Characterization of a Novel M<sub>1</sub> Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [<sup>3</sup>H]PT-1284

Smith, Deborah L.; Davoren, Jennifer E.; Edgerton, Jeremy R.; Lee, Che‐Wah; Neal, Sarah J.; Zhang, Lei; Grimwood, Sarah

doi:10.1124/mol.116.104737

Cited by 7 publications

(13 citation statements)

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“…PF-06764427 has been previously reported to be an M 1 selective PAM with activity in the a-LMA assay . In addition, PT-4186 (Compound 4), PT-8345 (Compound 7), PF-06764427, and PT-3214 (Compound 6) were shown to inhibit M 1 specific binding of [ 3 H]PT-1284 to allosteric binding sites on M 1 mAChR membranes with p K i values of 7.99, 7.28, 6.92, and 7.45, respectively, correlating well with FLIPR calcium mobilization EC 50 values …”

Section: Resultsmentioning

confidence: 94%

“…15 PF-06764427 has been previously reported to be an M 1 selective PAM with activity in the a-LMA assay. 15 In 25 FLIPR Potentiation Assay. FLIPR is commonly used to measure Gq-coupled GPCR functional activity associated with intracellular Ca 2+ release in stable cell lines.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Human M 1 , M 3 , and M 5 receptors stably expressed in Chinese hamster ovary (CHO) cells were obtained from Wyeth and HD Biosciences Co. Ltd. (Shanghai, China). Muscarinic receptor expression levels were determined using [ 3 H]- N -methylscopolamine binding, resulting in the following saturable binding capacity ( B max ) values: M 1 fluorometric imaging plate reader (FLIPR) assay, 384 fmol/mg of protein; M 1 IP 1 assay, 306.9 fmol/mg of protein; M 3 , 976.88 fmol/mg of protein; M 5 , not tested.…”

Section: Methodsmentioning

confidence: 99%

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Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

et al. 2016

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The rationale for using M selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M muscarinic acetylcholine receptor activation. Five brain penetrant M-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use.

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Section: Resultsmentioning

confidence: 94%

Section: ■ Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

et al. 2016

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“…Thus, high-affinity mGlu 2 NAMs may be better tools for further development of radiotracers due to their lack of affinity cooperativity with the endogenous ligand, glutamate. This may also apply more broadly to development of allosteric modulator radiotracers for other GPCRs since allosteric modulators of the class A receptors, M 1 , M 4 , and M 5 , all exhibit affinity cooperativity with the endogenous ligand and the binding of a recent M 1 PAM radioligand is dependent on agonist concentration (Bubser et al, 2014;Berizzi et al, 2016;Smith et al, 2016;Rook et al, 2017). The allosteric modulator mode of action may also be important for in vivo physiologic effects including adverse side effect profile.…”

Section: Discussionmentioning

confidence: 99%

“…6A). Previous studies of mGlu 2 and other GPCRs illustrated that the binding of such PAM radioligands is affected by concentration of an orthosteric agonist; however, whether the agonist affects the affinity and/or number of binding sites has not been consistent across radioligands even at mGlu 2 (Lavreysen et al, 2013;Doornbos et al, 2016;Smith et al, 2016). Thus, in further saturation binding studies, [ 3 H]-AZ12559322 affinity and number of binding sites were measured in the presence of different concentrations of the endogenous orthosteric agonist, glutamate (1-100 mM).…”

Section: Downloaded Frommentioning

confidence: 99%

Differential Pharmacology and Binding of mGlu₂ Receptor Allosteric Modulators

et al. 2018

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Allosteric modulation of metabotropic glutamate receptor 2 (mGlu) has demonstrated efficacy in preclinical rodent models of several brain disorders, leading to industry and academic drug discovery efforts. Although the pharmacology and binding sites of some mGlu allosteric modulators have been characterized previously, questions remain about the nature of the allosteric mechanism of cooperativity with glutamate and whether structurally diverse allosteric modulators bind in an identical manner to specific allosteric sites. To further investigate the in vitro pharmacology of mGlu allosteric modulators, we developed and characterized a novel mGlu positive allosteric modulator (PAM) radioligand in parallel with functional studies of a structurally diverse set of mGlu PAMs and negative allosteric modulators (NAMs). Using an operational model of allosterism to analyze the functional data, we found that PAMs affect both the affinity and efficacy of glutamate at mGlu, whereas NAMs predominantly affect the efficacy of glutamate in our assay system. More importantly, we found that binding of a novel mGlu PAM radioligand was inhibited by multiple structurally diverse PAMs and NAMs, indicating that they may bind to the mGlu allosteric site labeled with the novel mGlu PAM radioligand; however, further studies suggested that these allosteric modulators do not all interact with the radioligand in an identical manner. Together, these findings provide new insights into the binding sites and modes of efficacy of different structurally and functionally distinct mGlu allosteric modulators and suggest that different ligands either interact with distinct sites or adapt different binding poses to shared allosteric site(s).

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Molecular Imaging of Depressive Disorders

Ruhé

Frøkjær

Haarman

et al. 2020

PET and SPECT in Psychiatry

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This chapter summarizes findings of a large number of molecular imaging studies in the field of unipolar and bipolar depression (BD).Brain metabolism in depressed unipolar and bipolar patients is generally hypoactive in the middle frontal gyri, the pregenual and posterior anterior cingulate, the superior temporal gyrus, the insula, and the cerebellum, while hyperactivity exists in subcortical (caudate nucleus, thalamus), limbic (amygdala, anterior hippocampus), and medial and inferior frontal regions. Interestingly, after depletion of serotonin or noradrenalin/dopamine in vulnerable (recovered) major depressive disorder (MDD) patients, a similar response pattern in metabolism occurs.Findings on the pre-and postsynaptic dopaminergic system show indications that, at least in subgroups of retarded MDD patients, presynaptic dopaminergic markers may be decreased, while postsynaptic markers may be increased. The findings regarding serotonin synthesis, pre-and postsynaptic imaging can be integrated to a presumable loss of serotonin in MDD, while this remains unclear in BD. This reduction of serotonin and dopamine in MDD was recently summarized in a revised version of the monoamine hypothesis, which focuses more on a dysfunction at the level of the MAO enzyme. This should be addressed further in future studies. Nevertheless, it should be acknowledged that the serotonergic and dopaminergic systems appear adaptive; therefore, it remains difficult to distinguish state and trait abnormalities. Therefore, future longitudinal molecular imaging studies in the same subjects at different clinical mood states (preferably with different tracers and imaging modalities) are needed to clarify whether the observed changes in transporters and receptors are compensatory reactions or reflect different, potentially causal mechanisms. Several suggestions for future developments are also provided at the end of this chapter.

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Characterization of a Novel M₁ Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [³H]PT-1284

Cited by 7 publications

References 36 publications

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

Differential Pharmacology and Binding of mGlu₂ Receptor Allosteric Modulators

Molecular Imaging of Depressive Disorders

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Characterization of a Novel M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [3H]PT-1284

Cited by 7 publications

References 36 publications

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M1 Receptor Activation

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M1 Receptor Activation

Differential Pharmacology and Binding of mGlu2 Receptor Allosteric Modulators

Molecular Imaging of Depressive Disorders

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Characterization of a Novel M₁ Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [³H]PT-1284

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

Differential Pharmacology and Binding of mGlu₂ Receptor Allosteric Modulators