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This project is focused on investigation into mechanism of recognition and distinction of agonistic and antagonistic ligands by G-protein coupled receptors and elucidation of the molecular basis of their different biological effect (stimulation and inhibition). For this purpose the kinetics of agonist and antagonist simultaneous interaction with the receptor will be studied at wide concentration interval of both ligands and the mutual positioning of binding sites for agonists and antagonists will be established. Experiments will be made with muscarinic acetylcholine receptor (m2) and human P2Y1 purinoceptor and radioactive antagonists [3H]NMS and [35S]dATPalphaS will be used as "reporter" ligands. The former receptor is well characterized and is selected for devekolment and perfection of the kinetic approach by using carbachol, N-methylfurmethide and oxotremorine as muscarinic agonists. Properties of the latter receptor are not well understood and in this case we expect to demonstrate the advantages of the kinetic analysis and its implications for the effect-directed drug design, because the approach proposed describes the receptor-ligand interaction in terms of several kinetic parameters, instead of the dissociation constant used in classical receptor concepts. Besides the known P2Y1 agonists 2MeADP and ADP interaction of this receptor with novel non-phosphate adenosine nucleotide analogues, recently designed and synthesized in our laboratory, will be studied. The kinetic models for the two receptors will take into consideration both fast and slow steps of ligand binding and the presence of low and high affinity receptor states, and the project integrates the theoretical analysis of the receptor-ligand interaction process with massive kinetic experiments to contribute into the theory of receptor functioning. |