Kinetics of 5-HT_2BReceptor Signaling: Profound Agonist-Dependent Effects on Signaling Onset and Duration

Abstract

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 receptormediated 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-hydroxytryptamine 2B (5-HT 2B ) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Ga qmediated 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 signalregulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT 2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptordissociation 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-HT 2B 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.

Can residence time offer a useful strategy to target agonist drugs for sustained GPCR responses?

“…Using in vitro experiments this can be exhibited by 'wash-resistant' receptor activation, where signaling continues even after excess agonist is removed from the receptor by infinite dilution of the drug-containing buffer [8,9,[16][17][18][19][20][21][22]. It is anticipated that this could Thyroid-stimulating hormone SSIR >30 min a [9] Sphingosine-1 phosphate-1 SSIR; residence time and rebinding 300 min a Q6 [16,42] b2-adrenergic SSIR; exosite; membrane deposition >180 min a or >10 min b [25,27,43,44] Vasopressin-2 SSIR >40 min a [17] Glucagon-like peptide SSIR n.d. [37] Angiotensin-II SSIR >30 min b [45] Calcitonin a SSIR; residence time > 4320 min b [18] Serotonin 5-HT2B SSIR; residence time and rebinding >240 min a [19] GPR119…”

“…Anecdotal evidence suggests that agonist dissociation kinetics can regulate whether a ligand can evoke SSIR. For PTH receptor, calcitonin and serotonin 5-HT2B receptors the observation of SSIR appears to be characteristic of agonists with slow dissociation from these receptors, while being absent with rapidly dissociating ligands [8,18,19]. It is therefore tempting to speculate that long residence time is a general feature of SSIR agonists, although this remains to be characterized for other receptors.…”

“…However, additional evidence suggests that long residence time alone is not sufficient to support SSIR but that rebinding is also important. The duration of SSIR measured for 5-HT2B agonists is longer than their residence times [19], suggesting that multiple independent binding events are likely to be involved (i.e. a single binding event is not sufficiently long).…”

“…a single binding event is not sufficiently long). Moreover, SSIR observed for 5-HT2B Q4 and sphingosine-1 phosphate (S1PR) receptors can be reversed with antagonists [16,19]. This can be considered a hallmark of rebinding and thus indicates agonist dissociation events during the generation of sustained responses, assuming that the antagonists used are orthosteric and capable of passing cellular membranes to access intracellular receptor-binding sites.…”

“…Interestingly, long-and short-acting PTH receptor agonists have been proposed to have distinct potential clinical applications (hypoparathyroidism and osteoporosis, respectively) based on their differences to evoke SSIR [36], whereas SSIR of ergot 5-HT2B agonists has been postulated to confer cardiac valvulopathic side effects of these ligands [19]. Moreover, the clinical efficacy of the S1P1R immunomodulator drug FTY729P is proposed to be linked to its long residence time that promotes persistent receptor internalization and intracellular signaling [29].…”