title: Synthetic FXR agonist GW4064 is a modulator of multiple G protein-coupled receptors.
abstract: The synthetic nuclear bile acid receptor (farnesoid X receptor [FXR]) agonist GW4064 is extensively used as a specific pharmacological tool to illustrate FXR functions. We noticed that GW4064 activated empty luciferase reporters in FXR-deficient HEK-293T cells. We postulated that this activity of GW4064 might be routed through as yet unknown cellular targets and undertook an unbiased exploratory approach to identify these targets. Investigations revealed that GW4064 activated cAMP and nuclear factor for activated T-cell response elements (CRE and NFAT-RE, respectively) present on these empty reporters. Whereas GW4064-induced NFAT-RE activation involved rapid intracellular Ca(2+) accumulation and NFAT nuclear translocation, CRE activation involved soluble adenylyl cyclase-dependent cAMP accumulation and Ca(2+)-calcineurin-dependent nuclear translocation of transducers of regulated CRE-binding protein 2. Use of dominant negative heterotrimeric G-protein minigenes revealed that GW4064 caused activation of Gαi/o and Gq/11 G proteins. Sequential pharmacological inhibitor-based screening and radioligand-binding studies revealed that GW4064 interacted with multiple G protein-coupled receptors. Functional studies demonstrated that GW4064 robustly activated H1 and H4 and inhibited H2 histamine receptor signaling events. We also found that MCF-7 breast cancer cells, reported to undergo GW4064-induced apoptosis in an FXR-dependent manner, did not express FXR, and the GW4064-mediated apoptosis, also apparent in HEK-293T cells, could be blocked by selective histamine receptor regulators. Taken together, our results demonstrate identification of histamine receptors as alternate targets for GW4064, which not only necessitates cautious interpretation of the biological functions attributed to FXR using GW4064 as a pharmacological tool but also provides a basis for the rational designing of new pharmacophores for histamine receptor modulation.
authors.affiliation: Division of Biochemistry (N.S., M.Y., A.K.S., H.K., J.S.M., A.G., S.S.), Division of Endocrinology and Center for Anabolic Skeletal Targets in Health and Illness (S.K.D.D., N.C.), Division of Pharmacology (A.M., P.N.Y., K.J.), and Division of Molecular and Structural Biology (S.C., M.I.S.), Council of Scientific and Industrial Research-Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Singh
authors.forename: Manisha
authors.initials: M
authors.lastname: Yadav
authors.forename: Abhishek Kumar
authors.initials: AK
authors.lastname: Singh
authors.forename: Harish
authors.initials: H
authors.lastname: Kumar
authors.forename: Shailendra Kumar Dhar
authors.initials: SK
authors.lastname: Dwivedi
authors.forename: Jay Sharan
authors.initials: JS
authors.lastname: Mishra
authors.forename: Anagha
authors.initials: A
authors.lastname: Gurjar
authors.forename: Amit
authors.initials: A
authors.lastname: Manhas
authors.forename: Sharat
authors.initials: S
authors.lastname: Chandra
authors.forename: Prem Narayan
authors.initials: PN
authors.lastname: Yadav
authors.forename: Kumaravelu
authors.initials: K
authors.lastname: Jagavelu
authors.forename: Mohammad Imran
authors.initials: MI
authors.lastname: Siddiqi
authors.forename: Arun Kumar
authors.initials: AK
authors.lastname: Trivedi
authors.forename: Naibedya
authors.initials: N
authors.lastname: Chattopadhyay
authors.forename: Sabyasachi
authors.initials: S
authors.lastname: Sanyal
mesh_terms: D021581:Active Transport, Cell Nucleus; D019703:Calcineurin / Q000378:metabolism; D020013:Calcium Signaling; D002470:Cell Survival / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D056945:Hep G2 Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007555:Isoxazoles / Q000494:pharmacology*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D061986:MCF-7 Cells; D050778:NFATC Transcription Factors / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D019703:Calcineurin; D000242:Cyclic AMP; D020962:Heterotrimeric GTP-Binding Proteins; D007555:Isoxazoles; D008156:Luciferases; D050778:NFATC Transcription Factors; D054801:Phosphoinositide Phospholipase C; D018160:Receptors, Cytoplasmic and Nuclear; D043562:Receptors, G-Protein-Coupled; D000099150:Receptor, Farnesoid X-Activated; C412815:GW 4064