Discovery of an Ectopic Activation Site on the M₁Muscarinic Receptor

Abstract

Receptors have well-conserved regions that are recognized and activated by hormones and neurotransmitters. Most drugs bind to these sites and mimic or block the action of the native ligands. Using a high-throughput functional screen, we identified a potent and selective M 1 muscarinic receptor agonist from a novel structural class. Using a series of chimeric receptors, we demonstrated that this ligand activates the receptor through a region that is not conserved among receptor subtypes, explaining its unprecedented selectivity. This region of the receptor is distinct from the conserved region that is recognized by traditional ligands. The finding that receptors for small-molecule transmitters can have multiple, structurally distinct activation sites has broad implications for the study of receptor structure/function and the potential for the discovery of novel ligands with high selectivity.G-protein-coupled receptors that bind monoamine ligands (e.g., serotonin, adrenaline, dopamine, histamine, and acetylcholine) comprise the most intensively studied and exploited receptor family for the development of therapeutic agents by the pharmaceutical industry. The natural ligands for monoamine receptors are believed to bind a highly conserved pocket located deep within the transmembrane (TM)-spanning regions and to mediate receptor activation primarily through TM3, TM5, TM6, and TM7 (Spalding et al., 1994;Baldwin et al., 1997;Gether, 2000;Lu et al., 2001). Of the amino acids in these regions, 74% are identical in all five muscarinic receptor subtypes (Bonner et al., 1988). Potent small-molecule agonists are also believed to bind monoamine receptors through the same highly conserved regions (Strader et al., 1989(Strader et al., , 1991Wess et al., 1991;Page et al., 1995;Spalding et al., 1998;Ward et al., 1999;Allman et al., 2000).The muscarinic M 1 receptor has been targeted for the discovery of therapeutics for Alzheimer's Disease, and several companies have developed M 1 -selective agonists (e.g., Tecle et al., 1998;Wood et al., 1999;Bartolomeo et al., 2000;Wienrich et al., 2001). Many potent compounds came out of these programs, and several were shown to improve cognition in animals (WAY-132983 and CI-1017; Bartolomeo et al., 2000;Weiss et al., 2000) and people (Xanomeline, Bodick et al., 1997). However, many of the compounds also produced classic muscarinic side effects such as sweating, nausea and diarrhea (Bodick et al., 1997, Bartolomeo et al., 2000Thal et al., 2000). In vitro assays have shown that these compounds activate the M 1 , M 3 , M 4 , and M 5 muscarinic receptor subtypes at similar concentrations (Table 1 and Tecle Wood et al., 1999;Bartolomeo et al., 2000;Wienrich et al., 2001). This may be a direct result of the ligands activating the receptors through regions where the amino acid sequence is almost identical. Since drug interactions with nontarget receptor subtypes are often responsible for the unwanted side effects of commercial pharmaceuticals, there is strong motivation to design more selec...

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

“…Both AC-42 and 77-LH-28-1 potently stimulated intracellular calcium mobilization in CHO-hM 1 cells with pEC 50 values of 6.5 ± 0.1 and 8.1±0.3, respectively (Figure 2; Table 1). As shown previously, AC-42 does not display agonist activity at hM 2 -hM 5 receptor subtypes at concentrations up to 10 mM (Table 1; Figure 2; Spalding et al, 2002) and displays only weak antagonist activity at hM 2 , hM 3 and hM 4 receptor…”

“…The discovery of AC-42 as a selective, allosteric agonist of the M 1 mAChR (Spalding et al, 2002;Langmead et al, 2006), has opened up the possibility of designing agonists that display true selectivity over M 2 -M 5 mAChRs. However, studies to date with this compound have been limited to recombinantly expressed mAChRs.…”

“…In this respect, Spalding et al (2002) described a novel compound, AC-42, which activated the human recombinant M 1 mAChR but did not possess any significant agonist activity at the hM 2 -hM 5 receptors. Functional studies using chimeric receptors indicated that the N-terminus-transmembrane (TM)1 and extracellular loop 3-TM7 regions of the M 1 receptor are required for selective activation by AC-42: regions that are distinct from the orthosteric site of the M 1 mAChR deeper in the TM bundle.…”

Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties

“…Also in agreement with our results, truncated l-opioid receptors remain functional. Interestingly, this latter work (Chaturvedi et al 2000), together with a study on chimeric m1/m5 muscarinic receptors (Spalding et al 2002), pointed to the role of the N-terminal tails of both the l-opioid and muscarinic M1 receptors as determinants of binding affinity or activation selectivity of 'atypical' agonists. Such a contribution of the M1 receptor N-tail to the composition of an 'ectopic' activation site (Spalding et al 2002) has not been detected in the present study, probably due to the use of classical muscarinic ligands.…”

Novel N-Substituted Benzimidazolones as Potent, Selective, CNS-Penetrant, and Orally Active M1 mAChR Agonists

“…Virtual screening of the corporate compound collection against the allosteric binding site 11,12 of M 1 mAChR using an M 1 mAChR homology model, which was built on the basis of the crystal structure of bovine rhodopsin, 20 yielded about 1000 putative hits. Evaluation of these compounds in our human M 1 fluorometric imaging plate reader (FLIPR) assay, which measures compound induced Ca 2þ mobilization in Chinese hamster ovary (CHO) cells that stably expressed human M 1 mAChR, led to the identification of compound 1 as an M 1 mAChR agonist with moderate potency 21 (Figure 1 It is worth noting that compound 1 was also identified as a hit via high throughput screening (HTS) of the corporate compound collection that was carried out later.…”