THRX-198321 Is a Bifunctional Muscarinic Receptor Antagonist and β2-Adrenoceptor Agonist (MABA) That Binds in a Bimodal and Multivalent Manner

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 beta(2)-adrenoceptor (beta(2)AR) 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 (M-2 pK(I,App) = 10.57 +/- 0.09; M-3 pK(I,App) = 10.07 +/- 0.11) and beta(2)AR (pK(I,App) = 9.54 +/- 0.15), with potent mAChR antagonist (M-2 pK(I,Fn) = 9.69 +/- 0.23; M-3 pK(I,Fn) = 10.05 +/- 0.17) and beta(2)AR agonist (pEC(50) = 9.25 +/- 0.02) activities. Consistent with multivalent interactions, THRX-198321 binding affinity was >300-fold higher at mAChR and 29-fold higher at beta(2)AR relative to its monovalent fragments biphenyl carbamic acid piperidinyl ester (MA) and BA, respectively. THRX-198321 was a competitive antagonist at mAChR (M-2 pK(B) = 9.98 +/- 0.13; M-3 pK(B) = 10.31 +/- 0.89), whereas THRX-198321 agonist activity at beta(2)AR was competitively inhibited by propranolol. Interactions of THRX-198321 with an allosteric site on mAChR and a novel extracellular allosteric site on beta(2)AR, respectively, were detected by measuring THRX-198321-evoked changes in the dissociation rates for the orthosteric radioligands, [N-methyl-H-3]scopolamine methyl chloride (M-2 pEC(50,diss) = 6.73 +/- 0.10; M-3 pEC(50,diss) = 5.02 +/- 0.14) and [4,6-propyl-H-3]dihydroalprenolol (beta(2)AR pEC(50,diss) = 3.82 +/- 0.25). The carbostyril-linker fragment (BA-L) binds to the allosteric site of mAChR (M-2 pEC(50,diss) = 5.06 +/- 0.03; M-3 pEC(50,diss) = 4.15 +/- 0.25), whereas the MA fragment binds to the allosteric site of beta(2)AR (pEC(50,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 beta(2)AR, a phenomenon that may be unique to this class of molecule.