Molecular Determinants for the Interaction of the Valvulopathic Anorexigen Norfenfluramine with the 5-HT<sub>2B</sub>Receptor

Setola, Vincent; Dukat, Małgorzata; Glennon, Richard A.; Roth, Bryan L.

doi:10.1124/mol.104.009266

Cited by 63 publications

(56 citation statements)

References 38 publications

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“…Although mutations in the Ci2 domain did not affect the The depicted model of the rat 5-HT1A receptor was based on the highresolution crystal structure of bovine rhodopsin in its inactive state and homology with previously published 5-HT receptor models (Shapiro et al, 2002;Setola et al, 2005; see Materials and Methods) and was obtained using Protein Explorer (PE, http://www.proteinexplorer. org) and molecular mechanics energy calculations.…”

Section: Discussionmentioning

confidence: 99%

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Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

et al. 2006

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This study provides the first comprehensive evidence that the second intracellular loop C-terminal domain (Ci2) is critical for receptor-G protein coupling to multiple responses. Although Ci2 is weakly conserved, its role in 5-hydroxytryptamine-1A (5-HT1A) receptor function was suggested by the selective loss of G␤␥-mediated signaling in the T149A-5-HT1A receptor mutant. More than 60 point mutant 5-HT1A receptors in the ␣-helical Ci2 sequence ( 143 DYVNKRTPRR 152 ) were generated. Most mutants retained agonist binding and were tested for G␤␥ signaling to adenylyl cyclase II or phospholipase C and G␣i coupling to detect constitutive and agonist-induced G i /G o coupling. Remarkably, most point mutations markedly attenuated 5-HT1A signaling, indicating that the entire Ci2 domain is critical for receptor G-protein coupling. Six signaling phenotypes were observed: wild-type-like, G␣ i -coupled/weak G␤␥-coupled, G␤␥-uncoupled, G␤␥-selective coupled, uncoupled, and inverse coupling. Our data elucidate specific roles of Ci2 residues consistent with predictions based on rhodopsin crystal structure. The absolute coupling requirement for lysine, arginine, and proline residues is consistent with a predicted amphipathic ␣-helical Ci2 domain that is kinked at Pro150. Polar residues (Thr149, Asn146) located in the externally oriented positively charged face were required for G␤␥ but not G␣ i coupling, suggesting a direct interface with G␤␥ subunits. The hydrophobic face includes the critical Tyr144 that directs the specificity of coupling to both G␤␥ and G␣ i pathways. The key coupling residues Tyr144/Lys147 (Ci2) are predicted to orient internally, forming hydrogen and ionic bonds with Asp133/ Arg134 (Ni2 DRY motif) and Glu340 (Ci3) to stabilize the Gprotein coupling domain. Thus, the 5-HT1A receptor Ci2 domain determines G␤␥ specificity and stabilizes G␣ i -mediated signaling.

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Section: Discussionmentioning

confidence: 99%

“…A preliminary model of the rat 5-HT1A receptor (inactive state) was prepared using bovine rhodopsin as a template using methods similar to those described previously (Shapiro et al, 2002;Setola et al, 2005). The full details of the model will be described in a subsequent publication.…”

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confidence: 99%

Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

et al. 2006

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“…3 H]d-lysergic acid diethylamide (ϳ1 nM final; PerkinElmer Life and Analytical Sciences, Waltham, MA) to label 5-HT 2B receptors and varying concentrations of unlabeled ligand (spanning at least 5 orders of magnitude) to compete for radioligand binding (Setola et al, 2005) and 2) calcium flux assays (see Calcium Flux Assay) using varying concentrations of 5-HT and BW 723C86 to measure 5-HT 2B receptor activation. Cells were passaged fewer than 10 times beyond passage 0 to ensure stable 5-HT 2B receptor expression.…”

Section: Methodsmentioning

confidence: 99%

“…Cells stably expressing human 5-HT 2B receptors were generated using the FlpIn system (Invitrogen Corp., Carlsbad, CA). In brief, a cDNA for the human 5-HT 2B receptor was amplified by polymerase chain reaction from pUSF-5-HT 2B (Setola et al, 2005) using 5Ј and 3Ј BamHI linkers, and then subcloned into the BamHI site of pcDNA5.0/Frt to produce pFlpIn-5-HT 2B . Next, subconfluent (60%) FlpIn HEK293 cells in 10-cm dishes were cotransfected with 3 g of pFlpIn-5-HT 2B and 3 g of pOG5 (bearing the Frt recombinase; Invitrogen Corp.) using 30 l of FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN).…”

Section: Methodsmentioning

confidence: 99%

Parallel Functional Activity Profiling Reveals Valvulopathogens Are Potent 5-Hydroxytryptamine_2BReceptor Agonists: Implications for Drug Safety Assessment

Huang

Setola²,

Yadav³

et al. 2009

Mol Pharmacol

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Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine 2B (5-HT 2B ) receptor agonists. We have shown that activation of 5-HT 2B receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT 2B receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT 2B receptor agonists (hits); 14 of these had previously been identified as 5-HT 2B receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twentythree of the hits were then "functionally profiled" (i.e., assayed in parallel for 5-HT 2B receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC 50 data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT 2B receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease.In 1997, the anorexigen fenfluramine was voluntarily withdrawn from the U.S. market because of its association with valvular heart disease (VHD) and pulmonary hypertension

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“…Benfluorex, like dexfenfluramine and fenfluramine, is metabolised into active metabolite norfenfluramine [1]. It is well accepted that norfenfluramine induces hypophagia through serotonin interaction [13]. Activation of 5HT serotonin receptors is also the mechanism proposed to be involved in cardiovascular side-effects, including valvular heart disease, as underlined by ROTHMAN et al [14].…”

Section: Discussionmentioning

confidence: 99%

Fenfluramine-like cardiovascular side-effects of benfluorex

Boutet¹,

Frachon²,

Jobic³

et al. 2009

Eur Respir J

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Since 1976, benfluorex has been approved in Europe as a hypolipidemic and hypoglycemic drug, and is commonly used in the treatment of the metabolic syndrome. As a derivative of fenfluramine with an appetite suppressant action, benfluorex is preferentially used in overweight patients. In contrast to fenfluramine and dexfenfluramine, to date, benfluorex has not been reported to be associated with frequent cardiovascular side-effects.The present study reports five cases of severe pulmonary arterial hypertension and one case of valvular heart disease occurring in patients exposed to benfluorex. These individuals were middle age, diabetic females with a body mass index ranging 24.2-49 kg?m -2 . No definite causal effect for cardiovascular disease with benfluorex can be drawn from such case reports. However, as benfluorex, like dexfenfluramine and fenfluramine, is metabolised into active metabolite norfenfluramine, further extensive assessment of drug exposure in newly diagnosed pulmonary arterial hypertension or valvular heart disease patients is warranted.

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Molecular Determinants for the Interaction of the Valvulopathic Anorexigen Norfenfluramine with the 5-HT_2BReceptor

Cited by 63 publications

References 38 publications

Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

Parallel Functional Activity Profiling Reveals Valvulopathogens Are Potent 5-Hydroxytryptamine_2BReceptor Agonists: Implications for Drug Safety Assessment

Fenfluramine-like cardiovascular side-effects of benfluorex

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Molecular Determinants for the Interaction of the Valvulopathic Anorexigen Norfenfluramine with the 5-HT2BReceptor

Cited by 63 publications

References 38 publications

Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

Molecular Determinants in the Second Intracellular Loop of the 5-Hydroxytryptamine-1A Receptor for G-Protein Coupling

Parallel Functional Activity Profiling Reveals Valvulopathogens Are Potent 5-Hydroxytryptamine2BReceptor Agonists: Implications for Drug Safety Assessment

Fenfluramine-like cardiovascular side-effects of benfluorex

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Resources

About

Molecular Determinants for the Interaction of the Valvulopathic Anorexigen Norfenfluramine with the 5-HT_2BReceptor

Parallel Functional Activity Profiling Reveals Valvulopathogens Are Potent 5-Hydroxytryptamine_2BReceptor Agonists: Implications for Drug Safety Assessment