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Discovery of 8-Hydroxyquinoline as a Histamine Receptor 2 Blocker Scaffold

Histamine receptor 2 (HRH2) activation in the stomach results in gastric acid secretion, and HRH2 blockers are used for the treatment of peptidic ulcers and acid reflux. Over-the-counter HRH2 blockers carry a five-membered aromatic heterocycle, with two of them additionally carrying a tertiary amine that decomposes to N-nitrosodimethylamine, a human carcinogen. To discover a novel HRH2 blocker scaffold to serve in the development of next-generation HRH2 blockers, we developed an HRH2-based sensor in yeast by linking human HRH2 activation to cell luminescence. We used the HRH2-based sensor to screen a 403-member anti-infection chemical library and identified three HRH2 blockers, chlorquinaldol, chloroxine, and broxyquinoline, all sharing an 8-hydroxyquinoline scaffold, which is not found among known HRH2 antagonists. Critically, we validate their HRH2-blocking ability in mammalian cells. Molecular docking suggests that the HRH2 blockers bind the histamine binding pocket and structure–activity data point toward these blockers acting as competitive antagonists. Chloroxine and broxyquinoline are antimicrobials that can be found in the gastrointestinal tract at concentrations that would block HRH2, thus likely modulating gastric acid secretion. Taken together, this work demonstrates the utility of GPCR-based sensors for rapid drug discovery applications, identifies a novel HRH2 blocker scaffold, and provides further evidence that antimicrobials not only target the human microbiota but also the human host.


■ INTRODUCTION
The histamine receptor 2 (HR H2 ) is expressed in gastric parietal cells, and activation by histamine produced by enterochromaffin-like cells results in gastric acid secretion. 1 Gastric acid causes heartburn and acid reflux in 30% of the US population, 2 with these issues manifesting chronically as gastroesophageal reflux disease (GERD) for 18−27% of the population. 3 HR H2 antagonists, such as ranitidine (Zantac), cimetidine (Tagamet), famotidine (Pepcid), and nizatidine (Mylan), are used as over-the-counter medications to reduce gastric acid secretion in the treatment of peptidic ulcers and acid reflux. All four HR H2 blockers are composed of a five-membered aromatic heterocycle. Two of them, ranitidine and nizatidine, additionally contain a tertiary amine that decomposes to N-nitrosodimethylamine, 4,5 a human carcinogen, which has led to the recall of these drugs from the market 6 ( Figure 1A). The limited structural diversity among HR H2 blockers in clinical use is likely due to the drug discovery approach. Cimetidine was identified in the 1970s by synthesizing a series of histamine analogues and testing their effectiveness for blocking HR H2. 7 Ranitidine, famotidine, and nizatidine are variants of cimetidine. A new HR H2 blocker scaffold could aid in the structure−function understanding of HR H2 and serve as a starting point to develop next-generation treatment for peptidic ulcers and acid reflux.
A high-throughput drug discovery approach could be applied to identify novel HR H2 blocker scaffolds. Such an approach requires access to a robust and rapid HR H2 blocking assay. 8 While activation of HR H2 leads to cAMP accumulation in CHO cells 9 and transcriptional upregulation of antiinflammatory proteins in macrophages, 10 mammalian-based assays require 1−2 weeks from cell culture to assay results. The extended time length required for the current HR H2 activation assay, coupled to its potentially difficult adaptation to highthroughput screening, limits the discovery of new HR H2 blocker scaffolds.
Previously, we have engineered G-protein-coupled receptor (GPCR)-based sensors in yeast by expressing human GPCRs on the cell surface and coupling their activation to the yeast machinery, ultimately resulting in cell fluorescence 11 or luminescence. 12 By coupling human GPCRs to the yeast G α subunit, GPA1, we have generated sensors using olfactory receptors, 13 which natively couple to G αolf in olfactory neurons, and the serotonin receptor 4 (5-HTR 4 ), 12,14 which couples to G αs in mammalian cells. Given that HR H2 couples to G αs , we hypothesized that it would also couple to the yeast machinery via GPA1.
Here, we engineer an HR H2 -based sensor in yeast to aid in the discovery of a new HR H2 blocker scaffold. Hypothesizing that G αs -coupled human GPCRs can couple to the yeast machinery via GPA1 for the generation of sensors, we set out to couple HR H2 , a glucose-dependent insulinotropic receptor (GPR119), and a bile acid receptor (GPBAR1) to the yeast machinery. HR H2 and GPR119 coupled successfully to the yeast machinery; however, GPBAR1 did not. Next, we confirmed that the HR H2 -based sensor could detect the known HR H2 blocker famotidine. Then, we used the HR H2based sensor to screen a 403-member anti-infection chemical library for antimicrobial compounds that block HR H2 activation. We identified three antimicrobial agents, chloroxine, chlorquinaldol, and broxyquinoline, to act as HR H2 blockers in yeast. Interestingly, these compounds share an 8-hydroxyquinoline scaffold, which is not found among known HR H2 antagonists. Therefore, we validate chloroxine, chlorquinaldol, and broxyquinoline to also block HR H2 in mammalian cells. Molecular docking suggests the HR H2 blockers bind the HR H2 orthosteric site and initial structure−activity data suggest that HR H2 blockers act as competitive antagonists. The identification of 8-hydroxyquinoline as an HR H2 blocking scaffold Figure 1. Development of G αs -coupled GPCR-based sensors. (A) Over-the-counter HR H2 blockers: ranitidine (Zantac), cimetidine (Tagamet), famotidine (Pepcid), and nizatidine (Mylan). All blockers share a five-membered aromatic heterocycle (blue). Ranitidine and nizatidine contain a tertiary amine (red) that decomposes to N-nitrosodimethylamine, a human carcinogen. (B) Sequence alignment of the five human G α subunits and the yeast G α (GPA1). Uniprot codes: G αi (P63096), GPA1 (P08539), G αq (P50148), G α12 (Q03113), G αs (P63092), and G αolf (P38405). (C) Schematic of the GPCR-based sensor in yeast. Activation of the GPCR (blue) on the yeast cell surface couples to GPA1 (yellow) that activates the yeast mating pathway ultimately resulting in luciferase expression (purple). (D) Dose−response curve of the HR H2 -based sensor with histamine. (E) Dose−response curve of the glucose-dependent insulinotropic receptor (GPR119)-based sensor with oleoylethanolamide. All experiments were performed in triplicate. Shown are the mean and standard deviation.
has the potential to open the doors to the synthesis of nextgeneration HR H2 blockers.

■ RESULTS AND DISCUSSION
Construction of G αs -Based Sensors in Yeast. The Cterminus of the G α subunit plays a critical role in coupling GPCRs to the signaling pathway. 15 A sequence alignment of five human G α subtypes revealed that the C-termini of G αs and G αolf are highly conserved, with a 95% amino acid identity over the same range ( Figure 1B). Although least similar to GPA1 (22.5% identity over the final 40 C-terminal residues), both G αs -and G αolf -coupled GPCRs have been successfully connected to the yeast machinery via GPA1. 11,14 This is unsurprising as G αs -coupled receptors are known to show more promiscuous G-protein coupling. 15 To determine the generality of building G αs -coupled GPCRbased sensors in yeast, we swapped 5-HTR 4 from the previously developed 5-HTR 4 -based sensor 12 with three mammalian G αs -coupled GPCRs: HR H2 , GPR119, and GPBAR1 ( Figure 1C). GPR119 is expressed in pancreatic βcells, which secrete insulin upon activation by oleoylethanolamide (OEA), making GPR119 a pharmaceutical target for new antidiabetic drugs. 16 Of note, GPR119 has been previously coupled to the yeast machinery via a GPA1-G αs chimera. 17 GPBAR1 is overexpressed in macrophages and when activated reduces the expression of inflammatory genes. 18 HR H2 and GPR119 couple to the yeast machinery via GPA1. The HR H2 -based sensor results in a 9-fold increase in signal after activation upon the addition of 1 mM histamine ( Figure  1D). The GPR119-based sensor has a 7-fold increase in signal after activation upon the addition of 100 μM OEA ( Figure   1E). Histamine and OEA show limited activation of the sensor control strain, where the vector expressing the receptor has been swapped with an empty vector, confirming that the sensor activation is GPCR-dependent. GPBAR1 did not couple to GPA1 (Supporting Figure 1), underscoring the fact that GPCR-G α coupling is a complex multisurface process. 15 Of note, because the GPCR-based sensors are plasmid-based, we screened six distinct colonies to identify optimal biosensor response, with ≥50% of colonies giving robust agonist response. The difference in response is attributed to the fact that the GPCRs are expressed from a multicopy plasmid, leading to a different number of GPCRs trafficking to the membrane in each strain (Supporting Figure 2).
The HR H2 -based sensor in yeast could be used to identify HR H2 blockers by first activating the sensor with histamine followed by blocker addition. HR H2 blocker hits would then be validated in yeast, and ultimately in mammalian cells ( Figure  2A). First, the HR H2 -based sensor was validated by detecting famotidine, a known HR H2 blocker ( Figure 2B). Next, the HR H2 -based sensor was used to screen a 403-member antiinfection chemical library for HR H2 blockers ( Figure 2C). Twenty-one compounds reduced histamine-activated HR H2 signal by ≥50% (<0.5-fold activation compared to histamineonly signal). Some of the compounds that resulted in an increase in sensor luminescence turned out to be antifungal agents, including tioconazole, sulconazole, and bedaquiline. 19 The increase in the luminescence of tioconazole was corroborated via a dose−response curve (Supporting Figure  3). Thus, increased sensor luminescence was likely nonspecific.
Validation of HR H2 Blocker Hits in Yeast. To eliminate false positives, we run dose−response curves of the 21 HR H2 blocker hits (Supporting Figure 4). Seven of the 21 hits: chlorquinaldol, chloroxine, broxyquinoline, closantel, octeni- dine, cetylpyridinium, and enrofloxacin lowered the histamineactivated HR H2-based sensor signal in a dose-dependent manner. To ensure that the signal observed was GPCRdependent, we compared the decrease in luminescence signal from the histamine-activated HR H2 -based sensor to that of a control strain carrying an empty vector instead of the HR H2 in the presence of the 7 HR H2 blocker hits ( Figure 3A and Supporting Figure 5). Closantel and enroflaxin failed to lower histamine-activated HR H2 -based sensor signal. Cetylpyridinium, and octenidine, lowered histamine-activated HR H2based sensor signal in a dose-dependent manner. However, we discarded cetylpyridinium and octenidine as bona f ide HR H2 blockers as they are charged compounds with long chain hydrocarbon tails, which could embed themselves in the yeast membrane causing nonspecific cell toxicity, resulting in a reduction in the luminescent signal. Indeed, both compounds have been shown to be toxic to Saccharomyces cerevisiae. 20,21 Chlorquinaldol, chloroxine, and broxyquinoline all share an 8hydroxyquinoline scaffold, which has not been previously linked to HR H2 blockers 22 ( Figure 3A). Thinking that the 8hydroxyquinoline scaffold may be interfering with DNA replication or transcription, thus imparting toxicity to yeast, we measured their toxicity to yeast. For all three compounds, some reduction in cell growth can be observed above 0.1 μM. Chloroxine leads to a 50% reduction in cell growth at more than 1 μM. Chlorquinaldol and broxyquinoline have lower cell toxicity, leading to a 20% reduction in cell growth reduction at more than 100 μM ( Figure 3B).
Insight into 8-Hydroxyquinoline Binding. The 8hydroxyquinoline scaffold is intriguing as it lacks the basic amine group commonly found among aminergic GPCR antagonists. 23 To assess how the 8-hydroxyquinoline scaffold may be binding to HR H2 , we docked chlorquinaldol, chloroxine, and broxyquinoline to the AlphaFold model of HR H2 24,25 as there is no crystal structure available for HR H2 . Previously, Asp98, Asp186, and Thr190 have been experimentally determined to be important for HR H2 histamine binding; 26 thus, those residues were used to define the HR H2 orthosteric site. As shown in Figure 3C, the model suggests that the amino group of histamine forms a hydrogen bond with Asp98 (2.9 Å), which is consistent with previous experimental studies. 26 The protonated nitrogen in the imidazole ring forms a hydrogen bond with Tyr250 (2.9 Å), which is consistent with previous molecular dynamics simulations that involve Tyr250 in HR H2 agonist binding. 27 Confident that the model was docking histamine at the correct location, we docked chlorquinaldol, chloroxine, and broxyquinoline. Interestingly, the three 8-hydroxyquinoline antagonists bound slightly lower in the binding pocket. The hydroxyl group makes electrostatic interactions with Thr190 (2.7 Å), while the protonated nitrogen in the quinoline ring makes electrostatic interactions with Asp186 (2.7 Å) rather than the canonical Asp98. Taken together, these docking studies suggest that the 8-hydrox- yquinoline-based HR H2 blockers bind the HR H2 orthosteric site, and thus are likely competitive antagonists.
8-Hydroxyquinoline as a General HR H2 -Blocker Scaffold. To assess the generality of 8-hydroxyquinoline scaffold to block HR H2 , we scanned the anti-infection library for compounds containing 8-hydroxyquinoline that may have shown as false negative in our original screen. Cloxiquine, clioquinol, diiodohydroxyquinoline, and nitroxoline were the most similar compounds, all of them carrying the 8hydroxyquinoline core, and varying only in the number and identity of the halogen groups on the phenyl ring. Dose− response curves of cloxiquine, clioquinol, diiodohydroxyquinoline, and nitroxoline decrease the signal from the histamineactivated HR H2 -based sensor in a GPCR-dependent and dosedependent manner ( Figure 4A).
Insight into the 8-Hydroxyquinoline Mode of Action. Chlorquinaldol only differs from chloroxine by a methyl group at C 2 . In broxyquinoline, the chlorine atoms from chloroxine have been swapped with bromine. To gain further insight into the role of the C 2 position and the increasing size of the halogen groups at positions C 5 and C 7 , we run dose−response curves varying both blocker and histamine concentrations. As shown in Figure 4B, famotidine, a known reversible competitive antagonist, can be displaced by increasing concentration of histamine to recover full HR H2 receptor activation. A similar behavior is observed with chlorquinaldol. Chloroxine and broxyquinoline, however, cannot be competed out with increasing histamine concentration, hinting at a potential irreversible competitive antagonist behavior. An overlay of docked chlorquinaldol and chloroxine on HR H2 shows that the C 2 methyl is in a very crowded region, with Tyr182 and Phe254 approximately 3 Å away ( Figure 4C), which may facilitate chlorquinaldol displacement by histamine. Taken together, the data suggest that the methyl group at C 2 in Validation of HR H2 Blocker Hits in Mammalian Cells. As chlorquinaldol, chloroxine, and broxyquinoline were identified in yeast, we validated their ability to block HR H2 in mammalian cells ( Figure 5). In mammalian cells, HR H2 couples to G αs , and the addition of histamine results in an increase in cAMP levels. We expressed HR H2 in HEK293T cells and measured the decrease in cAMP levels of cells in the presence of histamine only, and histamine with different concentrations of chlorquinaldol, chloroxine, or broxyquinoline. As shown in Figure 5A, the addition of 1 μM histamine and 0.1 μM or more than 10 μM chlorquinaldol significantly reduces cAMP levels compared to cells activated with 1 μm histamine. At 1 μM, chlorquinaldol seems to also block HR H2 , albeit the data were too noisy to draw any conclusions (Supporting Figure 6). The noisiness of the data can be explained by the fact that the cells were transiently expressed with HR H2 and the cAMP sensor. In the presence of 1 μM histamine and either 10 μM broxyquinoline or 1 μM chloroxine, a decrease in cAMP levels was also observed ( Figure 5B,C). Importantly, none of the HR H2 blockers showed major toxicity to mammalian cells up to 1 mM concentration as measured using an MTT cell proliferation assay for cell viability ( Figure 5D). Indeed, the toxicity elicited by the three validated HR H2 blocker hits is comparable to that elicited by the known HR H2 blocker famotidine. Finally, we corroborated the identity of the chlorquinaldol, chloroxine, and broxyquinoline via proton and carbon nuclear magnetic resonance (Supporting Figures 7−12) Biological Relevance of Newly Identified HR H2 -Blockers. HR H2 is found in the parietal cell in the stomach, and both broxyquinoline and chloroxine can be found in the gastrointestinal tract at currently prescribed dosages. Chloroxine is used as an antidiarrhea medication in the treatment of intestinal microflora disorders, with a dosage of 250 mg, resulting in a theoretical maximum stomach concentration of 1.2 mM, more than 100 times the functional concentration shown here. Broxyquinoline (Intestopan) is an antiprotozooan and also used to treat diarrhea and inhibit cryptosporidium growth. 28 Broxyquinoline dosage is two 500 mg capsules, given three times a day. 29 If all broxyquinoline makes it to the stomach to interact with HR H2 expressing parietal cells, the receptors could experience broxyquinoline concentrations of up to 3.3 mM, more than 300 times the functional concentration shown here.

■ CONCLUSIONS
Taken together, HR H2 and GPR119 successfully coupled to the yeast machinery to generate high-throughput sensors. We use the HR H2 -based sensor in yeast to screen a 403-member antiinfection library leading to the discovery of chlorquinaldol, chloroxine, and broxyquinoline as HR H2 blockers in yeast. We show that 8-hydroxyquinoline is a general HR H2 blocker scaffold, and via computational docking studies using an HR H2 model, we put forth that 8-hydroxyquinoline binds at the same site as histamine, albeit making electrostatic contacts with Asp186 and Thr190 rather than Asp98 and Tyr250. Preliminary structure−activity relationship (SAR) studies suggest that the identified 8-hydroxyquinoline HR H2 blockers are competitive agonists, with potentially a different type of agonist behavior based on the moiety at the C 2 position. Future mutagenesis studies are needed to confirm the role of Asp186 and Thr190 in 8-hydroxyquinoline binding as well as confirm the HR H2 antagonism mechanism.
As the blockers were discovered using a synthetic yeast assay, we validated the HR H2 blocker hits in mammalian cells, establishing 8-hydroxyquinoline as a new scaffold of HR H2 blockers. This sets the stage for using the 8-hydroxyquinoline scaffold for the design of novel HR H2 therapeutics for the treatment of acid reflux without the cancer-causing moiety present on many current HR H2 blockers. Of note, the three HR H2 blockers identified in this work are antimicrobials. Broxyquinoline and chloroxine can be found in the gut at concentrations that block HR H2 at currently prescribed concentrations. Chlorquinaldol (Siosteran) is a topical antimicrobial agent. 30 The identification of antimicrobial-gut GPCR interactions shows that antimicrobials interact with human receptors and their activity in the gut may not be confined to only interactions with the microbiota.
Screening 403-Member Anti-Infection Library for HR H2 Blockers. The histamine sensing protocol was followed except as described. In a white, flat-bottomed 96-well plate, 188 μL of pH 7 SD (HL − ), 8 μL of cells, 2 μL of histamine (final concentration of 100 μM), 2 μL of anti-infection library compound (final concentration of 1 μM) were added. For the no chemical control, only 4 μL of DMSO was added and no histamine or compound was added. False-positive identification: Dose−response curves of the 21 HR H2 blocker hits were performed by following the library screening protocol using 100 μM histamine and 0.1−100 μM HR H2 blocker hit.
HR H2 Blocker Hits Validation in Yeast. Dose−response curves were performed by following the library screening protocol using 1 mM histamine and 10 −3 −10 2 μM of famotidine or the 7 HR H2 blocker hits. DMSO, the carrier solvent, was used as the no chemical control. The no receptor control strain was tested under the same conditions as the HR H2 sensor strain. The same protocol was used to validate cloxiquine, clioquinol, diiodohydroxyquinoline, and nitroxoline.
HR H2 Blocker Hits Validation in Mammalian Cells. HEK293T cells were grown in T75 flasks using growth medium (DMEM with GlutaMAX, 10% fetal bovine serum (FBS), and 1% pen/strep) at 37°C with 5% CO 2 until reaching 70−90% confluence. Cells were harvested using 0.05% trypsin-EDTA and diluted to a concentration of 1.5 × 10 5 cells/mL. The cells were seeded into a 96-well plate (1.5 × 10 4 cells/well) and incubated overnight. The cells were transiently transfected with PPY2295 and PPY2325 using FuGENE HD (Promega), and the plate was incubated overnight. The following day, the growth media was replaced with freshly made equilibrium media (CO 2 -independent medium with 10% FBS, and 5% GloSensor cAMP reagent). The plate was placed in the luminescent plate reader (BioTek Synergy 2), and the cells were equilibrated at 25°C for 2 h with readings every 15 min. The plate was then removed, and 2 μL of famotidine, chloroxine, chlorquinaldol, or broxyquinoline (final concentration 0.1 μM to 1 mM) was added. As the no chemical control, 2 μL of DMSO was used. The plate was incubated for 10 min. Next, 2 μL of histamine (final concentration 1 μM) was added and the plate was placed back into the plate reader. Luminescence was read every 2 min for 30 min.
Mammalian Toxicity Assay. Mammalian cell viability was assayed using an MTT Cell Proliferation Assay Kit (Cayman Chemical, 10009365). HEK293T cells were cultured in T75 flasks until reaching 70−90% confluence. Cells were trypsinized, counted, and replated in a clear, flat-bottom 96-well plate at a concentration of 5 × 10 2 cells/well, and incubated overnight (37°C with 5% CO 2 ). The cells were then incubated with 1 μm histamine and the HR H2 blocker hits (0.1 μM to 1 mM) or DMSO as a no chemical control. After 24 h, the directions from the MTT Assay Kit were followed. Briefly, 10 μL of MTT reagent was added to each well and the cells were incubated for 4 h. Next, 100 μL of crystal dissolving solution was added to each well and the cells were incubated for 18 h at 37°C with 5% CO 2 . Absorbance was read at 570 nm (BioTek Synergy 2) using default absorbance settings.