Ethan S. Burstein

The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine

RationaleClozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described.Objectives and methodsTo comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay.ResultsProfiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone.ConclusionsThe muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.

The Second Intracellular Loop of the m5 Muscarinic Receptor Is the Switch Which Enables G-protein Coupling

We have completed a systematic search of the intracellular loops of a muscarinic acetylcholine receptor for domains that govern G-protein coupling. A unique feature of the second intracellular (i2) loop was an ordered cluster of residues where diverse substitutions cause constitutive activation. A second group of residues in i2 was identified where mutations compromised receptor/G-protein coupling. The residues of each group alternate and are spaced three to four positions apart, suggesting an α-helical structure where these groups form opposing faces of the helix. We propose that the constitutively activating face normally constrains the receptor in the “off-state,” while the other face couples G-proteins in the “on-state.” Therefore, the i2 loop functions as the switch enabling G-protein activation.

Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H3 receptor

The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinct receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known H(3) agonists, while all agonists tested displayed increased potency at isoform 2 relative to isoform 1. Histamine, N(alpha)-methylhistamine, and R(-) and S(+)-alpha-methylhistamine are 16-23-fold more potent, while immepip and imetit are three to fivefold more potent. Antagonist experiments revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor activity, and identified a small number of antipsychotics that possess significant antagonist activity.

Pharmacological and Behavioral Profile of N-(4-Fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl) Carbamide (2R,3R)-Dihydroxybutanedioate (2:1) (ACP-103), a Novel 5-Hydroxytryptamine2A Receptor Inverse Agonist

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)2A receptor inverse agonist ACP-103 competitively antagonized the binding of [3H]ketanserin to heterologously expressed human 5-HT2A receptors with a mean pKi of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC50 of 8.7. ACP-103 demonstrated lesser affinity (mean pKi of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC50 7.1 in R-SAT) at human 5-HT2C receptors, and lacked affinity and functional activity at 5-HT2B receptors, dopamine D2 receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT2A receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT2A receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.

Trazodone kinetics: Effect of age, gender, and obesity

Single 25 mg intravenous and 50 mg oral doses of trazodone were given to 43 healthy subjects, divided into young men and women (aged 18 to 40 years) and elderly men and women (aged 60 to 76 years). Among men, trazodone volume of distribution (Varea) was increased in elderly vs. young subjects (1.15 vs. 0.89 L/kg; P < 0.05), and clearance decreased (1.65 vs. 2.31 ml/min/kg; P < 0.05), thereby increasing elimination half‐life (t½) in elderly men (8.2 vs. 4.7 hours; P < 0.001). Varea in women was also increased in the elderly (1.5 vs. 1.27 L/kg; P < 0.02), causing increased t½ (7.6 vs. 5.9 hours; P < 0.05), but clearance was unrelated to age. Absolute bioavailability of oral trazodone averaged 70% to 90% and was unrelated to age or sex. In 23 obese subjects (mean weight 112 kg) vs. 23 matched control subjects of normal weight (mean 65 kg), Varea was greatly increased (162 vs. 67 L; 1.43 vs. 1.04 L/kg; P < 0.001) and was highly correlated with body weight (r = 0.91). Clearance was unchanged between groups (146 vs. 136 ml/min), but the increased Varea caused prolonged t½ in obese subjects (13.3 vs. 5.9 hours; P < 0.001). Reduced clearance of trazodone among elderly men may indicate a need for dosage reduction during chronic therapy. In obese individuals, choice of dosage during chronic treatment should be based on ideal rather than total body weight.

Structural requirements of transmembrane domain 3 for activation by the M1 muscarinic receptor agonists AC-42, AC-260584, clozapine, and N-desmethylclozapine : Evidence for three distinct modes of receptor activation

Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M1 muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M1. In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M1 but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M1 receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.

Functional importance of the Ala(116)-Pro(136) region in the calcium-sensing receptor. Constitutive activity and inverse agonism in a family C G-protein-coupled receptor.

The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca2+ have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala116–Pro136 region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening assay. Selected mutants were characterized further in an inositol phosphate assay. The vast majority of the mutants tested displayed an increased affinity for Ca2+. Furthermore, 21 of the mutants showed increased basal activity in the absence of agonist. This constitutive activity was not diminished when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala116–Pro136 region is of key importance for the maintenance of the inactive conformation of CaR.

Structure of a G-protein-coupling Domain of a Muscarinic Receptor Predicted by Random Saturation Mutagenesis

The third intracellular loop (i3) plays a critical role in the coupling of many receptors to G-proteins. In muscarinic receptor subtypes, the N- and C-terminal regions (Ni3 and Ci3) of this loop are sufficient to direct appropriate G-protein coupling. The relative functional contributions of all amino acids within Ni3 was evaluated by constructing libraries of m5 muscarinic receptors containing random mutations in Ni3 and screening them using high throughput assays based on ligand-dependent transformation of NIH 3T3 cells. In receptors that retained a wild type phenotype, the pattern of functionally tolerated substitutions is consistent with the presence of three turns of an α helix extending from the transmembrane domain. All of the amino acid positions that tolerate radical substitutions face away from a conserved hydrophobic face that ends with an arginine, and helix-disrupting proline substitutions were not observed. All of the mutant receptors with significantly compromised phenotypes had amino acid substitutions in residues predicted to form the hydrophobic face. Similar data from the Ci3 region (Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R.(1995) J. Biol. Chem. 270, 3141-3146) are consistent with the presence of a single helical turn extending from the transmembrane domain, with an alanine that defines G-protein affinity. Functionally critical residues of Ni3 and Ci3 are predicted to be in close proximity where they form the G-protein-coupling domain.

AC-260584, an orally bioavailable M1 muscarinic receptor allosteric agonist, improves cognitive performance in an animal model

The recent discovery of allosteric potentiators and agonists of the muscarinic M(1) receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC(50) 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M(1) receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M(1) receptor over the M(2), M(3), M(4) and M(5) muscarinic receptor subtypes. Using GTPgammaS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M(1) receptor activation since it was not observed in M(1) knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M(1) receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M(1) selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimers disease.

Discovery of the first nonpeptide agonist of the GPR14/urotensin-II receptor: 3-(4-chlorophenyl)-3-(2- (dimethylamino)ethyl)isochroman-1-one (AC-7954).

A functional cell-based screen identified 3-(4-chlorophenyl)-3-(2-(dimethylamino)ethyl)isochroman-1-one hydrochloride (AC-7954, 1) as a nonpeptidic agonist of the urotensin-II receptor. Racemic 1 had an EC50 of 300 nM at the human UII receptor and was highly selective. Testing of the enantiopure (+)- and (-)- 1 revealed that the UII receptor activity of racemic 1 resides primarily in (+)-1. Being a selective nonpeptidic druglike UII receptor agonist, (+)-1 will be useful as a pharmacological research tool and a potential drug lead.