based ligand design as well as in GPCR activation studies.

Complementary three-dimensional quantitative structure-activity relationship modeling of binding affinity and functional potency: a study on alpha4beta2 nicotinic ligands.

Complementary 3D-QSAR modeling of binding affinity and functional potency is proposed as a tool to pinpoint the molecular features of the ligands, and the corresponding amino acids in the receptor, responsible for high affinity binding vs those driving agonist behavior and receptor activation. This approach proved successful on a series of nicotinic alpha(4)beta(2) ligands, whose partial/full agonist profile could be linked to the size of the scaffold as well as to the nature of the substituents.

Novel acetylcholine and carbamoylcholine analogues: development of a functionally selective alpha4beta2 nicotinic acetylcholine receptor agonist.

A series of carbamoylcholine and acetylcholine analogues were synthesized and characterized pharmacologically at neuronal nicotinic acetylcholine receptors (nAChRs). Several of the compounds displayed low nanomolar binding affinities to the alpha4beta2 nAChR and pronounced selectivity for this subtype over alpha3beta4, alpha4beta4, and alpha7 nAChRs. The high nAChR activity of carbamoylcholine analogue 5d was found to reside in its R-enantiomer, a characteristic most likely true for all other compounds in the series. Interestingly, the pronounced alpha4beta2 selectivities exhibited by some of the compounds in the binding assays translated into functional selectivity. Compound 5a was a fairly potent partial alpha4beta2 nAChR agonist with negligible activities at the alpha3beta4 and alpha7 subtypes, thus being one of the few truly functionally selective alpha4beta2 nAChR agonists published to date. Ligand-protein docking experiments using homology models of the amino-terminal domains of alpha4beta2 and alpha3beta4 nAChRs identified residues Val111(beta2)/Ile113(beta4), Phe119(beta2)/Gln121(beta4), and Thr155(alpha4)/Ser150(alpha3) as possible key determinants of the alpha4beta2/alpha3beta4-selectivity displayed by the analogues.

New alpha(1)-adrenoceptor antagonists derived from the antipsychotic sertindole - carbon-11 labelling and pet examination of brain uptake in the cynomolgus monkey.

Central alpha(1)-adrenergic receptors are potential targets for recently developed antipsychotic drugs. Two new 11C labeled potent and selective alpha(1)-adrenoceptor antagonists, 1- [2- [4-[1-(4-fluorophenyl)-5-(2-[(11)C]methyl-tetrazol-5-yl)-1H-indol-3-yl]-1-piperidinyl]ethyl]-imidazolidin-2-one ([(11)C]2) and 1- [2- [4-[1-(4-fluorophenyl)-5-(1-[(11)C]methyl-(1,2,3-triazol-4-yl)-1H-indol-3-yl]-1-piperidinyl]ethyl]-imidazolidin-2-one ([(11)C]3) were prepared and evaluated for imaging of central alpha(1)-adrenergic receptors in the cynomolgus monkey brain. For both compounds, the total brain radioactivity was only about 0.6% of the radioactivity injected i.v. There was no evident binding in regions known to contain alpha(1)-adrenoceptors. This observation suggests that the affinity of the radioligands in primates in vivo is not sufficient to provide a signal for specific binding that can be differentiated from the background. In addition, active efflux by P-glycoprotein may be responsible for the low total brain-uptake of the two radioligands. Both compounds showed a highly polarised and verapamile sensitive transport across monolayers of Caco-2 cells. The total brain-uptake of [(3)H]2 was 6 times higher in mdr1a(-/-) knock-out mice lacking the gene encoding P-glycoprotein compared to wild type mice. Pretreatment of one monkey with Cyclosporin A (15 mg/kg) resulted in 40% higher brain uptake for [(11)C]3 when compared with baseline. These observations support the view that efflux by P-glycoprotein can be of quantitative importance for the total brain-uptake of some PET radioligands.