Kinetics of 5-HT2B receptor signaling: profound agonist-dependent effects on signaling onset and duration.
The kinetics of drug-receptor interactions can profoundly influence in vivo and in vitro pharmacology. In vitro, the potencies of slowly associating agonists may be underestimated in assays capturing transient signaling events. When divergent receptor-mediated signaling pathways are evaluated using combinations of equilibrium and transient assays, potency differences driven by kinetics may be erroneously interpreted as biased signaling. In vivo, drugs with slow dissociation rates may display prolonged physiologic effects inconsistent with their pharmacokinetic profiles. We evaluated a panel of 5-hydroxytryptamine2B (5-HT2B) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Gαq-mediated activation of phospholipase C. In binding studies, ergot derivatives demonstrated slow receptor association and dissociation rates, resulting in significantly reduced potency in calcium assays relative to inositol phosphate accumulation assays. Ergot potencies for activation of extracellular signal-regulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptor-dissociation kinetics. Mechanistic studies indicated that persistent signaling originated from internalized or sequestered receptors. This study provides a mechanistic basis for the long durations of action in vivo and wash-resistant effects in ex vivo tissue models often observed for ergots. The 5-HT2B agonist activity of a number of ergot-derived therapeutics has been implicated in development of cardiac valvulopathy in man. The novel, sustained nature of ergot signaling reported here may represent an additional mechanism contributing to the valvulopathic potential of these compounds.
Departments of Receptor Pharmacology (D.J.U., X.C., J.G., T.L.A., S.C., H.T.-M.D., J.F., M.K.L., C.X., I.G.), Chemistry (D.J.B.), Drug Metabolism & Pharmacokinetics (C.C., A.J.M.S.), Arena Pharmaceuticals Inc., San Diego, California.
David J
DJ
Unett
Joel
J
Gatlin
Todd L
TL
Anthony
Daniel J
DJ
Buzard
Steve
S
Chang
Chuan
C
Chen
Xiaohua
X
Chen
Huong T-M
HT
Dang
John
J
Frazer
Minh K
MK
Le
Abu J M
AJ
Sadeque
Charles
C
Xing
Ibragim
I
Gaidarov
D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000662:Amphetamines / Q000493:pharmacokinetics; D019390:Arrestins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004876:Ergot Alkaloids / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D010643:Phenoxybenzamine / Q000494:pharmacology; D010766:Phosphorylation; D011869:Radioligand Assay; D044403:Receptor, Serotonin, 5-HT2B / Q000187:drug effects*; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D000071557:beta-Arrestins
D000317:Adrenergic alpha-Antagonists; D000662:Amphetamines; D019390:Arrestins; D004876:Ergot Alkaloids; D044403:Receptor, Serotonin, 5-HT2B; D017366:Serotonin Receptor Agonists; D000071557:beta-Arrestins; D010643:Phenoxybenzamine; D048049:Extracellular Signal-Regulated MAP Kinases; C015952:4-iodo-2,5-dimethoxyphenylisopropylamine; D002118:Calcium