Characterization of a CNS penetrant, selective M₁muscarinic receptor agonist, 77‐LH‐28‐1

Abstract

Background and purpose: M 1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimer's disease and schizophrenia. However, the discovery of subtypeselective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh-binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC-42 (4-nbutyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine), which bind to an allosteric site and selectively activate the M 1 mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs. Experimental approach: In this study, we have compared the pharmacological profile of AC-42 and a close structural analogue, 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) at human recombinant, and rat native, mAChRs by calcium mobilization, inositol phosphate accumulation and both in vitro and in vivo electrophysiology. Key results: Calcium mobilization and inositol phosphate accumulation assays revealed that both AC-42 and 77-LH-28-1 display high selectivity to activate the M 1 mAChR over other mAChR subtypes. Furthermore, 77-LH-28-1, but not AC-42, acted as an agonist at rat hippocampal M 1 receptors, as demonstrated by its ability to increase cell firing and initiate gamma frequency network oscillations. Finally, 77-LH-28-1 stimulated cell firing in the rat hippocampus in vivo following subcutaneous administration. Conclusions and implications: These data suggest that 77-LH-28-1 is a potent, selective, bioavailable and brain-penetrant agonist at the M 1 mAChR and therefore that it represents a better tool than AC-42, with which to study the pharmacology of the M 1 mAChR. (2008) 154, 1104-1115 doi:10.1038/bjp.2008 published online 5 May 2008 Keywords: muscarinic receptors; selective agonist; allosteric; AC-42; 77-LH-28-1; calcium mobilization; inositol phosphate; cell firing; network oscillations There is a wide array of pharmacological tools with which to study mAChRs. For example, N-methyl scopolamine, quinuclidinylbenzilate, pirenzepine and darifenacin are among numerous mAChR antagonists, and ACh and oxotremorine-M among mAChR agonists, which have been used in unlabelled and radiolabelled forms to characterize the localization, pharmacology and function of mAChRs. Unfortunately, most of these pharmacological tools exhibit poor selectivity between mAChR subtypes (Caulfield and Birdsall, 1998;Ellis, 2002). Those agents that do display high degrees of mAChR subtype selectivity are few in number and when discovered are often shown to interact with an allosteric, rather than the orthosteric, site as exemplified by the highly selective M 1 receptor peptide antagonist MT-7 (muscarinic toxin 7; Olianas et al., 2000). British Journal of PharmacologyTherefore, the identification of selective M 1 mAChR agonists would represent a significant advance in mAChR pharmacology and could offer t...

Contrasting Effects of Allosteric and Orthosteric Agonists on M1Muscarinic Acetylcholine Receptor Internalization and Down-regulation

“…The apparent binding affinity (pK B ) for each agonist is summarized in Table 1. Consistent with previous reports, both AC-42 and 77-LH-28-1 are able to displace specific [ 3 H]NMS binding from M 2 and M 3 mACh receptors (Spalding et al, 2002;May et al, 2007;Langmead et al, 2008a) with 77-LH-28-1 and AC-42 apparent affinities at the M 2 subtype being comparable with those observed at the M 1 mACh receptor (Table 1).…”

“…Recent studies have reported actions of AC-42 and 77-LH-28-1 at non-M 1 mACh receptor subtypes (May et al, 2007;Langmead et al, 2008a). Here, we have shown that both allosteric agonists can displace specific [ 3 H]NMS binding to the M 2 and M 3 mACh receptors (Table 1).…”

“…Here, we have shown that both allosteric agonists can displace specific [ 3 H]NMS binding to the M 2 and M 3 mACh receptors (Table 1). AC-42 and 77-LH-28-1 have been reported to act allosterically at the M 2 mACh receptor (May et al, 2007), but a similar mechanism of action has not been reported at the M 3 receptor (Langmead et al, 2008a). Oxo-M stimulated significant increases ERK1/2 phosphorylation (P Ͻ 0.001) via human M 1 , M 2 , and M 3 mACh receptor subtypes stably expressed in CHO cells (Fig.…”

Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

“…117 77-LH-28-1 (34, Figure 23), an M 1 mAChR-selective compound closely structurally related to the AC compounds, demonstrated agonistic behavior at rat hippocampal M 1 mAChRs in vitro and CNS penetration leading to the stimulation of rat hippocampal cell firing in vivo. 124 Furthermore, activation of M1 mAChRs by 77-LH-28-1 enhanced NMDA receptor activation in vitro, facilitating long-term potentiation. 125 Consistent with the interactions of AC-42, the mAChR orthosteric antagonists pirenzepine and scopolamine both produced a dextral displacement of the 77-LH-28-1 CRC; 124 however, 77-LH-28-1 has also been shown to compete with the prototypical muscarinic negative allosteric modulator C 7 /3-phth (at the NMS-occupied receptor), 115 adding weight to the theory that this scaffold interacts with portions of both orthosteric and allosteric sites on the M 1 mAChR.…”

Alzheimer’s Disease: Dawn of a New Era?

“…TBPB promotes the processing of APP towards the non-amyloidogenic pathway, decreases Aβ production in vitro, and has been shown to improve cognition in animal models (122). 77-LH-28-1 is another potent, selective and CNS penetrant allosteric M1 agonist which was a structural analog of AC-42 that was reported to improve cognition in animal models (123) as did AC-260584 (124). However, it is unclear if any of these agents are being pursued for clinical development.…”