THRX-198321 is a bifunctional muscarinic receptor antagonist and beta2-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 β(2)-adrenoceptor (β(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 β(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 β(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 β(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 β(2)AR was competitively inhibited by propranolol. Interactions of THRX-198321 with an allosteric site on mAChR and a novel extracellular allosteric site on β(2)AR, respectively, were detected by measuring THRX-198321-evoked changes in the dissociation rates for the orthosteric radioligands, [N-methyl-(3)H]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-(3)H]dihydroalprenolol (β(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 β(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 β(2)AR, a phenomenon that may be unique to this class of molecule.
Department of Molecular and Cellular Biology, Theravance, Inc., South San Francisco, CA 94080, USA. tsteinfeld@theravance.com
Tod
T
Steinfeld
Adam D
AD
Hughes
Uwe
U
Klein
Jacqueline A M
JA
Smith
Mathai
M
Mammen
D058666:Adrenergic beta-2 Receptor Agonists / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D002219:Carbamates / Q000378:metabolism / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D018721:Muscarinic Agonists / Q000378:metabolism / Q000494:pharmacology*; D010599:Pharmacokinetics; D015363:Quinolones / Q000378:metabolism / Q000494:pharmacology*; D011869:Radioligand Assay
D058666:Adrenergic beta-2 Receptor Agonists; D002219:Carbamates; D007295:Inositol Phosphates; D018721:Muscarinic Agonists; D015363:Quinolones; C557502:biphenyl-2-yl-carbamic acid 1-(9-(2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)ethylamino)nonyl)piperidin-4-yl ester; C002647:inositol 1-phosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP