Discovery of Natural Phenols as G Protein-Coupled Receptor-35 (GPR35) Agonists

We report the discovery and characterization of natural phenols as G protein-coupled receptor-35 (GPR35) agonists. Pharmacological characterization using label-free dynamic mass redistribution and Tango β-arrestin translocation assays revealed that GPR35-active natural phenols are divergent in their biased agonism

The simulated dose responses of distinct agonists as a function of spare receptors in the cell using the operational model.

<p>(a) epinephrine; (b) pindolol; (c) salbutamol; and (d) salmeterol. The spare receptors were normalized in percentage.</p

The DMR signals induced by distinct β₂AR agonists under distinct conditions.

<p>(a) salbutamol, (b) pindolol, and (c) salmeterol, under the 2 min pulse stimulation in comparison with the sustained stimulation conditions. (d) The amplitudes of both N-DMR and P-DMR (30 min post stimulation) of the 2^nd epinephrine stimulation induced DMR as a function of different agonists. For receptor desensitization studies, two steps were separated by ∼45 min. The initial stimulation duration with an agonist was 2 min, while the second stimulation was continuous. The concentration was 2 nM, 5 nM, 10 nM, and 100 nM for epinephrine, pindolol, salbutamol, and salmeterol, respectively. n = 3.</p

The DMR patterns of cells upon repeated stimulation with pindolol and epinephrine.

<p>Step 1: 2 min pulse stimulation in the absence and presence of an inhibitor, followed by perfusion with the assay buffer in the absence and presence of the respective inhibitor; and Step 2: continuous exposure to 2 nM epinephrine using perfusion. (a) DMR signals, and (b) DMR characteristics, later of which the P-DMR of the 1^st pindolol-induced DMR, and both N-DMR and P-DMR of the 2^nd EPI induced DMR were plotted as a function of inhibitors. The flow rate was 1 µl/min under all conditions. n = 2 to 4.</p

The simulated sensitivity of distinct agonists to loss of receptors (in percentage).

<p>(a) The effect of an EC₁₀₀ of each agonist (calculated where the total receptor is unchanged; <i>R_T</i> = 100) at different receptor numbers. Data were normalized to percentage of the response at the EC₁₀₀ where <i>R_T</i> = 100. (d) The function of <i>E_max</i> achievable by the agonists as receptor loss.</p

A microfluidic RWG biosensor array for characterizing ligand-directed desensitization of the β₂-adrenergic receptor.

<p>(a) Schematic of the biosensor microchamber, wherein an agonist solution, 2 µl in total, is perfused at a flow rate of 1 µl/min between two perfusion steps with the assay buffer, thus creating 2 min pulse stimulation to the cells located within the detection area (black box). During the time the agonist solution passing through the microchamber, small diffusion occurs. Since the detection area (0.2 mm) is smaller than the width of the agonist solution (∼2 mm for the 2 min pulse stimulation), the agonist concentration exposed to the cells within the detection area is considered to be constant. The white box indicates the location of the biosensor. (b) Structures of four β₂-agonists examined.</p

Receptor internalization and ERK1/2 phosphorylation are dependent on the agonist exposure time.

<p>(a)–(d) Confocal images of cells after immunostained with anti-β₂AR. The cells were first stimulated with 2 nM epinephrine for 1 min (a), 2 min (b), 5 min (c) and 30 min (d). After epinephrine removal, the cells were further incubated in medium at 37°C for 29 min (a), 28 min (b), 25 min (c) and 0 min (d), respectively, such that equal time post stimulation was achieved for all. The staining was performed using anti-β₂AR antibody and Alexa Fluor 488 goat anti-rabbit antibody. The nuclei were stained with DAPI. Confocal microscopy images were acquired on Zeiss confocal laser scanning microscope (oil-immersion, 63× objective). The green color is from the β₂AR, the blue from the nuclei. (e) The agonist exposure time and Ro31-8220 sensitivity of pERK stimulated by epinephrine in A431 cells. The cells were incubated with the vehicle (0 min) or 2 nM epinephrine for a specific time (1 min, 2 min, 30 min), followed by further incubation at 37°C for another time to ensure all reach 30 min post simulation. Ro31-8220 when used was presented throughout the incubation. Equal amounts of cell lysate were separated by SDS-PAGE and analyzed for pERK by Western blotting. Equivalent gel loading was confirmed by probing with antibodies against GAPDH.</p

The desensitization and resensitization patterns of quiescent A431 cells induced by epinephrine is sensitive to stimulation duration and several inhibitors.

<p>(a) The DMR signals upon repeated stimulations with epinephrine. After initial baseline (∼2 min), the cells were first stimulated with epinephrine (1^st EPI) for three different durations (1 min, 2 min, or 5 min), followed by perfusion with the assay vehicle for ∼35 min, and finally stimulated again with a continuous flow of epinephrine (2^nd EPI) for ∼30 min. (b) The amplitudes of both N-DMR and P-DMR of the 2^nd EPI induced DMR as a function of the 1^st stimulation conditions. (c) The epinephrine DMR in the absence and presence of dynasore. The cells were first stimulated with epinephrine for 2 min (1^st EPI) in the presence and absence of dynasore, followed by perfusion with the assay buffer in the absence and presence of dynasore, respectively. Afterwards, all the cells were repeatedly stimulated with epinephrine in the absence of dynasore (2^nd EPI). (d) The amplitudes of both N-DMR and P-DMR of the 2^nd EPI induced DMR as a function of inhibitors. Each inhibitor or their combinations were assayed in a manner similar to dynasore. The flow rate was 1 µl/min under all conditions. Inhibitor concentrations were 5 µM, 10 µM, 10 µM, and 50 µM for H-89, Ro31-8220, PP1, and dynasore, respectively. Epinephrine dose was 2 nM for all. n = 3.</p

Discovery of 2-(4-Methylfuran-2(5<i>H</i>)-ylidene)malononitrile and Thieno[3,2-<i>b</i>]thiophene-2-carboxylic Acid Derivatives as G Protein-Coupled Receptor 35 (GPR35) Agonists

Screening with dynamic mass redistribution (DMR) assays in a native cell line HT-29 led to identification of two novel series of chemical compounds, 2-(4-methylfuran-2(5<i>H</i>)-ylidene)malononitrile and thieno[3,2-<i>b</i>]thiophene-2-carboxylic acid derivatives, as GPR35 agonists. Of these, 2-(3-cyano-5-(3,4-dichlorophenyl)-4,5-dimethylfuran-2(5<i>H</i>)-ylidene)malononitrile (YE120) and 6-bromo-3-methylthieno[3,2-<i>b</i>]thiophene-2-carboxylic acid (YE210) were found to be the two most potent GPR35 agonists with an EC₅₀ of 32.5 ± 1.7 nM and 63.7 ± 4.1 nM, respectively. Both agonists exhibited better potency than that of zaprinast, a known GPR35 agonist. DMR antagonist assays, knockdown of GPR35 with interference RNA, receptor internalization assays, and Tango β-arrestin translocation assays confirmed that the agonist activity of these ligands is specific to GPR35. The present study provides novel chemical series as a starting point for further investigations of GPR35 biology and pharmacology