Structural pharmacology and therapeutic potential of 5-methoxytryptamines.

Psychedelic substances such as lysergic acid diethylamide (LSD) and psilocybin show potential for the treatment of various neuropsychiatric disorders1-3. These compounds are thought to mediate their hallucinogenic and therapeutic effects through the serotonin (5-hydroxytryptamine (5-HT)) receptor 5-HT2A (ref. 4). However, 5-HT1A also plays a part in the behavioural effects of tryptamine hallucinogens5, particularly 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a psychedelic found in the toxin of Colorado River toads6. Although 5-HT1A is a validated therapeutic target7,8, little is known about how psychedelics engage 5-HT1A and which effects are mediated by this receptor. Here we map the molecular underpinnings of 5-MeO-DMT pharmacology through five cryogenic electron microscopy (cryo-EM) structures of 5-HT1A, systematic medicinal chemistry, receptor mutagenesis and mouse behaviour. Structure-activity relationship analyses of 5-methoxytryptamines at both 5-HT1A and 5-HT2A enable the characterization of molecular determinants of 5-HT1A signalling potency, efficacy and selectivity. Moreover, we contrast the structural interactions and in vitro pharmacology of 5-MeO-DMT and analogues to the pan-serotonergic agonist LSD and clinically used 5-HT1A agonists. We show that a 5-HT1A-selective 5-MeO-DMT analogue is devoid of hallucinogenic-like effects while retaining anxiolytic-like and antidepressant-like activity in socially defeated animals. Our studies uncover molecular aspects of 5-HT1A-targeted psychedelics and therapeutics, which may facilitate the future development of new medications for neuropsychiatric disorders.

AlphaFold2 structures guide prospective ligand discovery.

AlphaFold2 (AF2) models have had wide impact but mixed success in retrospective ligand recognition. We prospectively docked large libraries against unrefined AF2 models of the σ2 and serotonin 2A (5-HT2A) receptors, testing hundreds of new molecules and comparing results with those obtained from docking against the experimental structures. Hit rates were high and similar for the experimental and AF2 structures, as were affinities. Success in docking against the AF2 models was achieved despite differences between orthosteric residue conformations in the AF2 models and the experimental structures. Determination of the cryo-electron microscopy structure for one of the more potent 5-HT2A ligands from the AF2 docking revealed residue accommodations that resembled the AF2 prediction. AF2 models may sample conformations that differ from experimental structures but remain low energy and relevant for ligand discovery, extending the domain of structure-based drug design.

Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A) receptor determine its activation and membrane-driven oligomerization properties.

From computational simulations of a serotonin 2A receptor (5-HT(2A)R) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT(2A)R, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.

Colocalisation of serotonin2A receptors with the glutamate receptor subunits NR1 and GluR2 in the dentate gyrus: an ultrastructural study of a modulatory role.

The serotonin(2A) receptor (5-HT(2A)R) is implicated in many neurological disorders and has a role in cognitive processes, reliant upon hippocampal glutamate receptors. Recent studies show that 5-HT(2A)R agonists and/or antagonists can influence cognitive function, suggesting a critical hippocampal role for these receptors, yet their cellular and subcellular distribution within this region has not been comprehensively analysed. Here, we have conducted an electron microscopic examination of 5-HT(2A)R distribution with the glutamate N-methyl-D-aspartate (NMDA) and amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor subunits NR1 and GluR2 in the hippocampal dentate gyrus (DG) in order to investigate whether 5-HT(2A)R location is compatible with a modulatory role over NMDA and/or AMPA receptor mediated neurotransmission. Of 5-HT(2A)R positive profiles, 56% were dendrites and 16% were dendritic spines. Labelling was both cytoplasmic and membranous. Spinous labelling was more frequently membranous at peri- and extra-synaptic sites, though was also associated with synaptic specialisations. Profiles displaying colocalisation of immunoreactivity for 5-HT(2A)Rs with NR1 or GluR2 were predominantly dendrites, representing 11% and 8% of 5-HT(2A)R positive profiles, respectively. Additionally, 12% of 5-HT(2A)R labelled profiles also displayed immunoreactivity for gamma-aminobutyric acid (GABA). These data indicate most 5-HT(2A)Rs are expressed on granule cell projections, with a smaller subpopulation expressed on GABAergic interneurons.

Possible autocrine regulation of chromaffin cell activity in adrenal glands of the frog by endothelin-1-induced serotonin release.

The aim of the present study was the demonstration of mechanisms of regulation of activity of chromaffin cells in the adrenal gland of Rana ridibunda (Anura-Amphibia). Previous studies have shown that endothelin-1 is an important factor for the maintenance of adrenal gland function. On the basis of these findings, frogs were injected with [Ala(1,3,11,15)]-endothelin-1 (0.025 mg/0.2 ml), which is a selective agonist of the endothelin B receptor, whereas control animals were injected with Ringer solution (0.2 ml). The adrenal glands were removed at 5, 20, and 60 min after injection and fixed, embedded in paraffin wax and stained by histological and immunohistochemical means, applied on adjacent 4-microm-thick sections. Sections were stained with hematoxylin and eosin for overall tissue analysis and, in parallel, serotonin was localized using the streptavidin-biotin complex technique while dopamine beta-hydroxylase and serotonin 2A receptors were shown by the peroxidase-antiperoxidase (PAP)-3,3'-diaminobenzidine tetrachloride (DAB) method. After injection of [Ala(1,3,11,15)]-endothelin-1, chromaffin cells secreted serotonin and synthesized dopamine beta-hydroxylase. In conclusion, these findings suggest that [Ala(1,3,11,15)]-endothelin-1 stimulates chromaffin cell activity in frog adrenal glands. Moreover, the presence of serotonin 2A receptors in chromaffin cells indicates that these cells are also targets for serotonin and that there is an autocrine signaling pathway in chromaffin cells. This is the first report providing data on the effects of endothelin-1 on chromaffin cells in frog adrenal glands.