ABSTRACT
There is a need for continued development of acetylcholinesterase (AChE) inhibitors that could prolong the life of acetylcholine in the synaptic cleft and also prevent the aggregation of amyloid peptides associated with Alzheimer's disease. The lack of a 3D-QSAR model which specifically deconvulates the type of interactions and quantifies them in terms of energies has motivated us to report a CoRIA model vis-à-vis the standard 3D-QSAR methods, CoMFA and CoMSIA. The CoRIA model was found to be statistically superior to the CoMFA and CoMSIA models and it could efficiently extract key residues involved in ligand recognition and binding to AChE. These interactions were quantified to gauge the magnitude of their contribution to the biological activity. In order to validate the CoRIA model, a pharmacophore map was first constructed and then used to virtually screen public databases, from which novel scaffolds were cherry picked that were not present in the training set. The biological activities of these novel molecules were then predicted by the CoRIA, CoMFA, and CoMSIA models. The hits identified were purchased and their biological activities were measured by the Ellman's method for AChE inhibition. The predicted activities are in unison with the experimentally measured biological activities.
Subject(s)
Acetylcholinesterase/chemistry , Alzheimer Disease/enzymology , Cholinesterase Inhibitors/chemistry , Molecular Docking Simulation/methods , Quantitative Structure-Activity Relationship , Acetylcholinesterase/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/prevention & control , Binding Sites , Cholinesterase Inhibitors/metabolism , Cholinesterase Inhibitors/therapeutic use , Donepezil , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/metabolism , Indans/chemistry , Indans/metabolism , Ligands , Molecular Conformation , Molecular Structure , Piperidines/chemistry , Piperidines/metabolism , Protein Binding , Protein Structure, Tertiary , Reproducibility of Results , ThermodynamicsABSTRACT
Glucose-dependent insulinotropic polypeptide (GIP), a gut peptide released in response to food intake brings about secretion of insulin in a glucose-dependent manner upon binding to its receptor, GIPR. GIP-GIPR has emerged as a new vista for anti-diabetic drug discovery and their interaction is being probed at the atomic level to aid rational drug design. In order to probe this interaction on cells, the current study attempts towards expressing 15N-labeled GIP using classical molecular biology tools. We have developed a methodology to obtain GIP devoid of extra amino acid(s); a prerequisite to the intended interaction study. The synthetic GIP cDNA with a Factor Xa protease site at the N-terminus of GIP was inserted in the vector pET32a(+); the fusion protein thus expressed was eventually cleaved to obtain GIP. After successful Factor Xa cleavage, the cleaved GIP was confirmed by western blot. Subsequently, the (15N)GIP was obtained using the aforementioned procedure and confirmed by MALDI-TOF.
