Serotonin receptors: The reason behind your happiness

Artwork of the brain depicting serotonin receptors made with tufting, stings shape forming and lino printing

What makes you happy? Good food, a good book or maybe exercising? Chemically, it all depends on the neurotransmitter, serotonin. This neurotransmitter is responsible for boosting your mood and low levels of serotonin are linked with depression. Serotonin, derived from the essential amino acid tryptophan, also plays important roles in cardiovascular functions, bowel movement and psychiatric disorders (1). 

 

But how does serotonin work? Serotonin, or 5-hydroxytryptamine (5-HT), functions by targeting various 5-HT receptors (5-HTRs) which are grouped into six classes of G protein-coupled receptors (GPCRs)- 5-HT1R, 5-HT2R, 5-HT4R, 5-HT5R, 5-HT6R and 5-HT7R and a class of cation-selective ligand-gated ion channels- 5-HT3R. 

 

The 5-HTRs are also important as drug targets for a broad range of diseases, such as obesity, migraine headaches, schizophrenia and depression. The 5-HTRs are also often involved in drug side-effects via unpredicted ‘off-target’ actions (2). A well-known example is that of the appetite suppressant drug, fenfluramine, which exerts its anti-obesity action by activating 5-HT2C receptors. This drug was banned because of a high rate of drug-induced valvular heart disease (VHD) which occurs due to the ‘off-target’ activation by fenfluramine and its active metabolite norfenfluramine at the closely related 5-HT2B receptor (5-HT2BR) (3-4).

 

The 5-HT2R class is essential for the various neurological and cardiovascular functions exerted by serotonin, and is further subdivided into 5-HT2A, 5-HT2B and 5-HT2C receptors. They are also involved in regulating abnormal serotonin release produced by recreational drugs, such as MDMA, more commonly known as ecstasy, and hallucinogens, such as lysergic acid diethylamide (LSD). We will focus on one of the members of this class, 5-HT2B receptors, which play several important functions including the regulation of serotonin release and thus  are crucial for maintaining serotonin levels in blood plasma. 

 

Functions of 5-HT2B receptors

Serotonin, or 5-hydroxytryptamine (5-HT), is derived from the amino acid tryptophan, upon hydroxylation at position 5 of the ring, followed by decarboxylation (5). In the cardiovascular tissue, 5-HT2BR is the most prevalent serotonin receptor and studies have revealed that these receptors are involved in several cardiovascular diseases such as cardiomyopathy and VHD (6).

 

A major role of 5-HT is to act as a neurotransmitter and the process is mediated by the 5-HT2BR. The 5-HT2BR has also been suggested to be involved in modulation of central dopamine activity, with important implications in neuropsychiatric disorders, especially in schizophrenia and drug addiction (7-8). Studies indicate that the 5-HT2BR may represent a potential pharmaceutical target for the treatment of drug addiction (9-10). Several medications, including anti-migraine drugs- methysergide and ergotamine, and anti-parkinsonian medications- pergolide, and cabergoline, act as agonists for the 5-HT2BRs and activate them resulting in VHD (11). 

 

Conformational states of 5-HT2B receptors


The 5-HT2BR belongs to the largest class of G-protein coupled receptors (GPCRs). The basic architecture of these receptors includes seven transmembrane helices, an eighth helix, three extracellular loops, an extracellular N-terminus, and an intracellular C-terminus (Figure 1) (12). 

 

Figure 1. A comparison of the different small-molecule-bound 5-HT2BR crystal structures- PDB IDs 4IB4 (5-HT2BR/ERG, green), 4NC3 (5-HT2BR/ERG, orange), 5TVN (5-HT2BR/LSD, lilac), 6DRX (5-HT2BR/lisuride, pink), 6DRY (5-HT2BR/methylergometrine, green), 6DRZ (5-HT2BR/methysergide, yellow), 6DS0 (5-HT2BR/LY266097, ochre).

Figure 1. A comparison of the different small-molecule-bound 5-HT2BR crystal structures- PDB IDs 4IB4 (5-HT2BR/ERG, green), 4NC3 (5-HT2BR/ERG, orange), 5TVN (5-HT2BR/LSD, lilac), 6DRX (5-HT2BR/lisuride, pink), 6DRY (5-HT2BR/methylergometrine, green), 6DRZ (5-HT2BR/methysergide, yellow), 6DS0 (5-HT2BR/LY266097, ochre).

 

Agonists that bind to GPCRs near the binding pocket, facilitate large-scale movements of the helices and cause rearrangements in highly conserved residues called ‘microswitches’. The Pro-Ile-Phe (P-I-F) motif forms an interface between helices V, III, and VI near the base of the ligand binding pocket.


All the different structures of  5-HT2BR were determined in the presence of various ligands. 5-HT2BR was crystallised for the first time in the presence of ergotamine (ERG), which is a precursor of the potent hallucinogen, LSD (13). Many other ergoline derivatives also bind to the 5-HT2BR but display very different functions. For example, while ERG has antimigraine effects, LSD has pronounced hallucinogenic effects. Till date, the structure of 5-HT2BR has been determined in the presence of eight small-molecules, including five representative ergolines - ergotamine (ERG), lysergic acid diethylamide (LSD), lisuride, methylergometrine, and methysergide, and one antagonist - LY266097 (14). These ligands prefer to bind at the orthosteric binding pocket (OBP), which is presumably the same pocket where the endogenous ligand 5-HT binds. Interestingly, the ligands ERG and LSD also interact with regions beyond the OBP, and into the extended binding pocket (EBP) (Figure 2). 

 

Figure 2. Receptor–ligand interactions in the orthosteric binding pocket (OBP) of the 5-HT2BR/LSD structure (PDB ID: 5TVN).
Figure 2. Receptor–ligand interactions in the orthosteric binding pocket (OBP) of the 5-HT2BR/LSD structure (PDB ID: 5TVN).

 

Binding of the different ergoline ligands causes different orientations of the indole nitrogens towards helix V residues along with the rotation of D135 side chains (Figure 3).

Figure 3. A comparison of the specific interactions in the OBP of ergoline ligand-bound structures: PDB IDs 4IB4 (5-HT2BR/ERG, green), 4NC3 (5-HT2BR/ERG, orange), 5TVN (5-HT2BR/LSD, lilac), 6DRX (5-HT2BR/lisuride, pink), 6DRY (5-HT2BR/methylergometrine, green), 6DRZ (5-HT2BR/methysergide, yellow), 6DS0 (5-HT2BR/LY266097, ochre). For clear visualisation, only the backbone of 5-HT2BR/ERG is shown as a ribbon cartoon.

Figure 3. A comparison of the specific interactions in the OBP of ergoline ligand-bound structures: PDB IDs 4IB4 (5-HT2BR/ERG, green), 4NC3 (5-HT2BR/ERG, orange), 5TVN (5-HT2BR/LSD, lilac), 6DRX (5-HT2BR/lisuride, pink), 6DRY (5-HT2BR/methylergometrine, green), 6DRZ (5-HT2BR/methysergide, yellow), 6DS0 (5-HT2BR/LY266097, ochre). For clear visualisation, only the backbone of 5-HT2BR/ERG is shown as a ribbon cartoon.

 

In the active state of the 5-HT2BR upon ligand binding, a chain of conformational rearrangements occurs in the P-I-F motif. There is an inward shift of P211, present in helix V, which is coupled with (i) a rotamer switch in I121, (ii) a large movement of the F282 side chain, and (iii) a corresponding rotation of helix VI on the cytoplasmic side. 

 

Out of the eight structures of the 5-HT2BR structures with different bound ligands, seven show very similar conformations, which include similar conformations of the PIF and NPxxY motifs and a slightly different conformation for the D(E)RY motif. The B-factor values suggest the D152 and R153 side chains are flexible with large B-factor values. The structure of the 5-HT2BR in complex with ERG-Fab (PDB id 5TUD) showed a more visible outward movement of helix VI compared to what was observed for the 5-HT2BR/ERG complex. In the case of the PIF motif, the 5-HT2B/ERG-Fab structure shows conformations similar to the active-state conformation. This leads to the conclusion that the 5-HT2BR/ERG-Fab structure is in an active state, whereas the other 5-HT2BR/ERG structures are in an intermediate state.

 

Pharmaceutical ligands targeting 5-HT2B receptors

 

Till date, few highly selective ligands for 5-HT2BRs have been discovered, although numerous non-selective compounds are known, especially molecules that bind to the 5-HT2B and 5-HT2C receptors concomitantly. Since it is well established that ligands that act as agonists for the 5-HT2BRs, also lead to drug-induced valvular heart disease, pharmaceutical ligand discovery has focused on the potential application of antagonists for this class of receptors. Recent research has focused on possible application of 5-HT2B antagonists as treatments for chronic heart disease (15).

 

MT-500 acts as an antagonist to the 5-HT2BR with a high affinity and high selectivity over other 5-HTR subtypes, especially 5-HT2AR and 5-HT2CR (16). There are several other  5-HT2BR antagonists that are currently under clinical trials, such as AM1125, developed by the company, AnaMar AB. Preclinical data showed that this compound had potential for the treatment of pulmonary fibrosis (17). AM1476, an orally administered 5-HT2BR antagonist with high selectivity, is currently in the late preclinical phase, being developed for the treatment of systemic sclerosis (18).  


Most of the 5-HT2BR binding ligands contain positively charged nitrogens or polar NH groups to form favourable salt bridge or hydrogen bond interactions with the carboxyl side chain of the residue D135 in helix III. Hydrophobic regions or aromatic rings of the ligand form π-π stacking or hydrophobic interactions with residues in the OBP, in helices III, V, VI and VII, as well as in the EBP, in helices III and VII. Considering all of the structures with ligands bound we can map out these preferences using a pharmacophore representation (Figure 4).

Figure 4. Key pharmacophore features of the 5-HT2BR based on PDB ID 5TVN. Positive electrostatic, hydrogen bond donor and hydrophobic pharmacophore features are coloured in blue, green, and cyan, respectively. (14)

Figure 4. Key pharmacophore features of the 5-HT2BR based on PDB ID 5TVN. Positive electrostatic, hydrogen bond donor and hydrophobic pharmacophore features are coloured in blue, green, and cyan, respectively. (14)

 

Conclusion and future directions

 

The 5-HT2BRs are extremely important since they have been implicated in various diseases, including cardiovascular diseases, fibrosis disorders, migraine, and neuropsychiatric disorders. A lot of work is ongoing into the interesting mechanism of these receptors. No structure has been solved yet for the 5-HT2BR in the inactive state. Also, many 5-HT2BR antagonists have been identified, with several candidates having advanced into clinical trials, but no approved drug currently exists for this receptor. In the future, there is huge potential for discovering new drugs that will target this receptor and help understand the therapeutic effect-related signalling.

 

About the artwork

 

This artwork, by Jennifer Gong, who is a Year 12 student at The Leys School, found the structure of the 5-HT2BR interesting and it acted as a base to develop her art. Although the artwork does not closely match the protein structure, it is the idea of presenting the inside of the brain, which is one of the organs that is affected by the hallucinogens, like LSD, that bind to the 5-HT2BR. She used different techniques to craft this piece, including tufting, stings shape forming and lino printing. Jennifer mentioned that this project helped her experience a thinking process for better combination of visual art techniques. 

 

View the artwork in the virtual 2023 PDB Art exhibition 

 

References

 

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