title: Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M(2) and M(4) receptors.
abstract: The M(4) receptor is a compelling therapeutic target, as this receptor modulates neural circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both antipsychotic and procognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. In this study, we present the pharmacological characterization of LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy] thieno[2,3-b]pyridine-2-carboxamide), a M(2)/M(4) receptor-selective positive allosteric modulator (PAM), chemically evolved from hits identified through a M4 allosteric functional screen. Although unsuitable as a therapeutic due to M(2) receptor cross-reactivity and, thus, potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity and broadens research capabilities through its development into a radiotracer. Characterization of LY2119620 revealed evidence of probe dependence in both binding and functional assays. Guanosine 5'-[γ-(35)S]-triphosphate assays displayed differential potentiation depending on the orthosteric-allosteric pairing, with the largest cooperativity observed for oxotremorine M (Oxo-M) LY2119620. Further [(3)H]Oxo-M saturation binding, including studies with guanosine-5'-[(β,γ)-imido]triphosphate, suggests that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein-coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M(4) receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately, it was the M(2) receptor pharmacology and PAM activity with iperoxo that made LY2119620 the most suitable allosteric partner for the M(2) active-state structure recently solved (Kruse et al., 2013), a structure that provides crucial insights into the mechanisms of orthosteric activation and allosteric modulation of muscarinic receptors.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (C.H.C., D.A.S., H.X., A.Q., C.C.F.); and Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C.).
authors.forename: Carrie H
authors.initials: CH
authors.lastname: Croy
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (C.H.C., D.A.S., H.X., A.Q., C.C.F.); and Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C.).
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Schober
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (C.H.C., D.A.S., H.X., A.Q., C.C.F.); and Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C.).
authors.forename: Hongling
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (C.H.C., D.A.S., H.X., A.Q., C.C.F.); and Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C.).
authors.forename: Anne
authors.initials: A
authors.lastname: Quets
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (C.H.C., D.A.S., H.X., A.Q., C.C.F.); and Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C.).
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (C.H.C., D.A.S., H.X., A.Q., C.C.F.); and Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (A.C.) felder@lilly.com.
authors.forename: Christian C
authors.initials: CC
authors.lastname: Felder
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects* / Q000502:physiology; D000495:Allosteric Site / Q000187:drug effects / Q000502:physiology; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D018721:Muscarinic Agonists / Q000494:pharmacology; D010095:Oxotremorine / Q000031:analogs & derivatives / Q000494:pharmacology; D043585:Receptor, Muscarinic M2 / Q000378:metabolism*; D043588:Receptor, Muscarinic M4 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D008024:Ligands; D018721:Muscarinic Agonists; D043585:Receptor, Muscarinic M2; D043588:Receptor, Muscarinic M4; D010095:Oxotremorine; C042743:oxotremorine M; D019204:GTP-Binding Proteins