Functional selectivity of hallucinogenic phenethylamine and phenylisopropylamine derivatives at human 5-hydroxytryptamine (5-HT) 2A and 5-HT2C receptors

Earlier research has established that 2,5-dimethoxy-4-substituted phenylisopropylamines (α‑methylated hallucinogenic amphetamine derivatives such as DOI, DOM, DOB) and their α‑demethylated phenethylamine counterparts (e.g., 2C‑I, 2C‑B, 2C‑D) act as agonists at human 5‑hydroxytryptamine (5‑HT) 2A and 5‑HT2C G protein‑coupled receptors. Most prior in vitro work focused on one downstream pathway, phospholipase C (PLC)–mediated inositol phosphate (IP) accumulation, despite evidence that these receptors also couple to additional effectors such as phospholipase A2 (PLA2)–mediated arachidonic acid (AA) release. Classical receptor theory assumed intrinsic efficacy to be a ligand constant independent of the effector pathway, but accumulating data across receptor families have undermined that view and motivated the concept of agonist‑directed trafficking or functional selectivity, whereby ligands stabilise distinct receptor conformations that preferentially engage particular signalling cascades. Moya and colleagues set out to test functional selectivity within two homologous series of hallucinogenic ligands by measuring both PLC and PLA2 responses in the same cell system expressing human 5‑HT2A or 5‑HT2C receptors. They compared relative efficacies and potencies of several phenylisopropylamines and their phenethylamine congeners across the two pathways, and they also examined whether in vitro differences at 5‑HT2A receptors corresponded with an established in vivo index of 5‑HT2A activation, the head‑shake behaviour in rats. The goal was to determine whether subtle structural changes, such as α‑methylation or different 4‑position substituents, produce pathway‑selective signalling profiles.

The investigators used two well characterised Chinese hamster ovary (CHO‑K1) cell lines that stably express human 5‑HT2C (CHO‑1C19) or 5‑HT2A (CHO‑FA4) receptors at approximately 200 fmol/mg protein; these lines show comparable maximal IP accumulation and AA release in response to 5‑HT. Cells were grown in serum‑free, supplemented media prior to assays and seeded into 24‑well plates. For simultaneous measurement of both pathways, cells were pre‑labelled with myo‑[3H]inositol (1 μCi/ml for 24 h) to monitor PLC‑mediated IP accumulation and with [3H]arachidonic acid (0.1 μCi/ml for 4 h) to monitor PLA2‑mediated AA release. Drugs were applied for 10 min and responses measured from the same wells. The compound set included multiple phenylisopropylamines (for example, DOI, DOM, DON, DOB, TMA, 2,5‑DMA) and corresponding phenethylamines (for example, 2C‑I, 2C‑B, 2C‑D, 2C‑N, 2C‑H, mescaline), drawn from previously characterised stocks and commercial sources. Concentration–response data were fitted by nonlinear regression to obtain Emax and EC50 (pEC50 reported in tables, where available). Statistical comparisons between IP and AA responses for the same drug used paired Student’s t tests; comparisons between drugs used unpaired t tests. Significance was taken at p < 0.05. To assess an in vivo correlate of 5‑HT2A activation, male Sprague–Dawley rats (180–280 g) were injected intraperitoneally with equimolar doses (8.4 μmol/kg) of each drug; groups comprised 8–10 animals. Head shakes, defined as rapid side‑to‑side rotations of the head and ears, were counted for 25 min beginning 5 min after injection by an observer blinded to treatment. Control animals received saline. In one experiment, DOI’s effect was pretreated with ketanserin (1 mg/kg) to confirm 5‑HT2A mediation. Behavioural data were analysed by analysis of variance followed by Newman–Keuls multiple comparison tests. The authors note that higher phenethylamine doses up to ~15 mg/kg were tested but did not increase head shakes and that still higher doses disrupted behaviour.

At the 5‑HT2C receptor, most compounds behaved as partial agonists for both PLC‑IP accumulation and PLA2‑AA release, but relative efficacies varied across drugs and between pathways. Some phenylisopropylamine/phenethylamine pairs showed higher efficacy for the α‑methylated phenylisopropylamine (for example, DOM versus 2C‑D), whereas other pairs (for example, DOB versus 2C‑B) showed similar efficacies. Rank orders of efficacy among phenylisopropylamines differed between IP accumulation and AA release, whereas phenethylamines generally displayed similar rank orders for both responses, with the exception of the least efficacious compound (2C‑H). Reported pEC50 values (summarised in tables) indicated that phenylisopropylamines were generally more potent than phenethylamines at 5‑HT2C receptors; however, because the cell system lacks receptor reserve, potencies did not differ between the two measured responses for a given compound. For the 5‑HT2A receptor, all tested compounds behaved as partial agonists in the two assays, but a clear pattern emerged: every phenylisopropylamine was more efficacious than its phenethylamine counterpart for both PLC and PLA2 responses. Some individual ligands displayed pathway selectivity: 2,5‑dimethoxyphenylisopropylamine (2,5‑DMA) activated only the PLC pathway in cells expressing either receptor subtype, whereas 2C‑N (2,5‑dimethoxy‑4‑nitrophenethylamine) elicited only PLA2‑AA release at the 5‑HT2A receptor and did not activate the PLC pathway. The authors note that accurate EC50 estimates for low‑efficacy phenethylamines are difficult to obtain and present pEC50 values with caution. In vivo, phenylisopropylamines produced robust head‑shake behaviour in rats that was significantly greater than saline and significantly greater than equimolar doses of the corresponding phenethylamines. Pretreatment with ketanserin (1 mg/kg) abolished DOI‑induced head shakes, consistent with mediation by 5‑HT2 receptors. Phenethylamines did not differ from saline in head shakes even when doses were increased up to about fivefold (≈15 mg/kg); still higher doses caused general behavioural disruption and were not informative. Overall, measured efficacies and behavioural potency trends were concordant for 5‑HT2A‑related endpoints.

The investigators interpret their findings as further support for the functional selectivity hypothesis: ligands can differentially activate distinct intracellular effector pathways coupled to the same receptor. They highlight examples in which small structural differences produced markedly different signalling profiles measured in the same cells. One illustrative comparison is DOM versus 2,5‑DMA at 5‑HT2C receptors: both are partial agonists for PLC‑IP (relative efficacies of about 85% and 65% of 5‑HT, respectively), yet DOM is a full agonist for PLA2‑AA release while 2,5‑DMA elicits virtually no AA response; the only structural difference is the presence or absence of a methyl substituent at the 4‑position. Similarly, 2C‑N (the 4‑nitro phenethylamine) produced PLA2‑selective activation at 5‑HT2A receptors and failed to activate PLC‑IP, a pattern consistent with previous observations in other models. The authors note that α‑methylation (phenylisopropylamines versus phenethylamines) generally increased intrinsic activity for the 5‑HT2A PLC pathway—consistent with prior reports and perhaps related to higher hallucinogenic potency of phenylisopropylamines—but this effect was not consistent at the 5‑HT2C receptor. Behavioural data showing head shakes induced by phenylisopropylamines but not by phenethylamines align with the lower in vitro efficacy of phenethylamines at 5‑HT2A receptors and support the role of 5‑HT2A activation in that behaviour. However, the authors caution that the in vivo results do not demonstrate pathway‑specific functional selectivity: further work is required to determine which intracellular pathway(s) mediate head shakes and other behavioural endpoints. They acknowledge that data on PLA2‑mediated responses to these ligand classes are limited and that other signalling routes (for example, phospholipase D) could be relevant to hallucinogenesis. The investigators suggest that identification of active‑state selective ligands may be a useful tool to probe signalling mechanisms and that exploiting ligand‑specific signalling properties could ultimately permit development of drugs with improved therapeutic selectivity and efficacy. The authors also emphasise that current pathway‑selective effects are unpredictable from structure alone and that further characterisation across pathways and models is needed.