Muscarinic receptors are known to play important biological roles and are drug targets for several human diseases. In a pilot study, novel muscarinic antagonists were synthesized and used as chemical probes to obtain additional information of the muscarinic pharmacophore. The design of these ligands made use of current orthosteric and allosteric models of drug-receptor interactions together with chemical motifs known to achieve muscarinic receptor selectivity. This approach has led to the discovery of several non-competitive muscarinic ligands that strongly bind at a secondary receptor site. These compounds were found to be non-competitive antagonists that completely abolished carbachol activation in functional assays. Several of these compounds antagonized functional response to carbachol with great potency at M1 and M4 than at the rest of receptor subtypes.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů chemická syntéza   chemie   metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• křečci praví MeSH
• lidé MeSH
• ligandy MeSH
• N-methylskopolamin chemická syntéza   chemie   metabolismus   MeSH
• pilotní projekty MeSH
• protein - isoformy chemie   genetika   metabolismus   MeSH
• pyridiny chemie   MeSH
• racionální návrh léčiv MeSH
• receptory muskarinové chemie   genetika   metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

• MeSH
• biologické modely MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• kinetika MeSH
• křečci praví MeSH
• lidé MeSH
• ligandy MeSH
• mutantní proteiny chemie   metabolismus   MeSH
• N-methylskopolamin chemie   metabolismus   MeSH
• proteinové domény MeSH
• receptory muskarinové chemie   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• simulace molekulární dynamiky MeSH
• tritium metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zrychlení MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH