title: NSC23766, a widely used inhibitor of Rac1 activation, additionally acts as a competitive antagonist at muscarinic acetylcholine receptors.
abstract: Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N(6)-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M2 muscarinic acetylcholine receptor (M2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly Giβγ-mediated, adenosine-induced activation of Gi protein-regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K(+) current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A1 receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca(2+) concentrations in human embryonic kidney 293 (HEK-293) cells expressing M1, M2, or M3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M2 and M3 mAChR crystal structures confirmed this interpretation.
authors.affiliation: Institute of Experimental Pharmacology and Toxicology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany (M.L., K.W., T.W.); DZHK (German Center for Cardiovascular Research), Partner Site, Heidelberg-Mannheim, Mannheim, Germany (M.L., D.D., T.W.); Department of Pharmacology, Institute of Clinical Medicine, Faculty of Medicine and K.G. Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway (K.A.K., F.O.V.); Institute of Pharmacology, Faculty of Medicine, University Duisburg-Essen, Essen, Germany, and Division of Experimental Cardiology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany (N.V., D.D.); and Computer-Aided Drug Design, Institute of Pharmacy, Free University of Berlin, Berlin, Germany (M.B., G.W.).
authors.forename: Magdolna
authors.initials: M
authors.lastname: Levay
authors.forename: Kurt Allen
authors.initials: KA
authors.lastname: Krobert
authors.forename: Karola
authors.initials: K
authors.lastname: Wittig
authors.forename: Niels
authors.initials: N
authors.lastname: Voigt
authors.forename: Marcel
authors.initials: M
authors.lastname: Bermudez
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wolber
authors.forename: Dobromir
authors.initials: D
authors.lastname: Dobrev
authors.forename: Finn Olav
authors.initials: FO
authors.lastname: Levy
authors.forename: Thomas
authors.initials: T
authors.lastname: Wieland
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D018727:Muscarinic Antagonists / Q000378:metabolism* / Q000494:pharmacology; D009924:Organ Culture Techniques; D011247:Pregnancy; D011743:Pyrimidines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D011976:Receptors, Muscarinic / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D000634:Aminoquinolines; D018727:Muscarinic Antagonists; C487513:NSC 23766; D011743:Pyrimidines; D011976:Receptors, Muscarinic; C499134:Rac1 protein, rat; D020830:rac1 GTP-Binding Protein