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Dive into the research topics where Tracy A. Spalding is active.

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Featured researches published by Tracy A. Spalding.


Psychopharmacology | 2004

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

David M. Weiner; Herbert Y. Meltzer; Isaac Veinbergs; E. M. Donohue; Tracy A. Spalding; T. T. Smith; N. Mohell; S. C. Harvey; Jelveh Lameh; Norman Nash; Kimberly E. Vanover; Roger Olsson; Karuna Jayathilake; Myung A. Lee; A. I. Levey; Uli Hacksell; Ethan S. Burstein; Robert E. Davis; Mark R. Brann

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.


Journal of Biological Chemistry | 1998

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

Ethan S. Burstein; Tracy A. Spalding; Mark R. Brann

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 Pharmacology | 2006

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

Tracy A. Spalding; Jian-Nong Ma; Thomas R. Ott; Mikael Friberg; Abhishek Bajpai; Stefania Risso Bradley; Robert E. Davis; Mark R. Brann; Ethan S. Burstein

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.


Journal of Biological Chemistry | 2000

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.

Anders A. Jensen; Tracy A. Spalding; Ethan S. Burstein; Paul O. Sheppard; Patrick J. O'Hara; Mark R. Brann; Povl Krogsgaard-Larsen; Hans Bräuner-Osborne

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.


Journal of Biological Chemistry | 1996

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

David C. Hill-Eubanks; Ethan S. Burstein; Tracy A. Spalding; Hans Bräuner-Osborne; Mark R. Brann

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.


Neuropharmacology | 2010

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

Stefania Risso Bradley; Jelveh Lameh; Linda Ohrmund; Thomas Son; Abhishek Bajpai; Derek Nguyen; Mikael Friberg; Ethan S. Burstein; Tracy A. Spalding; Thomas R. Ott; Hans H. Schiffer; Ali Tabatabaei; Krista McFarland; Robert E. Davis; Douglas W. Bonhaus

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.


ACS Chemical Neuroscience | 2013

Low Dose Bexarotene Treatment Rescues Dopamine Neurons and Restores Behavioral Function in Models of Parkinson's Disease

Krista McFarland; Tracy A. Spalding; David Hubbard; Jian-Nong Ma; Roger Olsson; Ethan S. Burstein

Nurr1 is a nuclear hormone receptor (NucHR) strongly implicated in the growth, maintenance, and survival of dopaminergic neurons. Nurr1 may be unable to bind ligands directly, but it forms heterodimers with other NucHRs that do. Using bioluminescence resonance energy transfer (BRET) assays to directly monitor interactions of Nurr1 with other NucHRs, we found the cancer drug bexarotene (Targretin, also LGD1069) displayed biased interactions with Nurr1-RXR heterodimers compared with RXR-RXR homodimers. Remarkably, at doses up to 100-fold lower than those effective in rodent cancer models, bexarotene rescued dopamine neurons and reversed behavioral deficits in 6-hydroxydopamine (6-OHDA) lesioned rats. Compared to the high doses used in cancer therapy, low doses of bexarotene have significantly milder side effects including a reduced increase in plasma triglycerides and less suppression of thyroid function. On the basis of extrapolations from rat to human doses, we hypothesize that low oral doses of bexarotene may provide an effective and tolerated therapy for Parkinsons disease (PD).


Current Pharmaceutical Design | 2006

Integrative Functional Assays, Chemical Genomics and High Throughput Screening: Harnessing Signal Transduction Pathways to a Common HTS Readout

Ethan S. Burstein; Fabrice Piu; Jian-Nong Ma; Jacques Weissman; Erika A. Currier; Norman Nash; David M. Weiner; Tracy A. Spalding; Hans H. Schiffer; Andria L. Del Tredici; Mark R. Brann

Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.


Life Sciences | 2001

Constitutively active muscarinic receptors.

Tracy A. Spalding; Ethan S. Burstein

Mutations that increase constitutive activity and alter ligand binding have been used to investigate the structure and mechanism of activation of muscarinic receptors. These data are reviewed with reference to the recently published three-dimensional structure of rhodopsin. Residues in TM3 and TM6 where amino acid substitutions increased constitutive activity align with residues within the core of the receptor. A nucleus of these residues is located immediately below the predicted binding site of acetylcholine. The i2 loop where mutations also increase constitutive activity was found to loop away from the i3 loop, which has been found to modulate G-protein coupling specificity.


Life Sciences | 2001

Exploring the potential for subtype-selective muscarinic agonists in glaucoma

Daniel W. Gil; Tracy A. Spalding; Alex Kharlamb; Niels Skjaerbaek; Allan K. Uldam; Carol Trotter; Donghui Li; Elizabeth Woldemussie; Larry A. Wheeler; Mark R. Brann

Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.

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Ethan S. Burstein

ACADIA Pharmaceuticals Inc.

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Erika A. Currier

ACADIA Pharmaceuticals Inc.

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Jian-Nong Ma

ACADIA Pharmaceuticals Inc.

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David M. Weiner

ACADIA Pharmaceuticals Inc.

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Niels Skjaerbaek

ACADIA Pharmaceuticals Inc.

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Norman Nash

ACADIA Pharmaceuticals Inc.

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Mikael Friberg

ACADIA Pharmaceuticals Inc.

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Fabrice Piu

ACADIA Pharmaceuticals Inc.

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Jacques Weissman

ACADIA Pharmaceuticals Inc.

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