Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Probe Summary
Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels, and Transporters, David Weaver ..more
Depositor Specified Assays
Assay Provider: P. Jeffery Conn
Assay Provider Affiliation: Vanderbilt University
Grant Title: Discovery of novel allosteric modulators of the M1 muscarinic receptor
Grant Number: 1 R03 MH077606-01
Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels, and Transporters, David Weaver
Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig Lindsley
The M1 muscarinic receptor is thought to be an important therapeutic target. Agents that enhance cholinergic transmission or activate muscarinic acetylcholine receptors (mAChRs) have been developed to ameliorate the loss of cognitive function in patients with Alzheimer's Disease (AD). While cholinergic agents have been partially successful in improving cognitive function in AD patients, the most exciting findings coming from clinical studies with these agents have been the demonstration of efficacy in reducing psychotic symptoms in patients with AD and other neurodegenerative disorders. Interestingly, the M1/M4 preferring mAChR agonist, xanomeline, also induces a robust antipsychotic effect in schizophrenic patients, suggesting that mAChR agonists may have broad utility in reducing psychotic symptoms in patients suffering from schizophrenia and certain neurodegenerative disorders. Evidence suggests that the antipsychotic effects of cholinergic agents may be mediated by the M1 mAChR subtype. However, previous compounds developed to selectively activate M1 receptors have failed in clinical development due to a lack of true specificity for M1 and adverse effects associated with activation of other mAChR subtypes. Furthermore, the lack of highly selective compounds has made it impossible to definitively determine whether the behavioral and clinical effects of these compounds are mediated by M1 and the M4 receptor subtype is also a viable candidate for mediating the antipsychotic effects. Previous attempts to develop compounds that are highly selective for M1 or other specific mAChR subtypes have failed because of the high conservation of the Ach binding site and difficulty in developing truly specific compounds (Bonner et al. 1987, 1988; Felder et al. 2000; Bymaster et al. 2003). The lack of highly selective compounds has made it impossible to definitively determine the behavioral and clinical effects of these receptors. In numerous Phase II and III clinical trails, pan-mAChR agonists were shown to improve cognitive performance in AD patients, but the GI-and/or cardiovascular side effects, resulting from activation of peripheral mAChRs, were deemed intolerable and the trials were discontinued (Eglen et al. 2001). Importantly, several pan-mAChR agonists demonstrated decline of A-beta-42 in the cerebral spinal fluid of AD patients, suggesting that mAChR activation has the potential to be disease modifying as well as providing palliative cognitive therapy (Bodick et al. 1997). More recent studies in 3xTg-AD mice further support a disease modifying role for mAChR activation, and several Ph III trials demonstrated that mAChR activation lowered AB42 in patients (Caccamo et al. 2006). Interestingly, the M1/M4 preferring xanomeline, in addition to improving cognitive performance, had robust therapeutic effects on the psychotic symptoms and behavioral disturbances associated with AD and recently published clinical trial data indicates efficacy in schizophrenic patients (Bodick et al. 1997; Shekhar et al. 2008). Probes developed from these efforts will greatly advance the current state of the art by aiding in the understanding of M1's role in cell-based physiology and may extend the clinical understanding of psychotic and cognitive symptoms associated with neurodegenerative disorders like Alzheimer's Disease and schizophrenia. A cell-based fluorometric calcium assay was developed for high throughput screening. This assay was used to identify compounds with high selectivity for the M1 receptor subtype that act at an allosteric site on the receptor, thus providing increased specificity for this single receptor subtype. It is anticipated that these compounds will provide important tools for the study of muscarinic receptor function in the CNS.
For the discovery of novel allosteric agonists of the M1 muscarinic receptor, we completed the primary screen (AID 626) using a real-time cell-based assay against the full 65K library. The assay performed very well (Z' averaged 0.7) and we identified approximately 2179 putative agonist primary hits. The compounds clustered nicely into different structural classes with multiple representative analogs in each cluster. The agonist compounds were chosen as the focus of this probe effort, but we previously filed a probe report for the first truly selective small molecule M1 antagonist, SID 56373925. For the confirmation screen, 1665 agonist hits were reordered from Biofocus-DPI. The compounds were tested in duplicate against M1/CHO cells (AID 1488), and in parallel, counter screened against M4/Gqi5 CHO cells (AID 1741). Compounds showing initial selectivity for M1 over M4 were tested in triplicate in a 10 point dose-response series against the muscarinic panel (M1-M5). Compounds showing selectivity for M1 were then tested as a concentration series in the presence of atropine. Pubchem CID 644390 and CID 647412 were selective (>50-fold) for M1 vs. M4 with M1 EC50s from the DPI DMSO stock of ~ 1 uM and sensitive to block by atropine.
We then resynthesized the two HTS leads to confirm structure and activity. The resynthesized CID 644390 and CID 647412 confirmed, with M1 EC50s of 804 nM and 1.74 uM, respectively (AID 1744). Importantly, both compounds proved to be highly selective versus M2-M5, affording no activation at concentrations exceeding 50 uM. We employed an iterative parallel synthesis approach for the optimization of CID 644390 and CID 647412, as the scaffolds were modular and readily amenable to this approach. CIDs 25010774 and 25010775 resulted which were completely selective versus M2-M5 (AID 1744, EC50s>50uM) and possessed submicromolar potencies (152+8.4 nM and 198+13.2 nM, respectively).
To confirm that these compounds are indeed allosteric agonists, we evaluated them on a Y381A mutant M1 cell line (AID 1743). Allosteric agonists of M1 can be differentiated from orthosteric agonists by their ability to activate the receptor in which there is a single point mutation (Y381A) in the orthosteric binding site that renders the receptor insensitive to acetylcholine or orthosteric agonists. The Y381A mutation causes three order of magnitude right-shift in the ACh CRC and TBPB, the prototypical M1 allosteric agonist, retains some efficacy on this line. In contrast, the initial HTS leads (CID 644390 and CID 647412) and the two probe candidates (CID 25010774 and CID 25010775) remain fully efficacious on this mutant line. In fact, the EC50s for CID 25010774 and CID 25010775 shift less than 2-fold (Y381A M1 EC50s = 304+56 nM and 379+91 nM, respectively). These data, coupled with competition binding studies with [3H]-NMS (AID 1470 (M1), 1767 (M2), 1764 (M3), 1757 (M4), 1508 (M5)), clearly indicate that these new M1 agonists activate the receptor through binding at an allosteric site.
The Lead Profiling Screen (68 GPCRs, ion channels and transporters) from MDS Pharma (AID 1788) was performed. CID 25010775 (SID 56353039) possessed superior ancillary profile with only three activities >50% at 10 uM, and was thus declared an MLPCN probe.
CID 25010775 has the following IUPAC nomenclature: ethyl 4-(2-methylbenzamido)ethylamino) piperidine-1-carboxylate.
Finally, a brain/plasma study was conducted to determine if our M1 allosteric agonist probe (CID 25010775) was centrally penetrant after systemic dosing. A brain/plasma ratio of 4.2 was observed, with the compound preferentially portioning into the brain with levels in the uM range for up to 5 hours post dose - >8-fold above the EC50 for M1 activation. Importantly, the animals were closely monitored, and were healthy, with no signs of classical pan-mAChR activation (SLUD : salivation, lacramation, urination and defecation) indicating that the in vitro mAChR selectivity profile was mirrored in vivo. Thus, CID 25010775 has utility as both an in vitro and in vivo probe for selective M1 activation by an allosteric mechanism.
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Shekhar, A.; Potter, W.Z.; Lightfoot, J.; Lienemann, J.; Dube, S.; Mallinckrodt, C.; Bymaster, F.P.; McKinzie, D.L.; Felder, C.C. Selective muscarinic receptor agonist xanomeline as a novel treatment approach for schizophrenia Am. J. Psychiatry 2008, 165, 1033-1039.
Data Table (Concise)