TY - JOUR T1 - THRX-198321 Is a Bifunctional Muscarinic Receptor Antagonist and β<sub>2</sub>-Adrenoceptor Agonist (MABA) That Binds in a Bimodal and Multivalent Manner JF - Molecular Pharmacology JO - Mol Pharmacol SP - 389 LP - 399 DO - 10.1124/mol.110.069120 VL - 79 IS - 3 AU - Tod Steinfeld AU - Adam D. Hughes AU - Uwe Klein AU - Jacqueline A. M. Smith AU - Mathai Mammen Y1 - 2011/03/01 UR - http://molpharm.aspetjournals.org/content/79/3/389.abstract N2 - Biphenyl-2-yl-carbamic acid 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)-ethylamino]-nonyl}-piperidin-4-yl ester (THRX-198321) is a single molecule composed of a muscarinic acetylcholine receptor (mAChR) antagonist moiety, represented by the fragment MA, linked by a C9 polymethylene chain to a β2-adrenoceptor (β2AR) agonist moiety, represented by the fragment 8-hydroxy-5-((R)-1-hydroxy-2-methylamino-ethyl)-1H-quinolin-2-one (BA). THRX-198321 exhibited high affinity for mAChR (M2 pKI,App = 10.57 ± 0.09; M3 pKI,App = 10.07 ± 0.11) and β2AR (pKI,App = 9.54 ± 0.15), with potent mAChR antagonist (M2 pKI,Fn = 9.69 ± 0.23; M3 pKI,Fn = 10.05 ± 0.17) and β2AR agonist (pEC50 = 9.25 ± 0.02) activities. Consistent with multivalent interactions, THRX-198321 binding affinity was &gt;300-fold higher at mAChR and 29-fold higher at β2AR relative to its monovalent fragments biphenyl carbamic acid piperidinyl ester (MA) and BA, respectively. THRX-198321 was a competitive antagonist at mAChR (M2 pKB = 9.98 ± 0.13; M3 pKB = 10.31 ± 0.89), whereas THRX-198321 agonist activity at β2AR was competitively inhibited by propranolol. Interactions of THRX-198321 with an allosteric site on mAChR and a novel extracellular allosteric site on β2AR, respectively, were detected by measuring THRX-198321-evoked changes in the dissociation rates for the orthosteric radioligands, [N-methyl-3H]scopolamine methyl chloride (M2 pEC50,diss = 6.73 ± 0.10; M3 pEC50,diss = 5.02 ± 0.14) and [4,6-propyl-3H]dihydroalprenolol (β2AR pEC50,diss = 3.82 ± 0.25). The carbostyril-linker fragment (BA-L) binds to the allosteric site of mAChR (M2 pEC50,diss = 5.06 ± 0.03; M3 pEC50,diss = 4.15 ± 0.25), whereas the MA fragment binds to the allosteric site of β2AR (pEC50,diss = 3.60 ± 0.18). Collectively, these observations suggest that THRX-198321 exhibits a multivalent bimodal orientation in the orthosteric and allosteric binding pockets of mAChR and β2AR, a phenomenon that may be unique to this class of molecule. ER -