Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity
To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1-M5) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems (Langmead et al. 2008). Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia (Felder et al. more ..
To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1-M5) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems (Langmead et al. 2008). Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia (Felder et al. 2000; Clader and Wang 2005; Wess et al. 2007; Langmead et al. 2008). For example, clinical trials with xanomeline, a M1/M4-preferring orthosteric agonist, demonstrated efficacy as both a cognition-enhancing agent and an antipsychotic agent (Bodick et al. 1997a; Bodick et al. 1997b; Shekhar et al. 2008). However, a long standing question concerns whether or not the antipsychotic efficacy or antipsychotic-like activity in animal models is mediated by activation of M1, M4, or a combination of both receptors. Data from mAChR knockout (KO) mice led to the suggestion that a selective M1 agonist would be beneficial for cognition, whereas an M4 agonist would provide antipsychotic activity for the treatment of schizophrenia (Gomeza et al. 1999; Gomeza et al. 2001; Anagnostaras et al. 2003; Tzavara et al. 2004). This proposal is further supported by recent studies demonstrating that M4 receptors modulate the dynamics of cholinergic and dopaminergic neurotransmission and that loss of M4 function results in a state of dopamine hyperfunction (Tzavara et al. 2004). These data, coupled with findings that schizophrenic patients have altered hippocampal M4 but not M1 receptor expression (Raedler et al. 2007), suggest that selective activators of M4 may provide a novel treatment strategy for schizophrenia patients.
Unfortunately, xanomeline lacks true M1/M4 specificity and also has significant affinity and efficacy at M2 and M3 mAChRs. Thus xanomeline, like many other cholinergic agents including acetylcholinesterase (AChE) inhibitors, displays significant adverse effects including bradycardia, GI distress, excessive salivation, and sweating, which are thought to be primarily due to activation of peripheral M2 and M3 mAChRs (Bymaster et al. 2003; Wess et al. 2007; Langmead et al. 2008). Due to the high sequence homology and conservation of the orthosteric ACh binding site among the mAChR subtypes, development of chemical agents that are selective for a single mAChR subtype has been largely unsuccessful, and in the absence of highly selective activators of M4, it has been impossible to test the role of selective M4 activation. However, in recent years, we have been highly successful in the identification of multiple highly selective allosteric activators for M1 (Bridges et al. 2008; Jones et al. 2008; Ma et al. 2009; Marlo et al. 2009; Shirey et al. 2009; Bridges et al. 2010) and M4 (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). Though we are encouraged by these results, unfortunately, highly selective centrally penetrant activators of either M1 or M4 remain unavailable, making it impossible to determine the in vivo effects of selective activation of these receptors. Future compounds developed should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress M4 biology, which will undoubtedly allow the intense study of M4 activation in multiple areas of neuroscience.
Potency and efficacy of compounds will be determined by performing concentration-response curves (CRCs, 10 points, ranging from approximately 30 uM-1 nM at 0.3% final DMSO concentration) at human M4 using a calcium assay in which the cells also express the chimeric G protein Gqi5 to couple M4 to calcium mobilization (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). PAMs with EC50 values < 1 uM versus human M4 will next be evaluated for potency versus rat M4 in an analogous calcium assay utilizing Gqi5. Following potency evaluation at human and rat M4, PAMs with EC50 values less than 1 uM at rat M4 will be evaluated for their ability to left-shift the CRC of acetylcholine (ACh) for rat M4 in a calcium assay and will be examined for their selectivity for M4 relative to other mAChR subtypes (Brady et al. 2008; Shirey et al. 2008; Kennedy et al. 2009). PAMs with a fold-shift of the ACh CRC of > 5 will then be evaluated for Tier 1 DMPK assays including plasma protein binding (PPB), intrinsic clearance (Obach 1997; Obach 1999), and inhibition of cytochrome p450 enzymes (CYP) (Youdim et al. 2008; Engers et al. 2009; Lebois et al. 2011; Jones et al. 2012). Next, compounds demonstrating PPB > 0.01 fraction unbound (Fu), a clean CYP profile, and moderate clearance will be evaluated for CNS exposure and Plasma:Brain levels using an in vivo snapshot PK paradigm (Frick et al. 1998; Engers et al. 2009; Lebois et al. 2011; Jones et al. 2012). Novel M4 PAMs showing a CNS exposure > PAM EC50, a Brain:Plasma ratio of >0.5, and at least a 10-fold or greater selectivity versus other mAChR subtypes will next be evaluated in a preclinical model of antipsychotic drug action: amphetamine-induced hyperlocomotion (AHL) (Brady et al. 2008; Jones et al. 2008). M4 PAMs demonstrating activity in AHL will have their ancillary pharmacology fully evaluated and those compounds that show no significant off-target activity and suitable solubility will be declared an MLPCN probe. The ultimate goal of this project from the PI's perspective is to generate compounds which should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress M4 biology.
1. Anagnostaras, S. G., G. G. Murphy, et al. (2003). "Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice." Nat Neurosci 6(1): 51-8.
2. Bodick, N. C., W. W. Offen, et al. (1997a). "Effects of xanomeline, a selective muscarinic receptor agonist, on cognitive function and behavioral symptoms in Alzheimer disease." Arch Neurol 54(4): 465-73.
3. Bodick, N. C., W. W. Offen, et al. (1997b). "The selective muscarinic agonist xanomeline improves both the cognitive deficits and behavioral symptoms of Alzheimer disease." Alzheimer Dis Assoc Disord 11 Suppl 4: S16-22.
4. Brady, A. E., C. K. Jones, et al. (2008). "Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats." J Pharmacol Exp Ther 327(3): 941-53.
5. Bridges, T. M., A. E. Brady, et al. (2008). "Synthesis and SAR of analogues of the M1 allosteric agonist TBPB. Part I: Exploration of alternative benzyl and privileged structure moieties." Bioorg Med Chem Lett 18(20): 5439-42.
6. Bridges, T. M., J. Phillip Kennedy, et al. (2010). "Chemical lead optimization of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) lead. Part II: development of a potent and highly selective M1 PAM." Bioorg Med Chem Lett 20(6): 1972-5.
7. Bymaster, F. P., P. A. Carter, et al. (2003). "Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis, and pilocarpine-induced seizure activity." Eur J Neurosci 17(7): 1403-10.
8. Clader, J. W. and Y. Wang (2005). "Muscarinic receptor agonists and antagonists in the treatment of Alzheimer's disease." Curr Pharm Des 11(26): 3353-61.
9. Engers, D. W., C. M. Niswender, et al. (2009). "Synthesis and evaluation of a series of heterobiarylamides that are centrally penetrant metabotropic glutamate receptor 4 (mGluR4) positive allosteric modulators (PAMs)." J Med Chem 52(14): 4115-8.
10. Felder, C. C., F. P. Bymaster, et al. (2000). "Therapeutic opportunities for muscarinic receptors in the central nervous system." J Med Chem 43(23): 4333-53.
11. Frick, L. W., K. K. Adkison, et al. (1998). "Cassette dosing: rapid in vivo assessment of pharmacokinetics." Pharmaceutical Science & Technology Today 1(1): 12-18.
12. Gomeza, J., L. Zhang, et al. (1999). "Enhancement of D1 dopamine receptor-mediated locomotor stimulation in M(4) muscarinic acetylcholine receptor knockout mice." Proc Natl Acad Sci U S A 96(18): 10483-8.
13. Gomeza, J., L. Zhang, et al. (2001). "Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice." Life Sci 68(22-23): 2457-66.
14. Jones, C. K., A. E. Brady, et al. (2008). "Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats." J Neurosci 28(41): 10422-33.
15. Jones, C. K., M. Bubser, et al. (2012). "The metabotropic glutamate receptor 4-positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine 2A antagonist in preclinical rodent models of Parkinson's disease." J Pharmacol Exp Ther 340(2): 404-21.
16. Kennedy, J. P., T. M. Bridges, et al. (2009). "Synthesis and structure-activity relationships of allosteric potentiators of the m(4) muscarinic acetylcholine receptor." ChemMedChem 4(10): 1600-7.
17. Langmead, C. J., J. Watson, et al. (2008). "Muscarinic acetylcholine receptors as CNS drug targets." Pharmacol Ther 117(2): 232-43.
18. Lebois, E. P., G. J. Digby, et al. (2011). "Development of a highly selective, orally bioavailable and CNS penetrant M1 agonist derived from the MLPCN probe ML071." Bioorg Med Chem Lett 21(21): 6451-5.
19. Ma, L., M. A. Seager, et al. (2009). "Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation." Proc Natl Acad Sci U S A 106(37): 15950-5.
20. Marlo, J. E., C. M. Niswender, et al. (2009). "Discovery and characterization of novel allosteric potentiators of M1 muscarinic receptors reveals multiple modes of activity." Mol Pharmacol 75(3): 577-88.
21. Obach, R. S. (1997). "Nonspecific binding to microsomes: impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of warfarin, imipramine, and propranolol." Drug Metab Dispos 25(12): 1359-69.
22. Obach, R. S. (1999). "Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes." Drug Metab Dispos 27(11): 1350-9.
23. Raedler, T. J., F. P. Bymaster, et al. (2007). "Towards a muscarinic hypothesis of schizophrenia." Mol Psychiatry 12(3): 232-46.
24. Shekhar, A., W. Z. Potter, et al. (2008). "Selective muscarinic receptor agonist xanomeline as a novel treatment approach for schizophrenia." Am J Psychiatry 165(8): 1033-9.
25. Shirey, J. K., A. E. Brady, et al. (2009). "A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning." J Neurosci 29(45): 14271-86.
26. Shirey, J. K., Z. Xiang, et al. (2008). "An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission." Nat Chem Biol 4(1): 42-50.
27. Tzavara, E. T., F. P. Bymaster, et al. (2004). "M4 muscarinic receptors regulate the dynamics of cholinergic and dopaminergic neurotransmission: relevance to the pathophysiology and treatment of related CNS pathologies." FASEB J 18(12): 1410-2.
28. Wess, J., R. M. Eglen, et al. (2007). "Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development." Nat Rev Drug Discov 6(9): 721-33.
29. Youdim, K. A., R. Lyons, et al. (2008). "An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point." J Pharm Biomed Anal 48(1): 92-9.
Categorized Comment - additional comments and annotations
Assay Test Type: In vitro
From MLP Probe Report:
Probe count: 3
MLP Probe ML# for probe 1: ML108
PubChem Substance ID (SID) for probe 1: 85163688
PubChem Compound ID (CID) for probe 1: 864492
Probe type for probe 1: Modulator
IC50/EC50 (nM) for probe 1: 370
Target for probe 1: M4 (gi: 52426748)
Disease relevance for probe 1: Neurodegenerative disorders
Anti-target for probe 1: M1, M2, M3, M5, PanLabs
Fold selectivity for probe 1: >100
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK50687/ (ID: 2376420)
Grant number for probe 1: MH077607-01
MLP Probe ML# for probe 2: ML173
PubChem Substance ID (SID) for probe 2: 92392533
PubChem Compound ID (CID) for probe 2: 45142486
Probe type for probe 2: Agonist
IC50/EC50 (nM) for probe 2: <2440
Target for probe 2: rM4; hM4 (gi: 52426748)
Disease relevance for probe 2: Alzheimer's
Anti-target for probe 2: M1, M2, M3, M5
Fold selectivity for probe 2: >100
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK143196/ (ID: 3033902)
Grant number for probe 2: MH077607-01
MLP Probe ML# for probe 3: ML293
PubChem Substance ID (SID) for probe 3: 134220214
PubChem Compound ID (CID) for probe 3: 56592940
Probe type for probe 3: PAM
IC50/EC50 (nM) for probe 3: 1030
Target for probe 3: M4 (gi: 52426748)
Disease relevance for probe 3: Alzheimer's disease
Anti-target for probe 3: hM1-3; hM5
Fold selectivity for probe 3: >20
NCBI Book chapter link for probe 3: http://www.ncbi.nlm.nih.gov/books/NBK143538/ (ID: 3036596)
Grant number for probe 3: MH077607-01
PubMed Publication ID (PMID) for probe 1: 18772318
PubMed Publication ID (PMID) for probe 3: 22738637
NCBI Book chapter title for probe 1: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM)
NCBI Book chapter title for probe 2: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activity
NCBI Book chapter title for probe 3: Discovery of a novel structural class of M4 positive allosteric modulators: Characterization of ML293, N-(4-methoxy-7-methylbenzo[d]thiazol-2-yl)isonicotinamide, with CNS exposure in rats