Extraoral bitter taste receptors as mediators of off-target drug effects

Abstract

We present a novel hypothesis that could explain many off-target effects of diverse pharmaceuticals. Specifically, we propose that any drug with a bitter taste could have unintended actions in the body through stimulation of extraoral type 2 taste receptors (T2Rs). T2Rs were first identified in the oral cavity, where they function as bitter taste receptors. However, recent findings indicate that they are also expressed outside the gustatory system, including in the gastrointestinal and respiratory systems. T2R ligands include a diverse array of natural and synthetic compounds, many of which are toxins. Notably, many pharmaceuticals taste bitter, with compounds such as chloroquine, haloperidol, erythromycin, procainamide, and ofloxacin known to activate T2Rs. Bitter-tasting compounds can have specific physiological effects in T2R-expressing cells. For example, T2Rs are found in some gastrointestinal endocrine cells, including those that secrete the peptide hormones (e.g., ghrelin and glucagon-like peptide-1) in response to stimulation by bitter-tasting compounds. In the respiratory system, stimulation of T2Rs expressed in respiratory epithelia and smooth muscle has been implicated in protective airway reflexes, ciliary beating, and bronchodilation. If our hypothesis is confirmed, it would offer a new paradigm for understanding the off-target actions of diverse drugs and could reveal potential new therapeutic targets.—Clark, A. A., Liggett, S. B., Munger, S. D. Extraoral bitter taste receptors as mediators of off-target drug effects.

The off-target effects of pharmaceuticals can have significant consequences for patients. Exciting new treatments can emerge from the recognition that a drug has a positive, but unanticipated, impact on physiology. By contrast, otherwise efficacious therapies have been taken off the market or may never progress through the development and approval processes in the first place, because they exhibit life-threatening side effects. Because of the potential to design new drugs that mimic positive off-target effects as well as the need to prevent negative outcomes that limit the repertoire of usable therapeutic agents, it is imperative that we understand the molecular mechanisms underlying off-target drug actions.

Notably, many common drugs are bitter tasting and are thus effective ligands for type 2 taste receptors (T2Rs; a.k.a. bitter taste receptors), which are expressed in specialized sensory cells of the gustatory system (1–4). Surprisingly, it now appears that T2Rs may also have important physiological roles in other areas of the body, including in the respiratory and gastrointestinal (GI) systems (5–9). The observation that T2R expression is not restricted to the gustatory system (10, 11) suggests the novel hypothesis that the same receptors that underlie bitter taste in the mouth also mediate the off-target actions of numerous drugs.

T2RS AND THE DETECTION OF BITTER-TASTING COMPOUNDS

Compounds that elicit a taste perception do so via their activation of sensory cells within the gustatory epithelium of the tongue, soft palate, and oropharynx (10, 12). Individual taste cells are dedicated to only one of the five taste qualities (sweet, bitter, umami, salty, and sour), and the stimulus selectivity of a taste cell is dictated by the taste receptors it expresses (10, 12). Bitter taste is evoked by perhaps tens of thousands of synthetic and natural compounds (1) that are agonists for one or more of the 25 T2Rs encoded in the human genome (10, 13–15).

The T2Rs (encoded by the TAS2R genes) are G-protein-coupled receptors (GPCRs) and are found in vertebrates from fish to humans (10). Fish (e.g., zebrafish, Medaka, and pufferfish) and birds (e.g., chickens) have relatively few (<10) intact Tas2r genes, while the family is significantly expanded in tetrapods (e.g., mice have 35 intact Tas2rs, while Xenopus have 49); humans have 25 putatively functional TAS2Rs, as well as 11 TAS2R pseudogenes (2–4, 13–17). T2Rs appear to vary greatly in their stimulus tuning (1, 10, 18): some are activated by only a few compounds (1, 19, 20), while others respond to an array of structurally diverse molecules (1, 21, 22). Bitter taste is thought to serve a sentinel function to prevent the ingestion of toxins, and a premium is placed on detecting, rather than discriminating, bitter-tasting compounds so that potential toxins are spit out before swallowing. Many toxins produced by plants to protect themselves against predation, including glucosinolates in Brassica vegetables and quinine in cinchona tree bark, taste bitter to humans because they activate T2Rs on the tongue (1, 23). However, while bitter taste may have evolved to protect us, we humans regularly choose to ingest or inhale natural and synthetic bitter-tasting compounds in foods (e.g., broccoli and soy), beverages (e.g., coffee and beer), and medications. Numerous drugs taste bitter and activate T2Rs, including the analgesic acetaminophen, the fluoroquinolone antibiotic ofloxacin, the antiasthmatic cromoglicic acid, and the antithyroid drugs methimazole and propylthiouracil (Table 1 and refs. 1, 24, 25).

Table 1.

Bitter-tasting drugs and other bioactive compounds and their cognate human T2Rs

Drug	Action	Responsive T2R isoforms	Ref.
Acetaminophen	Analgesic	39	1
Aloin	Laxative	31, 43	1, 20
Azathioprine	Immunosuppressive	4, 10, 14, 46	1
Carisoprodol	Muscle relaxant	14, 46	1
Chloramphenicol	Antibiotic	1, 8, 10, 39, 43, 46	1
Chloroquine	Antimalarial	3, 7, 10, 39	1, 18
Colchicine	Gout	4, 39, 46	1
Cromoglicic acid	Mast cell stabilizer	7, 20, 43	1
Dapsone	Topical Antibacterial	4, 10, 40	1
Dextromethorphan	Antitussive	1, 10	1
Diphenhydramine	Antihistamine	14, 40	1
Diphenidol	Antiemetic	1, 4, 7, 10, 13, 14, 16, 20, 30, 31, 38, 39, 40, 43, 46	1
Erythromycin	Antibiotic	10	1
Famotidine	Gastric acid Inhibitor	10, 31	1
Flufenamic acid	Anti-inflamatory	14	1
Haloperidol	Antipsychotic	10, 14	1
Hydrocortisone	Glucocorticoid	46	1
Methimazole	Antithyroid	38	1
Noscapine	Antitussive	14	1
Orphenadrine	Antispasmodic	14, 46	1
Papaverine	Antispasmodic	7, 10, 14, 31,46	1, 18
Pirenzapine	Gastric acid Inhibitor	9	24
Propylthiouracil	Antithyroid	38	1, 3, 23
Procainamide	Antiarrhythmic	9	24
Ofloxacin	Antibiotic	9	24
Quinine	Antimalarial	4, 7, 10, 14, 31, 39, 40, 43, 46	1

T2RS ARE EXPRESSED OUTSIDE THE GUSTATORY SYSTEM

Although T2Rs were first discovered in the gustatory system, they are broadly expressed in multiple tissues and organs that play critical roles in disease response and in the maintenance of homeostasis (Table 2). T2Rs are reported in diverse cell types of the respiratory system [including ciliated respiratory epithelial cells, brush cells, solitary chemosensory cells (SCCs), and airway smooth muscle; refs. 6–8, 26] as well as in endocrine cells of the GI tract (9, 24, 27). The Gene Expression Omnibus collection (http://www.ncbi.nlm.nih.gov/geo) indicates that T2Rs are expressed in additional human tissues, including brain, heart, skeletal muscle, endometrium, liver, and omental adipose tissue. Like many other GPCRs, T2Rs are typically expressed at low levels. However, because GPCRs utilize a powerful intracellular amplification cascade for signaling, high expression levels are not required for significant physiological effects. Due to their widespread distribution and many nongustatory functions (see below), we suggest T2Rs comprise a previously unrecognized “chemosensory network” that can be intentionally targeted with new drugs or that could represent the basis of off-target effects of existing therapeutic agents.

Table 2.

Cell types expressing T2Rs

System	Tissue or cell type	Species	T2Rs expressed (T2Rx)a,b
Taste	Taste papillae	Human	1, 3, 4, 5, 7, 8, 9, 10, 13, 14, 16, 19, 20, 30, 31, 38, 39, 40, 41, 42, 43, 45, 46, 50, 60
	Taste papillae	Mouse	102, 103, 104, 105, 106, 107, 108, 109, 110, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 129, 130, 131, 134, 135, 136, 137, 138, 139, 140, 143, 144
Respiratory	Smooth muscle	Human	1, 3, 4, 5, 8, 9, 10, 13, 14, 19, 20, 30, 31, 42, 45, 46, 50
	Epithelial cells	Human	1, 3, 4, 7, 8, 9, 10, 13, 14, 16, 38, 43, 46
	Solitary chemosensory cells	Mouse	108, 119
Gastrointestinal	Stomach/duodenum	Rat	r1, r2, r3, r4, r5, r6, r7, r8, r9, r10, r12, r16, r34, r38
		Mouse	108, 134, 138
	Cecum/colon	Human	3, 4, 5, 9, 10, 13, 20, 30, 31, 38, 39, 40, 42, 43, 46, 50, 60
	Gastrointestinal cell lines
	NCI-H716 cells	Human	9, 38
	HuTu-80	Human	4, 5, 14, 16, 20, 30, 31, 38, 39, 40, 46, 50, 60
	STC-1 cells	Mouse	108, 134, 135, 137, 138, 144
	Ar42J	Rat	r16, r34, r38
Nervous	Brain neurons/glia	Rat	r107 (a.k.a. r10), r4, r38

^aT2R isoform numbers (x) are for the associated species.

^bSee Behrens and Meyerhof (10) for a review of T2R expression studies as well as a detailed discussion of T2R nomenclature.

In the respiratory system, T2Rs are implicated in several distinct roles. In the upper airways, T2R-expressing SCCs are associated with trigeminal nerve fibers and regulate protective airway reflexes in response to irritants in mice (8, 28). In the lower airways, stimulation of T2R-expressing cholinergic brush cells influences respiratory rate in mice (26). Human respiratory epithelial cells respond to bitter-tasting compounds with increased beating of motile cilia (6), potentially facilitating the clearance of pathogens or irritants. Finally, T2Rs are found on human and mouse airway smooth muscle, with bitter-tasting compounds evoking smooth muscle relaxation and bronchodilation (7, 29). Together, these findings suggest a role for T2Rs in clearing the airway of toxins or cellular debris that results from infections.

T2Rs are also expressed in the GI system, but again the tissue-specific roles need refinement. T2R or associated signaling molecules are found primarily in brush cells and in various endocrine cells of the mouse stomach and intestine (10). Amplification of T2R cDNAs from stomach, intestine, and enteroendocrine cell lines is consistent with this localization, as is the expression of the taste-related G protein α-gustducin (9, 10, 24, 27, 30). Bitter-tasting compounds can stimulate the secretion of gut hormones (e.g., cholecystokinin and glucagon-like peptide-1) from human and mouse enteroendocrine cell lines (24, 27) and can promote ghrelin release in vivo after an oral gavage (30). Thus, T2Rs may also regulate metabolism, satiety, gastric emptying, and the processing and absorption of ingested foods and pharmaceuticals.

All T2Rs are expressed in taste cells of the human gustatory epithelium, and every T2R-expressing taste cell expresses multiple, though perhaps not all, T2Rs (31). This broad expression is consistent with the need of the gustatory system to act as a broadly tuned and sensitive detector of potential toxins. However, to what extent nongustatory systems express the full T2R repertoire is unclear. Quantitative PCR results from human airway smooth muscle cDNA suggests that only about two-thirds of the T2R repertoire is present in that cell type and that broadly (e.g., T2R10 and T2R14), intermediate (e.g., T2R31), and narrowly (e.g., T2R5) tuned receptors are among the most highly expressed isoforms (7). Other tissues and cell types might be expected to express only a small subset of T2Rs that are most relevant to their particular physiology. It is imperative to quantify T2R expression in extraoral tissues if we are to elucidate their normal physiological roles outside of the gustatory system.

THE HYPOTHESIS

We hypothesize that extraoral T2Rs mediate many off-target effects, whether deleterious or beneficial, associated with numerous bitter-tasting pharmaceuticals.

TESTING THE HYPOTHESIS

If our hypothesis is correct, we reason that the following five criteria would be met for a drug of interest.

1) The drug of interest, or its metabolites, must activate ≥1 T2Rs at physiologically relevant concentrations. Effective activation of T2Rs is thought to require midnanomolar to micromolar concentrations of an agonist (1, 32). Thus, we expect that drug concentrations at the tissue or cell-type of interest should generally be in this range to exert effects through T2Rs. Indeed, the bioavailability of many drugs as well as their potential to accumulate in target tissues may be limiting factors in those compounds' ability to evoke physiological responses of T2R-expressing tissues. We caution, though, that both the T2R efficacies of many bitter compounds and the off-target tissue concentrations of many drugs are unknown.

2) T2R activation must lead to relevant cellular- or systems-level responses. For example, T2R-mediated release of gut insulinotropic hormones (9, 24) should impact glucose homeostasis in vivo.

3) T2R antagonists should reduce or eliminate the off-target effects of T2R agonists. While pan-T2R antagonists have not been identified, natural and synthetic T2R antagonists have been reported (33, 34) and could be used to antagonize subsets of T2Rs. For example, 4-(2,2,3-trimethylcyclopentyl)butanoic acid is a robust antagonist of several human T2Rs including T2R40 and T2R43 (33), while 3β-hydroxydihydrocostunolide and 3β-hydroxypelenolide, both found in wormwood, act as an antagonist or a weak partial agonist, respectively, for the broadly-tuned receptor T2R46 (34). The identification of these few inhibitors of ligand-dependent T2R activation suggest it will be possible to design effective antagonists for both broadly and narrowly tuned T2Rs.

4) Off-target effects should be reduced or absent in model systems where T2R expression can be decreased or eliminated (e.g., Tas2r-knockout mice).

5) T2R-mediated effects should be altered in individuals with nonfunctional alleles of the relevant TAS2R gene. Indeed, multiple haplotypes have been reported for all human TAS2Rs (35). Several of these variants have been shown to disrupt responses to specific compounds such as phenylthiocarbamide and aloin (20, 24, 36, 37) and thus provide the human equivalent of a knockout mouse. This genetic variation could help explain well-known interindividual variations in off-target effects outside the gustatory system. However, the overlapping specificities of many T2Rs (1) could mask the functional loss of a particular TAS2R gene.

Therefore, a combination of in vitro and in vivo studies, both in humans and in model systems, is required to fully test our hypothesis. High throughput screening strategies for identifying T2R ligands and antagonists have been successfully employed in industry and academia (1, 20, 24, 33), but there has not been a systematic focus on pharmaceuticals as potential T2R ligands. Localization studies are problematic because only a single antisera has been validated as T2R-specific (38). Thus, new specific T2R antisera must be developed and their specificity rigorously verified. We caution that T2Rs could be expressed in the affected organ or in neural or endocrine tissues that regulate the affected organ. In other words, T2R-mediated effects could be direct or indirect. For example, T2R agonists promote local airway dilation in isolated trachea (7). By contrast, SCC-associated trigeminal neurons likely mediate the respiratory depression and inflammatory responses seen after SCC stimulation with T2R agonists (28). In addition, T2R-mediated effects could be modulated by neural or endocrine factors.

Candidate gene studies in human populations could probe associations between TAS2R gene variants and patients' sensitivity to the off-target effects of individual drugs. Luckily, TAS2Rs are small genes with a single coding exon (10), factors that should facilitate genetic analyses. However, because many T2Rs respond to multiple ligands (1), individual bitter compounds often activate more than one T2R (1), and many individuals will carry multiple alleles for specific TAS2Rs, reduced sensitivity to particular compounds may be difficult to assess. Psychophysical assessments of taste responses, which have proven valuable in identifying genotype/phenotype associations, may also be useful tools for evaluating the physiological relevance of specific alleles (20, 25, 36). Animal models must be developed to assess the effects of Tas2r deletion or overexpression on physiological responses to ingested and inhaled compounds. The broad tuning and overlapping ligand specificities of many T2Rs (1) suggests that knocking out individual Tas2r genes will minimally informative for most T2R agonists (39). Instead, chromosomal engineering approaches that can take advantage of the relatively tight clustering of most Tas2rs on mouse chromosome 6 (10) could be employed to delete almost all Tas2rs in a single mouse. Alternatively, nonmammalian models such as fish or birds that possess a small T2R repertoire may be useful for assessing the contributions of these receptors in various tissues. Finally, transgenic expression of human T2Rs with no functional mouse orthologue (e.g., the β-d-glucopyranoside–responsive human receptor T2R16) to create “humanized” mice can permit in vivo testing of human receptors in an animal model (39). Such in vivo studies would likely precede, or at least parallel, clinical studies in humans.

CONCLUSIONS

We predict that any T2Rs could mediate unanticipated physiological changes in any T2R-expressing tissue exposed to significant concentrations of a bitter-tasting drug. T2Rs in respiratory and GI tissues would be particularly available to ingested or inhaled compounds, but drugs absorbed into the bloodstream could reach T2Rs in almost any tissue in the body. Our hypothesis, if supported, has important implications for drug development and medical practice. For example, routine testing of new drugs for their efficacy as T2R ligands might be recommended. Highly effective T2R agonists would warrant added caution because of the potential for unintended actions. Of course, T2R agonists or antagonists might also be valuable therapeutics, especially if they are selective for one or just a few T2Rs. Bitter-tasting phytochemicals (40), some of which are suspected to have biological activity, could similarly exert their physiological actions via extraoral T2Rs if target concentrations are high enough. Because individual tissues may express only a subset of T2Rs, adjusting the route of drug delivery may avoid potential complications or facilitate targeting to the desired site of action. Because of the significant genetic variation in human TAS2Rs, multiple T2R variants would need to be examined, and the use of T2R ligands as therapeutics tailored to a patient's individual genetic profile.