2B Determined: The Future of the Serotonin Receptor 2B in Drug Discovery

Abstract

The cardiotoxicity associated with des-ethyl-dexfenfluramine (norDF) and related agonists of the serotonin receptor 2B (5-HT_2B) has solidified the receptor’s place as a traditional “antitarget” in drug discovery. Conversely, a growing body of evidence has highlighted the utility of 5-HT_2B antagonists for the treatment of pulmonary arterial hypertension (PAH), valvular heart disease (VHD) and related cardiopathies. In this Perspective, we summarize the link between the clinical failure of fenfluramine-phentermine (fen-phen) with the subsequent research on the role of 5-HT_2B in disease progression, as well as the development of drug-like and receptor subtype-selective 5-HT_2B antagonists. Such agents represent a promising class for the treatment of PAH and VHD, but their utility has been historically understudied due to the clinical disasters associated with 5-HT_2B. Herein, it is our aim to examine the current state of 5-HT_2B drug discovery, with an emphasis on the receptor’s role in the central nervous system (CNS) versus the periphery, as well as known and marketed compounds with 5-HT_2B antagonist activity as part of their broader polypharmacology.

Graphical Abstract

1. INTRODUCTION

1.1. Background and Characterization of 5-HT_2B.

Serotonin, 5-hydroxytryptamine (5-HT), is the endogenous ligand for the 5-HT receptor family, where it acts as a neurotransmitter and growth factor through various signaling pathways. Two superfamilies mediate the physiological actions of serotonin: G protein-coupled receptors (GPCRs) and ligand-gated ion channels, comprising fourteen total receptors between both families. The ligand-gated ion channels are currently comprised of one family: 5-HT₃. The GPCR superfamily includes 5-HT₁, 5-HT₂, and 5-HT_4–7 (Table 1), and was initially split into two distinct groups: the ‘D’ receptors for their irreversible interaction with the antagonist dibenzyline and the ‘M’ receptors for their ability to be blocked by morphine.¹ A 1979 study on brain homogenates identified distinct serotonin receptors: 5-HT₁ and 5-HT₂. 5-HT₁ was reported to have a higher affinity for serotonin, and 5-HT₂ had a high affinity for certain antagonists correlating with the ‘D’-type receptors previously described.² The 5-HT_2B subtype was first characterized in an organ bath studying the 5-HT-induced contraction of rat stomach fundus. The receptor was originally known as “5-HT_2F^” for “stomach fundus” but was later changed to 5-HT_2B to match the proposed nomenclature.³ Following the discovery and characterization of 5-HT_2B, the receptor has been implicated in many important roles within the cardiovascular system, central nervous system (CNS), and gastrointestinal (GI) tract.

Table 1.

5-HT Receptor Classification.^a

Family	Potential	Type	Mechanism of Action
5-HT1	Inhibitory	Gi/Go protein-coupled	Decrease intracellular concentrations of cAMP
5-HT2	Excitatory	Gq11 protein-coupled	Increase intracellular concentrations of IP3, DAG, and calcium
5-HT3	Excitatory	Ligand-gated ion channel	Depolarization of plasma membrane
5-HT4	Excitatory	Gs protein-coupled	Increase intracellular concentrations of cAMP
5-HT5	Inhibitory	Gi/Go protein-coupled	Decrease intracellular concentrations of cAMP
5-HT6	Excitatory	Gs protein-coupled	Increase intracellular concentrations of cAMP
5-HT7	Excitatory	Gs protein-coupled	Decrease intracellular concentrations of cAMP

^a.Adapted from Reference 4.

1.2. Relationship to Other Serotonin Receptors.

Currently, the seven receptor subtypes are separated by their primary signaling pathways (Table 1).⁴ The 5-HT₂ family is G_q/11-coupled, which activates various signaling molecules and intracellular calcium release from the endoplasmic reticulum. The family is divided into three distinct subtypes: 5-HT_2A, 5-HT_2B, and 5-HT_2C. While 5-HT_2A and 5-HT_2C are more closely related, 5-HT_2B shares similar sequence homology with both 5-HT_2A and 5-HT_2C, with up to 79% similarity within the transmembrane domain and 50% overall.⁵ There is high homology between 5-HT_2B across species compared to the human receptor: rat (79%), mouse (82%), dog (83%), and pig (95%).^6–8

1.3. Roles of 5-HT_2B in Physiological Processes.

The primary physiological effects of 5-HT_2B are mediated through the canonical G_q11 protein signaling pathway (calcium release and activation of secondary signaling molecules, see Figure 1).^9–11 Receptor expression can be found throughout the body, with the highest expression levels in the liver, kidneys, stomach fundus, and gut. 5-HT_2B has relatively moderate expression in the cardiovascular system, and low expression within the CNS.¹² Within the GI tract, 5-HT_2B is responsible for gut motility and hypersensitivity of colonic smooth muscle.¹³ Within the CNS, 5-HT_2B is thought to be involved in sleep initiation as well as regulation of the central respiratory system and blood volume.^14,15 Cardiovascular expression and activation of 5-HT_2B can lead to myofibroblast proliferation and valvular heart disease (VHD) by increasing valve area and causing poor valve closure, which will be discussed in this Perspective.¹⁶ It is because of this expression in the cardiovascular system that 5-HT_2B is considered a prototypical “antitarget” in medicinal chemistry programs.

Figure 1.

(A) Crystal structure of the human 5-HT_2B receptor, PDB: 5TVN. (B) Signaling Pathways Associated with 5-HT_2B (Adapted from “Activation of Protein Kinase C (PKC)”, by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates).

2. ANTITARGETS

2.1. The “Antitarget” Designation.

In pharmacology, an “antitarget” is broadly defined as any biological target that “[is] detrimental towards progression of [a] compound towards becoming a drug.”¹⁷ A ligand’s activity at an antitarget falls under the broader umbrella of “off-target” activity, which is generally unanticipated at the outset of a drug discovery program (it is not explicitly designed but need not necessarily be detrimental).^17,18 While select antitargets garner the majority of attention in the medicinal chemistry literature, and indeed seem to be encountered more frequently in drug discovery programs, the full list of known antitargets is broad and diverse (and certainly not comprehensive).¹⁸ In short, any biological target that, upon engagement by a ligand, has the potential to induce adverse drug reactions (ADRs) can be classified as an antitarget.¹ As will be further discussed, such a classification is often dependent on the specific mode of pharmacology of the ligand at the target in question (i.e. activation vs. inhibition).^17,19

The labelling of a specific biological target as an antitarget also need not be absolute. A given target may be unofficially reexamined and reclassified over time, and an antitarget designation is often program specific. For example, agonist activity at the serotonin receptor 2A (5-HT_2A) is associated with visual hallucinations and psychedelic experiences, and indeed many of the classical psychedelics are robust 5-HT_2A agonists (lysergic acid diethylamide (LSD), psilocin, etc.).²⁰ However, given the recent resurgence of this class of molecules in the context of psychedelic-assisted therapy,²¹ in which the overt psychedelic effects are postulated by many to be at least partly responsible for the observed efficacy,²² a blanket classification of 5-HT_2A as an antitarget seems inappropriate. For indications unrelated to psychedelic-assisted therapy, however, such effects would almost certainly be undesired.

2.2. Examples of Antitargets.

In addition to 5-HT_2A and other GPCRs,²³ the current list of biological targets deemed “anti” is broad and includes kinases,²⁴ ion channels,²⁵ cytochrome P450s,²⁶ and efflux pumps.²⁷ Perhaps the most well-known and frequently encountered antitarget in the drug discovery literature is the human Ether-à-go-go-Related Gene, or the hERG channel.^28–30 hERG is a potassium ion channel expressed in cardiac tissue, and plays a critical role in the regulation of the heart’s electrical activity.³¹ Specifically, disruption of hERG is associated with the development of potentially fatal Long QT Syndrome (LQTS), an unnatural lengthening of the QT cardiac repolarization interval.³²

hERG has become a canonical antitarget amongst drug discovery scientists.³³ Activity at the channel is routinely screened at early stages of the drug discovery pipeline, and strategic medicinal chemistry is implemented (if necessary) to avoid hERG inhibition for next generation molecules. This is due in no small part to the channel’s promiscuity; hERG-biased pharmacophores are routinely encountered in drug-like chemotypes,³⁴ and compounds across a broad range of indications have been pulled from the market following observations of hERG-related cardiac abnormalities.^35,36 Other potential antitargets, however, are less ubiquitous and therefore are not always a component of routine counter-screening. Subsequently, many undiscovered or poorly characterized targets likely exist that have the potential to become as notorious as hERG, and conversely, screening may eventually be deprioritized for other putative antitargets as their roles in physiological processes become clearer. A selection of notable biological antitargets, their associated risks, and exemplary ligands is summarized in Table 2.

Table 2.

Selected Antitargets and Associated Ligands.

Biological Target	Classification	Associated Deleterious Effects	Mode of Pharmacology	Example Ligand(s)
5-HT2A	GPCR	Hallucinations, psychedelic-experiences, changes in perception20–22	Agonism	LSD Psilocin
5-HT 2B	GPCR	Pulmonary arterial hypertension, valve disease 37,38	Agonism	Fenfluramine Norfenfluramine MDMA
μOR	GPCR	Bradypnea, hypoxemia39	Agonism	Morphine Fentanyl Carfentanil
M2	GPCR	Reduction in heart rate23,40	Agonism	Oxotremorine M (non-selective)
hERG	Ion Channel	LQTS31–36	Inhibition	Cisapride
CaV1.2	Ion Channel	LQTS, Brugda Syndrome25	Inhibition	Verapamil (non-selective)
CYP3A4	Cytochrome P40	Various drug-drug interactions26,41	Inhibition	Mibefradil
MDR1 (ABCB1)	Efflux Pump	Multiple drug resistance (chemotherapy)42,43	Efflux substrate	Verapamil (inhibitor) Paclitaxel (substrate)

To reiterate, an antitarget label does not completely preclude the utility of a target (many of our most important and useful drugs target 5-HT_2A, calcium channels, and the mu opioid receptor (μOR), Table 2). As will be further discussed, a target’s physiological location (central vs. peripheral tissue) can also be deeply important concerning the manifestation of ADRs.

3. 5-HT_2B AS AN ANTITARGET

3.1. Fen-Phen and Related Compounds.

It is now well established that excessive activation of 5-HT_2B can lead to an increased risk for a number of cardiopathies including pulmonary arterial hypertension (PAH)³⁷ and valvular heart disease (VHD).³⁸ The wealth of available literature demonstrating this link^{21,22,44–47} is directly related to the 1997 withdrawal of the combination anti-obesity regimen fenfluramine/phentermine (fen-phen, Figure 2), which was associated with PAH and VHD in humans. In the original press release, the FDA stated that the basis for the withdrawal was “based on new findings from doctors who have evaluated patients taking these two drugs with echocardiograms, a special procedure that can test the functioning of heart valves. These findings indicate that approximately 30 percent of patients who were evaluated had abnormal echocardiograms, even though they had no symptoms. This is a much higher than expected percentage of abnormal test results.”⁴⁸ The year prior, Connolly et al. identified a patient population of 24 women treated with fen-phen who developed VHD despite no history of cardiac disease.⁴⁵ Additional studies from around this time demonstrated that a regimen of fenfluramine or its (S)-enantiomer dexfenfluramine (DF, Figure 2), increased the risk of developing PAH by a factor anywhere between 3.7 and 23-fold.^49,50 A large population-based study of patients previously taking either fenfluramine, DF, or phentermine revealed several cases of idiopathic valvular disorders in patients taking fenfluramine or DF (with no cases noted for the phentermine population).⁴⁷ Cumulatively, these studies strongly suggest that fenfluramine (and DF) were the agents responsible for the observed cardiopathies.

Figure 2.

Chemical Structures of Key 5-HT_2B Agonists

3.2. Molecular Pharmacology.

Although DF itself binds only weakly to the 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors, its primary metabolite, N-des-ethyl DF (norDF, Figure 2) is a high affinity 5-HT_2B ligand with selectivity relative to 5-HT_2A and 5-HT_2C (5-HT_2B K_i = 11.2 ± 4.3 nM).³⁸ In functional assays, norDF is a potent agonist that stimulates phosphoinositide hydrolysis, intracellular Ca²⁺ levels, and the MAPK cascade (EC₅₀ = 23–24 nM in IP hydrolysis and Ca²⁺ mobilization assays).^38,51 Phentermine, by contrast, has no appreciable 5-HT_2B binding affinity up to 10 μM, and is primarily a dopamine-releasing agent.³⁸ A convergent body of evidence indeed suggests that the progression of both PAH and VHD are associated with highly increased 5-HT_2B receptor expression levels, and that 5-HT_2B activation is essential for disease progression.^{37,38,46,52,53} Rodent studies have since recapitulated the DF-induced development of PAH observed in human subjects,^37,52,53 and additional studies have noted similar cardiopathies, primarily VHD, for other 5-HT_2B agonists including MDMA,⁵⁴ pergolide,^54,55 and methysergide^38,56. Taken together, these results strongly indicate substantial risks for treatments involving 5-HT_2B agonists, and it has been recommended that all serotonergic drugs be screened for this functional profile.^38,54 (Several widely used 5-HT_2A agonists including DMT, LSD, psilocin and related phenethylamines and tryptamines are relatively non-selective relative to 5-HT_2B; the increasingly prevalent use of such compounds will need to be reconciled with the risks associated with 5-HT_2B activation).^57–59

Unsurprisingly, 5-HT_2B is now widely regarded as one of the primary antitargets in drug development pipelines, but it is critical that a compound’s mode of pharmacology at the receptor be fully understood before de-prioritization is initiated. A variety of receptor profiling tools, in silico cheminformatics assays, 5-HT_2B functional assays, and suggested safety margins have been recommended toward this end.⁶⁰ Currently, there is no evidence to suggest a role for 5-HT_2B antagonists in the development of PAH and VD, and the paucity of such compounds, particularly those highly selective for 5-HT_2B, have not indicated a potential for mechanism-based toxicity (cardiac or otherwise) associated with 5-HT_2B inhibition. Moreover, as will be discussed in the following sections, such agents have the potential to be disease-modifying treatment strategies for these and related cardiac disorders.⁷

4. 5-HT_2B ANTAGONISTS AND ANIMAL MODELS

4.1. SB-204741.

Work to elucidate fen-phen’s off-target 5-HT_2B agonism led to the hypothesis that 5-HT_2B antagonism is a potential therapeutic for cardiopulmonary diseases. Of the multitude of selective and non-selective 5-HT_2B antagonists, SB-204741 was the first synthesized (1994) and is widely used; it has a high affinity for 5-HT_2B (pK_i = 7.95) and high selectivity (>135) compared to 5-HT_2C, the receptor most closely matching 5-HT_2B in morphology.^61,62 One of the first animal studies in which SB-204741 was utilized involved antagonizing individual 5-HT₂ receptors to investigate renal sympathoinhibition and mean arterial blood pressure following intracerebroventricular administration of quipazine, a 5-HT₂ agonist, in rats¹⁵. SB-204741 has been used to study the blockade of 5-HT_2B in the context of several cardiopulmonary diseases such as pulmonary hypertension,^37,63,64 myocardial infarction,⁶⁵ and calcific aortic valve disease,^66,67 with encouraging results in the prevention of disease progression.

4.2. Gene Editing.

In addition to the administration of antagonists, mouse models that target 5-HT_2B through genetic ablation have confirmed the receptor’s role in cardiopulmonary disease. In one report, the 5-HT_2B allele was rendered nonfunctional in embryonic stem cells through the interruption of the protein reading frame; this was done by introducing the bacterial neo gene in exon 2 of the 5-HT_2B gene sequence.⁶⁸ Ablation of both copies of the 5-HT_2B gene results in viable offspring, with mutant mice growing to adulthood; however, due to its importance in heart development, 5-HT_2B mutant mice demonstrate ventricular hypoplasia, myocyte disarray, and ventricular dilation.^68,69

The gene editing technology “Cre-Lox” allows for the knockout of both 5-HT_2B alleles in a time- and site-specific manner through homologous recombination. It relies upon the recognition of specific DNA sequences called loxP sites by the enzyme Cre recombinase, which is activated by a tissue-specific promoter that itself is induced exogenously by a stimulus, such as tamoxifen or doxycycline.⁷⁰ Tissue-specific promoters such as Transcription factor 21(Tcf21)^Cre, Periostin (Pstn)^Cre, and Angiopoietin-1 receptor gene (Tie2)^Cre are useful tissue-specific promoters that provide cell-lineage tracing capabilities, as Tcf21 is expressed in resident fibroblasts, Pstn is expressed in myofibroblasts, and Tie2 is expressed in endothelial cells.^71–73 5-HT_2B conditional knockout models have been used to investigate cardiopulmonary fibrosis in diseases such as myocardial infarction⁶⁵ and PAH.⁶⁴

4.3. Relevant Signaling Pathways.

5-HT_2B agonism results in downstream activation of the signal transduction pathway Ras/Raf/mitogen-activated protein kinase (MAPK), leading to an increase in rate of cell division and proliferation.^9,74 Cytoplasmic tyrosine kinases (e.g., Src) are key mediators of G protein-coupled receptor (GPCRs) signaling to the MAPK pathway.^9,75 Additionally, transforming growth factor-β1 (TGF-β1) is a cytokine that mediates fibroblast proliferation, extracellular matrix (ECM) deposition, and myofibroblast differentiation⁷⁶ through SMADS, the substrates for TGF-β1 receptors. TGF-β1 and its fibrotic activity is upregulated upon 5-HT_2B agonism due to signaling pathway crosstalk via Src phosphorylation.⁶⁷

With 5-HT_2B leading to increased stromal fibroblast and myofibroblast proliferation, combined with the increased deposition of collagen into the ECM,⁷⁷ this provides a rational hypothesis for the pathophysiology of many cardiopulmonary diseases involving 5-HT_2B signaling (Figure 3). In PAH, the core etiology is unchecked muscularization of the pulmonary arterioles; the tissue-specific promoter Tie2^Cre allows for targeted 5-HT_2B ablation in the bone marrow-derived proangiogenic cells (PACs) and results in normalized arteriole compliance.⁶⁴ Conditional 5-HT_2B ablation in myocardial infarction using tissue-specific Cre promoters demonstrated that resident fibroblasts and myofibroblasts were the main culprit of detrimental scar thickness and heart contractility.⁶⁵ Similarly, calcific aortic valve disease is characterized by fibrotic deposition on aortic valve leaflet cusps by aortic valve interstitial cells (AVICs), and antagonism of 5-HT_2B opposed AVIC activation through a myofibroblast, and therefore TGF-β1, mechanism.^66,67 Taken together, these studies indicate that 5-HT_2B inactivation is an attractive strategy for modifying cardiopulmonary fibrotic disease.

Figure 3.

(A) 5-HT_2B-mediated recruitment of proangiogenic cells in pulmonary arterial hypertension leads to muscularization of pulmonary arterioles, shown by proliferation of pulmonary artery smooth muscle cells (PASMCs). (B) Valve remodeling in calcific aortic valve disease is driven by aortic valve interstitial cells (AVICs) that increase deposition of collagen and glycosaminoglycans (GAG) into the ECM; this stiffens the aortic valve leaflets and decreases valve compliance. It is unknown whether proangiogenic cells play a role in remodeling the valve ECM. (C) Myofibroblasts are responsible for ECM stiffening and scar tissue formation of the infarct zone in myocardial infarction, causing cardiac tissue deterioration, decreased compliance, and decreased cardiac output. Retrieved from https://app.biorender.com

5. DEVELOPMENT OF NEXT GENERATION 5-HT_2B ANTAGONISTS

5.1. Preclinical Compounds.

From a medicinal chemistry perspective, a number of interesting 5-HT_2B structure-activity relationship (SAR) studies are reported in the literature, with programs ranging from early lead optimization^61,78–81 through clinical development.^82,83 In both cases, the optimization for selectivity relative to 5-HT_2A and 5-HT_2C is a critical parameter, and in many programs has proven challenging to attain for both receptors simultaneously.^61,81,84 In the 1990s, a series of reports detailing compounds derived from yohimbine,⁸¹ and substituted indoles and indolines^61,78–81 described reasonable (~100 fold) selectivity for 5-HT_2A and/or 5-HT_2C (see Figure 4 for a selection of reported 5-HT_2B chemotypes). In the latter class, the previously discussed isothiazole SB-204741 is considered to be the first reported selective 5-HT_2B antagonist (Figure 4).⁶¹ While these early reports are admirably thorough with respect to SAR and 5-HT_2A/2C selectivity profiling, they contain limited information regarding pharmacokinetics (PK) and broader ancillary pharmacology. Bonhaus et al. subsequently reported a series of naphthylpyrimidines, including RS-127445, which displays improved 5-HT_2B selectivity (~1,000 fold against a broader off-target profile), albeit with limited oral bioavailability in rats (Figure 4).⁸⁴ Additional reports of selective 5-HT_2B antagonists with more detailed PK profiles have slowly started to emerge in the literature.^85,86 More recently, members of the 2-thiazoline pulicatin class of natural products (and synthetic derivatives) were reported to have high selectivity for 5-HT_2B relative to a broader panel of serotonin receptor subtypes (Figure 4).⁸⁷ As before, drug development-enabling PK information is not reported. In 2023, Schieferdecker and Vock reported detailed 5-HT_2B pharmacophore models which are likely to aid in the development of next-generation ligands with robust subtype selectivity.⁸⁸

Figure 4.

Chemical Structures of Selected 5-HT_2B Antagonists

5.2. Clinical Compounds.

With respect to the clinical development of more advanced molecules, thiophenylpyrimidine PRX-08066 (Figure 4) is a potent and selective 5-HT_2B antagonist developed by Epix Pharmaceuticals, which was shown to be highly effective in the treatment of drug-induced PAH and VHD in rats.⁸² As of 2009, a Phase 2, “3-month open label study to evaluate the safety and efficacy of PRX-08066 in patients with pulmonary hypertension and COPD” was terminated for undisclosed reasons.⁸⁹ Terguride, a potent ergoline 5-HT_2A/5-HT_2B dual antagonist,⁸³ was granted orphan drug status for PAH treatment as of 2008,⁹⁰ although clinical development was ultimately discontinued by 2011 due to lack of efficacy.^91,92 In the absence of more concrete findings, it is difficult to speculate on the reasons for theses terminations (in the case of terguride the issue is thought to be related to appropriate plasma exposure levels),⁹² but there is clearly a need for a more thorough discussion on PK, exposure, and tolerability of clinical-stage 5-HT_2B antagonists in the literature. These data will be critical for the design of future clinical trials with next generation molecules.

6. 5-HT_2B POLYPHARMACOLOGY

6.1. Marketed 5-HT_2B Antagonists.

Many currently marketed drugs (as well as widely studied compounds in late stage clinical development) display robust 5-HT_2B antagonism in radioligand binding assays as part of their broader polypharmacological profile. This is primarily true of antipsychotic medications, although examples of antidepressants, antihypertensives, antiparkinsonians, and antisedatives with 5-HT_2B activity are also known. A search of the National Institute of Mental Health’s Psychoactive Drug Screening Program (NIMH-PDSP)⁹³ K_i Database for 5-HT_2B ligands with K_i’s < 100 nM returns over 500 unique results, one of which, aripiprazole, is still among the top 100 pharmaceuticals in terms of yearly sales (Table 3).⁹⁴ Although many of these compounds are promiscuous with respect to additional CNS receptor targets, it seems clear that 5-HT_2B antagonist activity (which should of course be rigorously characterized during development) should not preclude a compound from advancement to the clinic.

Table 3.

Selected 5-HT_2B Binding Affinity Data for Notable Psychoactive Compounds

Compound	Classification	5-HT2B Ki (nM)	Species	Radioligand
Mianserin95	antidepressant	9.0	Human	[3H]-5HT
Trazodone38	antidepressant	74	Human	[3H]-5HT
Cyproheptadine96	antihistamine	1.5	Human	[3H]-5HT
Ketanserin97	antihypertensive	2.4	Bovine	[3H]-ketanserin
Lisuride96	antiparkinsonian	1.1	Human	[3H]-5HT
Amisulpride98	antipsychotic	13	Human	[3H]-LSD
Aripiprazole99	antipsychotic	0.36	Human	[3H]-LSD
Asenapine93	antipsychotic	0.21	Human	[3H]-LSD
Chlorpromazine97	antipsychotic	6.0	Bovine	[3H]-ketanserin
Clozapine95	antipsychotic	7.2	Human	[3H]-5HT
Lurasidone93	antipsychotic	24	Human	[3H]-LSD
Olanzapine100	antipsychotic	12	Human	[3H]-5HT
Quetiapine93	antipsychotic	86	Human	[3H]-LSD
Risperidone100	antipsychotic	29	Human	[3H]-5HT
Spiperone97	antipsychotic	0.8	Bovine	[3H]-ketanserin
Xanomeline101	antipsychotic	20	Human	[3H]-5HT
Yohimbine95	antisedative (veterinary)	43	Human	[3H]-5HT

While many of the examples reported here fall into similar indication classes, a couple of examples warrant further discussion. Lisuride is a potent dopamine agonist and a synthetic ergoline derivative, a class to which cabergoline and pergolide also belong. Of these and related dopamine agonists, only cabergoline and pergolide (5-HT_2B agonists) were associated with VHD after long term use; 5-HT_2B antagonists (i.e. lisuride) demonstrate no such association. Indeed, lisuride has been prescribed for decades without a single known VHD report.^102,103 Although a lack of association does not necessarily demonstrate prevention, examples also exist of marketed drugs with 5-HT_2B antagonism as part of their polypharmacology that explicitly reverse drug-induced VHD. Cyproheptadine, a first-generation tricylic antihistamine with potent 5-HT_2B antagonist activity (K_i = 1.5 nM)⁹⁶, has been shown to reverse pergolide-induced valvulopathy in rats.¹⁰⁴ Future analyses of patient populations taking one or more of these compounds will be important to further understand the potential for drug repurposing toward the prevention or treatment of VHD and related disorders.

It is worth noting that the compounds in Table 3 represent only molecules that are known to be psychoactive (CNS-penetrant). As will be discussed in the next section, it will be important to understand the potential risks associated with centrally-mediated 5-HT_2B antagonism with the development of any next generation therapeutic.

7. NOVEL 5-HT_2B ANTAGONISTS FOR PAH TREATMENT

7.1. VU6047534 and Analogs.

Our group has recently disclosed a potent and highly selective 5-HT_2B antagonist, VU6047534, which possesses rodent PK properties suitable for proof-of-concept studies (Figure 5). Structurally, VU6047534 is derived from the SB204741-like series of urea-indoles, but is cyclized to give a substituted thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione backbone. Encouragingly, VU6047534 demonstrated robust efficacy in Sugen-hypoxia mouse models of PAH prevention and treatment, as well as the prevention of right ventricle hypertrophy in Sugen-hypoxia and pulmonary arterial banding models. VU6047534 also displayed no significant off-target responses in the Eurofins Panlabs ancillary pharmacology screen of 68 common membrane proteins, ion channels and transporters (including the hERG channel),¹⁰⁵ and was clean with respect to cytochrome P450 inhibition across 4 major isoforms (1A2, 2C9, 2D6, 3A4 IC₅₀s >30 μM).¹⁰⁶

Figure 5.

Development of Selective and Peripherally-Restricted 5-HT_2B Antagonists for In Vivo Studies

Although VU6047534 displays negligible brain exposure in mice (Figure 5), this compound is predicted to have moderate brain exposure in human subjects due to a relative lack of P-glycoprotein (P-gp)-mediated efflux (3.7 efflux ratio, P_appA-B = 18.0 10⁻⁶ cm/s). Centrally-mediated 5-HT_2B antagonism is thought to be associated with a variety of adverse effects including depression, aggression, impulsivity, and suicidality. The presence of a relatively common 5-HT_2B stop codon exclusive to Finnish populations has been associated with these types of psychiatric diseases, highlighting the potential dangers associated with a centrally-penetrant antagonist.^107–109

An important caveat, however, is that all of the drugs listed in Table 3 are known to be CNS-active, and many are routinely and safely taken by millions across the globe. While the majority of these compounds tend to be promiscuous with respect to off-target activity at additional CNS receptors, it is tempting to speculate that CNS-penetrant compounds with robust 5-HT_2B antagonist activity may be well tolerated with long term use (at least for a majority of the population). Further research in this area is clearly needed to understand this apparent discrepancy.

Subsequent SAR on the VU6047534 scaffold, specifically the exploration of polar indole N-substitutions, yielded next generation molecules with comparable potency and selectivity profiles that are predicted to be robust P-gp efflux substrates (VU6055320; 69.4 efflux ratio, P_appA-B = 0.35 10⁻⁶ cm/s).¹⁰⁶ Further preclinical characterization (and assessment for efficacy in similar rodent models) will be needed for these next generation 5-HT_2B antagonists, and such studies are ongoing in our laboratories. Additionally, we have recently disclosed results from a high-throughput screen (HTS) aimed at identifying additional chemical matter for the development of structurally orthogonal 5-HT_2B antagonists.¹¹⁰ Our HTS campaign led to the immediate identification of potent and selective compounds (5-HT_2B IC₅₀s in the low nanomolar range; <50% inhibition of 5-HT2_A/2C at 10 μM). Furthermore, selected compounds from the most potent reconfirmed hits were selected for profiling in the P-gp assay, with exemplary compounds showing a low potential for brain exposure in human subjects.¹¹⁰

8. CONCLUSIONS AND PERSPECTIVES

While 5-HT_2B has historically been viewed as an antitarget by medicinal chemists, the assessment of a compound’s mode of pharmacology at the receptor is crucial. The difference between 5-HT_2B agonism and antagonism could mean the difference between a cardiotoxic agent (fen-phen) and a disease modifying treatment for PAH, VHD, and related disorders. It is our hope that the chemical scaffolds described herein will provide a useful platform for drug discovery scientists interested in this field, as there still exists an enormous unmet need to develop a 5-HT_2B antagonist with the full package of properties suitable for clinical development. Strategies for 5-HT_2B inactivation also need not be limited to simple orthosteric antagonists; the development of negative allosteric modulators (NAMs) and 5-HT_2B-specific protein degraders could also prove viable. Regardless of the approach, it is our belief that the future for drug discovery at this receptor is bright.

• Significance: Antagonists of the serotonin receptor 2B (5-HT_2B) are a promising and underexplored potential treatment for pulmonary arterial hypertension (PAH) and valvular heart disease (VHD).

• Impact: 5-HT_2B antagonists are disease modifying with respect to PAH and VHD in preclinical species, and could translate to a first in class treatment in human subjects.

• Innovation: Structurally-novel 5-HT_2B antagonists with favorable selectivity and pharmacokinetic (PK) profiles are being profiled for clinical development.

ABBREVIATIONS USED

5-HT

5-hydroxytryptamine

5-HT_2A

serotonin receptor 2A

5-HT_2B

serotonin receptor 2B

5-HT_2C

serotonin receptor 2C

ADRs

adverse drug reactions

AVICs

aortic valve interstitial cells

CNS

central nervous system

DF

dexfenfluramine

DMT

dimethyltryptamine

ECM

extracellular matrix

FDA

Food and Drug Administration

GI

gastrointestinal

GPCR

G protein-coupled receptor

hERG

human ether-à-go-go-related gene

i.p.

intraperitoneal

IP

inositol monophosphate

K_i

inhibition constant

K_p

total brain:total plasma ratio

LQTS

long QT syndrome

LSD

lysergic acid diethylamide

MAPK

mitogen-activated protein kinase

MDMA

3,4-methyl enedioxy methamphetamine

μOR

mu opioid receptor

NIMH-PDSP

National Institute of Mental Health’s Psychoactive Drug Screening Program

norDF

des-ethyl-dexfenfluramine

PACs

proangiogenic cells

PAH

pulmonary arterial hypertension

P-gp

P-glycoprotein

PK

pharmacokinetic

Pstn

periostin

SAR

structure-activity relationship

Tcf21

transcription factor 21

TGF-β1

transforming growth factor-β1

Tie2

angiopoietin-1 receptor gene

VHD

valvular heart disease

Biographies

Aaron M. Bender earned his Ph.D. in medicinal chemistry from the University of Michigan in 2016, and subsequently joined the Warren Center for Neuroscience Drug Discovery (WCNDD) at Vanderbilt University as a postdoctoral scholar. Upon completion of his postdoctoral training, Aaron remained at Vanderbilt and is currently an Assistant Director of Medicinal Chemistry at the WCNDD. His research interests are in medicinal chemistry, particularly the development of therapeutics targeting G protein-coupled receptors, and organic methodology.

Lauren C. Parr received her B.A from the University of Kansas in 2019 and M.S. from the University of Florida in 2022. She is currently a Ph.D. student in Dr. Craig W. Lindsley’s Lab at Vanderbilt University focusing on the molecular mechanisms of kinases and development of small-molecule inhibitors.

William B. Livingston received his B.S. degree from the Jacobs School of Engineering at the University of California San Diego in 2022. He then joined the Merryman Mechanobiology Laboratory in 2022 as a pre-doctoral student in the Biomedical Engineering Department at Vanderbilt University, where he is currently investigating the role of 5-HT2B in hypertrophic cardiomyopathy.

Craig W. Lindsley is the William K. Warren, Jr. Chair in Medicine and a University Professor of Pharmacology, Chemistry and Biochemistry at Vanderbilt University. Dr. Lindsley is also the director of the Warren Center for Neuroscience Drug Discovery (WCNDD) at Vanderbilt, and the Editor-in-Chief of the Journal of Medicinal Chemistry. His research interests include the development of novel treatment strategies for schizophrenia, Parkinson’s disease, and other brain disorders, as well as disorders of the cardiovascular system.

W. David Merryman W. David Merryman received his Ph.D. degree from the University of Pittsburgh in 2007 and is currently the Walters Family Professor in the Department of Biomedical Engineering, and Professor of Pharmacology, Medicine, and Pediatrics at Vanderbilt University. His research interests are cardiopulmonary and renal mechanobiology with a particular focus on developing new therapeutic strategies.