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 (HRH2) 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]

HRH2 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 HRH2 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 A). The limited structural diversity among HRH2 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 HRH2.[7] Ranitidine, famotidine, and nizatidine are variants of cimetidine. A new HRH2 blocker scaffold could aid in the structure–function understanding of HRH2 and serve as a starting point to develop next-generation treatment for peptidic ulcers and acid reflux.

Figure 1

Development of Gαs-coupled GPCR-based sensors. (A) Over-the-counter HRH2 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 Gα subunits and the yeast Gα (GPA1). Uniprot codes: Gαi (P63096), GPA1 (P08539), Gαq (P50148), Gα12 (Q03113), Gαs (P63092), and Gαolf (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 HRH2-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.

A high-throughput drug discovery approach could be applied to identify novel HRH2 blocker scaffolds. Such an approach requires access to a robust and rapid HRH2 blocking assay.[8] While activation of HRH2 leads to cAMP accumulation in CHO cells[9] and transcriptional upregulation of anti-inflammatory 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 HRH2 activation assay, coupled to its potentially difficult adaptation to high-throughput screening, limits the discovery of new HRH2 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 Gα subunit, GPA1, we have generated sensors using olfactory receptors,[13] which natively couple to Gαolf in olfactory neurons, and the serotonin receptor 4 (5-HTR4),[12,14] which couples to Gαs in mammalian cells. Given that HRH2 couples to Gαs, we hypothesized that it would also couple to the yeast machinery via GPA1.

Here, we engineer an HRH2-based sensor in yeast to aid in the discovery of a new HRH2 blocker scaffold. Hypothesizing that Gαs-coupled human GPCRs can couple to the yeast machinery via GPA1 for the generation of sensors, we set out to couple HRH2, a glucose-dependent insulinotropic receptor (GPR119), and a bile acid receptor (GPBAR1) to the yeast machinery. HRH2 and GPR119 coupled successfully to the yeast machinery; however, GPBAR1 did not. Next, we confirmed that the HRH2-based sensor could detect the known HRH2 blocker famotidine. Then, we used the HRH2-based sensor to screen a 403-member anti-infection chemical library for antimicrobial compounds that block HRH2 activation. We identified three antimicrobial agents, chloroxine, chlorquinaldol, and broxyquinoline, to act as HRH2 blockers in yeast. Interestingly, these compounds share an 8-hydroxyquinoline scaffold, which is not found among known HRH2 antagonists. Therefore, we validate chloroxine, chlorquinaldol, and broxyquinoline to also block HRH2 in mammalian cells. Molecular docking suggests the HRH2 blockers bind the HRH2 orthosteric site and initial structure–activity data suggest that HRH2 blockers act as competitive antagonists. The identification of 8-hydroxyquinoline as an HRH2 blocking scaffold has the potential to open the doors to the synthesis of next-generation HRH2 blockers.

Results and Discussion

Construction of Gαs-Based Sensors in Yeast

The C-terminus of the Gα subunit plays a critical role in coupling GPCRs to the signaling pathway.[15] A sequence alignment of five human Gα subtypes revealed that the C-termini of Gαs and Gαolf are highly conserved, with a 95% amino acid identity over the same range (Figure B). Although least similar to GPA1 (22.5% identity over the final 40 C-terminal residues), both Gαs- and Gαolf-coupled GPCRs have been successfully connected to the yeast machinery via GPA1.[11,14] This is unsurprising as Gαs-coupled receptors are known to show more promiscuous G-protein coupling.[15]

To determine the generality of building Gαs-coupled GPCR-based sensors in yeast, we swapped 5-HTR4 from the previously developed 5-HTR4-based sensor[12] with three mammalian Gαs-coupled GPCRs: HRH2, GPR119, and GPBAR1 (Figure C). GPR119 is expressed in pancreatic β-cells, 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-Gαs chimera.[17] GPBAR1 is overexpressed in macrophages and when activated reduces the expression of inflammatory genes.[18]

HRH2 and GPR119 couple to the yeast machinery via GPA1. The HRH2-based sensor results in a 9-fold increase in signal after activation upon the addition of 1 mM histamine (Figure D). The GPR119-based sensor has a 7-fold increase in signal after activation upon the addition of 100 μM OEA (Figure E). 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-Gα 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 HRH2-based sensor in yeast could be used to identify HRH2 blockers by first activating the sensor with histamine followed by blocker addition. HRH2 blocker hits would then be validated in yeast, and ultimately in mammalian cells (Figure A). First, the HRH2-based sensor was validated by detecting famotidine, a known HRH2 blocker (Figure B). Next, the HRH2-based sensor was used to screen a 403-member anti-infection chemical library for HRH2 blockers (Figure C). Twenty-one compounds reduced histamine-activated HRH2 signal by ≥50% (<0.5-fold activation compared to histamine-only 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.

Figure 2

Applying the HRH2-based sensor for HRH2 blocker discovery. (A) Workflow for the discovery of HRH2 blockers. The HRH2-based sensor in yeast is activated by histamine. HRH2 blockers are identified by activating the sensor with histamine and screening a 403-member anti-infection chemical library for a decrease in sensor signal. The HRH2 blocker hits are validated in yeast and mammalian cells. (B) Validation of the HRH2-based sensor in yeast using the known HRH2 blocker famotidine. Black line: HRH2-based sensor in the presence of histamine (1 mM) and famotidine (10–3–102 μM). Red line: control strain, i.e., yeast sensor expressing an empty plasmid instead of HRH2 under the same conditions. “H” is the sensor signal in the presence of 1 mM histamine only. “D” is the sensor signal in the presence of the carrier solvent DMSO only. All experiments were performed in triplicate. Shown are the mean and standard deviation. (C) Screening of 403-member anti-infection chemical library for the identification of HRH2 blockers. Blue squares: chemicals that show >50% reduction in HRH2-based sensor signal (dashed line 0.5). Pink squares: sample compounds that show increased fluorescence.

Validation of HRH2 Blocker Hits in Yeast

To eliminate false positives, we run dose–response curves of the 21 HRH2 blocker hits (Supporting Figure 4). Seven of the 21 hits: chlorquinaldol, chloroxine, broxyquinoline, closantel, octenidine, cetylpyridinium, and enrofloxacin lowered the histamine-activated HRH2-based sensor signal in a dose-dependent manner. To ensure that the signal observed was GPCR-dependent, we compared the decrease in luminescence signal from the histamine-activated HRH2-based sensor to that of a control strain carrying an empty vector instead of the HRH2 in the presence of the 7 HRH2 blocker hits (Figure A and Supporting Figure 5). Closantel and enroflaxin failed to lower histamine-activated HRH2-based sensor signal. Cetylpyridinium, and octenidine, lowered histamine-activated HRH2-based sensor signal in a dose-dependent manner. However, we discarded cetylpyridinium and octenidine as bona fide HRH2 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 8-hydroxyquinoline scaffold, which has not been previously linked to HRH2 blockers[22] (Figure A). Thinking that the 8-hydroxyquinoline 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 B).

Figure 3

Validation of the HRH2 blocker hits from the anti-infection library in yeast. (A) Dose–response curve of the HRH2-based sensor with chlorquinaldol, chloroxine, and broxyquinoline. Black line: HRH2-based sensor in the presence of histamine (1 mM) and HRH2 blocker hits (10–3–102 μM). Red line: Control strain, i.e., yeast sensor expressing an empty plasmid instead of HRH2 under the same conditions. “H” is the sensor signal in the presence of histamine (1 mM) only. “D” is the sensor signal in the presence of the carrier solvent DMSO only. The 8-hydroxyquinoline scaffold is in blue. Dose–response curves of closantel, octenidine, cetylpyridinium, and enroflaxacin can be found in Supporting Figure 5. (B) Toxicity assessment of chlorquinaldol, chloroxine, and broxyquinoline to yeast; * represents statistically significantly different cell growth (P < 0.005). All experiments were performed in triplicate. Shown are the mean and standard deviation. (C) Docking of histamine (blue), chlorquinaldol (magenta), chloroxine (yellow), and broxyquinoline (pink) in a model of the HRH2 receptor (gray) showing with key residues D98, Y250, D186, and T190 (green) and electrostatic interactions (dotted yellow lines).

Insight into 8-Hydroxyquinoline Binding

The 8-hydroxyquinoline 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 HRH2, we docked chlorquinaldol, chloroxine, and broxyquinoline to the AlphaFold model of HRH2[24,25] as there is no crystal structure available for HRH2. Previously, Asp98, Asp186, and Thr190 have been experimentally determined to be important for HRH2 histamine binding;[26] thus, those residues were used to define the HRH2 orthosteric site. As shown in Figure C, 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 HRH2 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-hydroxyquinoline-based HRH2 blockers bind the HRH2 orthosteric site, and thus are likely competitive antagonists.

8-Hydroxyquinoline as a General HRH2-Blocker Scaffold

To assess the generality of 8-hydroxyquinoline scaffold to block HRH2, 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 8-hydroxyquinoline 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 histamine-activated HRH2-based sensor in a GPCR-dependent and dose-dependent manner (Figure A).

Figure 4

8-Hydroxyquinoline as a general HRH2 blocker scaffold. (A) HRH2-dependent decrease in sensor signal in the presence of other 8-hydroxyquinoline-containing compounds found in the anti-infection library. Black line: HRH2-based sensor in the presence of histamine (1 mM) and 8-hydroxyquinoline-containing compounds (10–3–102 μM). Red line: control strain, i.e., yeast sensor strain expressing an empty plasmid instead of HRH2 under the same conditions. “H” is the sensor signal in the presence of histamine (1 mM) only. “D” is the sensor signal in the presence of the carrier solvent DMSO only. (B) Dose–response curves of the HRH2-based sensor in the presence of various concentrations of histamine and famotidine, chlorquinaldol, chloroxine, and broxyquinoline. The 8-hydroxyquinoline scaffold is in blue. The C2 methyl in chlorquinaldol is in pink. All experiments were performed in triplicate. Shown are the mean and standard deviation. (C) Docking overlay of chlorquinaldol (magenta) and chloroxine (yellow) on HRH2. Residues T190 and D186 (green) have electrostatic interactions with 8-hydroxyquinoline. Residues F254 and Y182 (cyan) are in close proximity to C2 methyl in chlorquinaldol.

Insight into the 8-Hydroxyquinoline Mode of Action

Chlorquinaldol only differs from chloroxine by a methyl group at C2. In broxyquinoline, the chlorine atoms from chloroxine have been swapped with bromine. To gain further insight into the role of the C2 position and the increasing size of the halogen groups at positions C5 and C7, we run dose–response curves varying both blocker and histamine concentrations. As shown in Figure B, famotidine, a known reversible competitive antagonist, can be displaced by increasing concentration of histamine to recover full HRH2 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 HRH2 shows that the C2 methyl is in a very crowded region, with Tyr182 and Phe254 approximately 3 Å away (Figure C), which may facilitate chlorquinaldol displacement by histamine. Taken together, the data suggest that the methyl group at C2 in the 8-hydroxyquinoline scaffold may be sufficient to alter the antagonism mechanism.

Validation of HRH2 Blocker Hits in Mammalian Cells

As chlorquinaldol, chloroxine, and broxyquinoline were identified in yeast, we validated their ability to block HRH2 in mammalian cells (Figure ). In mammalian cells, HRH2 couples to Gαs, and the addition of histamine results in an increase in cAMP levels. We expressed HRH2 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 A, 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 HRH2, 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 HRH2 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 B,C). Importantly, none of the HRH2 blockers showed major toxicity to mammalian cells up to 1 mM concentration as measured using an MTT cell proliferation assay for cell viability (Figure D). Indeed, the toxicity elicited by the three validated HRH2 blocker hits is comparable to that elicited by the known HRH2 blocker famotidine. Finally, we corroborated the identity of the chlorquinaldol, chloroxine, and broxyquinoline via proton and carbon nuclear magnetic resonance (Supporting Figures 7–12)

Figure 5

Validation of the HRH2 blocker hits in mammalian cells. (A–C) Dose–response curves of mammalian cells (HEK293T) cells co-transfected with HRH2 and cAMP sensor in the presence of histamine (1 μM) and various concentrations of (A) chlorquinaldol, (B) chloroxine, and (C) broxyquinoline. (A, C) Average and standard deviation of three independent transformants. (B) Average and standard deviation of three independent transformants in the case of “1 μM histamine” and two independent transformants in the case of “1 μM chloroxine and 1 μM histamine”. (D) Cell viability assessment of mammalian cells in the presence of chloriquinaldol, chloroxine, broxyquinoline, famotidine, or the carrier solvent DMSO using an MTT cell proliferation assay.

Biological Relevance of Newly Identified HRH2-Blockers

HRH2 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 HRH2 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, HRH2 and GPR119 successfully coupled to the yeast machinery to generate high-throughput sensors. We use the HRH2-based sensor in yeast to screen a 403-member anti-infection library leading to the discovery of chlorquinaldol, chloroxine, and broxyquinoline as HRH2 blockers in yeast. We show that 8-hydroxyquinoline is a general HRH2 blocker scaffold, and via computational docking studies using an HRH2 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 HRH2 blockers are competitive agonists, with potentially a different type of agonist behavior based on the moiety at the C2 position. Future mutagenesis studies are needed to confirm the role of Asp186 and Thr190 in 8-hydroxyquinoline binding as well as confirm the HRH2 antagonism mechanism.

As the blockers were discovered using a synthetic yeast assay, we validated the HRH2 blocker hits in mammalian cells, establishing 8-hydroxyquinoline as a new scaffold of HRH2 blockers. This sets the stage for using the 8-hydroxyquinoline scaffold for the design of novel HRH2 therapeutics for the treatment of acid reflux without the cancer-causing moiety present on many current HRH2 blockers. Of note, the three HRH2 blockers identified in this work are antimicrobials. Broxyquinoline and chloroxine can be found in the gut at concentrations that block HRH2 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.

Methods

Materials

The anti-infection chemical library (L3100) was purchased from Selleck Chemicals. Luciferase expression was assayed using the NanoGlo Luciferase Assay System (Promega N1120). Histamine dihydrochloride (Sigma H7250), famotidine (TCI F0530), oleolyethanolamide (Cayman 90265), lithocholic acid (Cayman 20253), chlorquinaldol (Selleck S4192), chloroxine (Selleck S1839), and broxyquinoline (Selleck S4195) were purchased from the specified vendors. HEK293T cells (CRL-11268) were obtained from ATCC.

Gs-Coupled GPCR-Based Sensor Construction

HRH2 (Uniprot P2501), GPR119 (Uniprot Q8TDV5), and GPBAR1 (Uniprot Q8TDU6) were codon-optimized for S. cerevisiae, commercially synthesized (Thermo Fisher), and cloned into pESC-HIS3-PTEF-PADH (pKM111)[10] at BamHI/SacII to generate pESC-HIS3-PTEF-HRH2 (pRLH16), pESC-HIS3-PTEF-GPR119 (pRLH15), and pESC-HIS3-PTEF-GPBAR1 (pRLH14), respectively. Constructs were sequence verified using primers EY46–2/NK12. To generate the GPCR-based sensor strains, pRLH16, pRLH15, and pRLH14 were co-transformed with pRS415-Leu2-PFIG1-NanoLuc (pEY15)[11] into PPY140 (S. cerevisiae W303 Δfar1, Δste2, Δsst2)[10] to generate PPY2171, PPY2172, and PPY2173, respectively. The no receptor control strain (PPY1809) was generated via co-transformation of pEY15 and pKM111 into PPY140.[11]

Histamine, Oleoylethanolamide, and Lithocholic Acid Sensing

An overnight culture of PPY2171, PPY2172, PPY2173, or PPY1809 was used to inoculate 50 mL of synthetic complete medium with 2% glucose lacking histidine and leucine (SD(HL–)) to an OD600 = 0.06. After 18 h at 15 °C (150 rpm), the cultures were centrifuged (3500 rpm, 10 min), and resuspended in SD(HL–) to an OD600 = 1. In a white, flat-bottomed 96-well plate, 190 μL of pH 7 SD (HL–), 8 μL of cells, and 2 μL of either histamine, oleoylethanolamide, or lithocholic acid (final concentration 0–104 μM), or DMSO as control were added. After chemical incubation (2.5 h, 30 °C, 250 rpm), 20 μL of a 1:100 mixture of NanoLuc substrate to NanoLuc buffer were added, and the reaction was incubated for 30 min (30 °C, 250 rpm). Luminescence was read in a Biotek Synergy 2 using default settings.

Screening 403-Member Anti-Infection Library for HRH2 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 HRH2 blocker hits were performed by following the library screening protocol using 100 μM histamine and 0.1–100 μM HRH2 blocker hit.

HRH2 Blocker Hits Validation in Yeast

Dose–response curves were performed by following the library screening protocol using 1 mM histamine and 10–3–102 μM of famotidine or the 7 HRH2 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 HRH2 sensor strain. The same protocol was used to validate cloxiquine, clioquinol, diiodohydroxyquinoline, and nitroxoline.

Yeast Toxicity Assay

To measure chloroxine, chlorquinaldol, and broxyquinoline toxicity to yeast, an overnight culture of PPY2171 was diluted to OD600 = 1 in fresh SD(HL-). Histamine (10 μL, final concentration 1 mM) and HRH2 blocker hit (10 μL, final concentration 10–3–102 μM) or DMSO as no chemical control was added to the cells. After chemical incubation (2.5 h, 30 °C, 250 rpm), absorbance (OD600) was measured.

Docking of HRH2 Blockers in the HRH2 Model

The HRH2 structure was obtained from AlphaFold (UniProt P25021).[24,25] Structure data files for histamine (ZINC388081), chlorquinaldol (ZINC119403), chloroxine (ZINC1131), and broxyquinoline (ZINC1064) were obtained from the ZINC15 database.[31] Hydrogens were added using CACTUS structure file generator (https://cactus.nci.nih.gov/translate/), and ChemDraw was used to protonate the nitrogen in the imidazole ring of chlorquinaldol, chloroxine, and broxyquinoline. The grid box (binding pocket) was defined by Asp98, Asp186, and Thr190.[26] Each chemical was then docked using AutoDock 4.2.6 with results visualized in AutoDockTools1.5.7[32] and Pymol.

HRH2 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% CO2 until reaching 70–90% confluence. Cells were harvested using 0.05% trypsin-EDTA and diluted to a concentration of 1.5 × 105 cells/mL. The cells were seeded into a 96-well plate (1.5 × 104 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 (CO2-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 × 102 cells/well, and incubated overnight (37 °C with 5% CO2). The cells were then incubated with 1 μm histamine and the HRH2 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% CO2. Absorbance was read at 570 nm (BioTek Synergy 2) using default absorbance settings.

Determining HRH2-Blocker Hit Mode of Action

The chemical library screening protocol was followed except that the HRH2-based sensor was activated with histamine (10–2–103 μM) prior to the addition of chlorquinaldol, chloroxine, broxyquinoline, or famotidine (0, 1, 10, 50 nM).

Structural Characterization of Chlorquinaldol, Chloroxine, and Broxyquinoline

Chlorquinaldol: 1H NMR (500 MHz, DMSO) δ 8.37 (dd, J = 8.6, 4.8 Hz, 1H), 7.73 (d, J = 3.9 Hz, 1H), 7.63 (dd, J = 8.7, 3.5 Hz, 1H), 2.76 (s, 3H). 13C NMR (126 MHz, DMSO) δ 159.50, 148.91, 138.90, 133.28, 127.18, 124.53, 123.55, 119.51, 115.89, 25.06. Chloroxine: 1H NMR (500 MHz, DMSO) δ 9.01 (dd, J = 4.2, 1.5 Hz, 1H), 8.53 (dd, J = 8.5, 1.5 Hz, 1H), 7.84 (s, 1H), 7.77 (dd, J = 8.5, 4.2 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 150.40, 149.74, 139.45, 133.39, 128.34, 125.32, 123.71, 119.39, 116.06. Broxyquinoline: 1H NMR (500 MHz, DMSO) δ 8.98 (dd, J = 4.1, 1.5 Hz, 1H), 8.46 (dd, J = 8.6, 1.5 Hz, 1H), 8.08 (s, 1H), 7.79 (dd, J = 8.5, 4.2 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 151.57, 150.34, 139.44, 135.93, 133.77, 126.97, 124.18, 109.29, 105.60.