Development of an Affinity-Based Probe to Profile Endogenous Human Adenosine A3 Receptor Expression

Abstract

The adenosine A₃ receptor (A₃AR) is a G protein-coupled receptor (GPCR) that exerts immunomodulatory effects in pathophysiological conditions such as inflammation and cancer. Thus far, studies toward the downstream effects of A₃AR activation have yielded contradictory results, thereby motivating the need for further investigations. Various chemical and biological tools have been developed for this purpose, ranging from fluorescent ligands to antibodies. Nevertheless, these probes are limited by their reversible mode of binding, relatively large size, and often low specificity. Therefore, in this work, we have developed a clickable and covalent affinity-based probe (AfBP) to target the human A₃AR. Herein, we show validation of the synthesized AfBP in radioligand displacement, SDS-PAGE, and confocal microscopy experiments as well as utilization of the AfBP for the detection of endogenous A₃AR expression in flow cytometry experiments. Ultimately, this AfBP will aid future studies toward the expression and function of the A₃AR in pathologies.

Figure 1

(A) Previous work: covalent hA₃AR antagonist LUF7602 and control compound LUF7714; (B) this work: AfBP LUF7960; (C) strategy to label the hA₃AR with an AfBP. First, the AfBP is added to cells or membrane fractions to allow irreversible bond formation between receptor and probe. Click reagents are then added to install a detection moiety onto the probe-bound receptor. Lastly, cells are further processed for detection, dependent on the detection method of interest. The image of the hA₃AR was generated using Protein Imager, using the structure of the hA₃AR (AF-P0DMS8-F1) as predicted by Alphafold.^,

Scheme 1. Synthesis of hA₃AR-Targeting Affinity-Based Probes

Reagents and conditions: (a) DBU, 1-bromopropane, MeCN, 70 °C, 1 h, quant.; (b) Pd(OH)₂/C, NH₄HCO₂, EtOH, 80 °C, 7 days, 53%; (c) K₂CO₃, DMF, rt, overnight, 29%; (d) tert-butyl (3-bromopropyl)carbamate, K₂CO₃, DMF, 100 °C, 2 h, 97%; (e) TFA, CHCl₃, 60 °C, overnight, 90%; (f) (i) BBr₃ 1 M in DCM, CHCl₃, 50 °C, 6 days; (ii) 4-fluorosulfonyl benzoic acid, EDC·HCl, DIPEA, DMF, rt, two days, 13% over two steps; (g) propargyl bromide (80% in toluene), K₂CO₃, DMF, rt, overnight, 30%; (h) Pd(OH)₂/C, NH₄HCO₂, EtOH, 80 °C, 7 days; (i) tert-butyl (3-bromopropyl)carbamate, K₂CO₃, DMF, 0–40 °C, 6 days, 25%; (j) (i) propargyl bromide (80% in toluene), DBU, MeCN, rt, overnight; (ii) TFA, DCM, rt, 2 h, 67%; (k) 4-fluorosulfonyl benzoic acid, EDC·HCl, DIPEA, DMF, rt, 3 h, 13%.

Figure 2

Putative binding modes of compounds 5 (A), 9 (B), and 13 (C) in an Alphafold model of the hA₃AR (AF-P0DMS8-F1-model_v4).^, The extracellular side of the receptor is located at the top of the images, while the intracellular side is at the bottom. All three compounds show a hydrogen bond interaction with the conserved N250 and π–π stacking with Phe168, two well-known interactions in ligand recognition of adenosine receptors. The alkyne group, indicated with an arrow in each panel, fits into the binding pocket on each exit vector, and the binding orientation of the core compound, as published previously by our group, is maintained.

Figure 3

Wash-out assay reveals covalent binding of all three probes to the hA₃AR. CHO cells membranes stably transfected with the hA₃AR were pre-incubated with 1% DMSO (vehicle), 1 μM non-covalent control compound LUF7714, or 1 μM compounds 5, 9, or 13. The samples were washed for either 0 or 4 times, before being exposed to [³H]PSB-11 in a radioligand displacement assay. Data is expressed as the percentage of the vehicle group (100%) and represents the mean ± SEM of three individual experiments performed in duplicate. ****p < 0.0001 determined by a two-way ANOVA test using multiple comparisons.

Figure 4

Labeling of the hA₃AR by the synthesized affinity-based probes. (A) Labeling of proteins by 5, 9, and 13. Membrane fractions from CHO cells stably overexpressing the hA₃AR were pre-incubated for 30 min with the antagonist (PSB-11, 1 μM final concentration) or 1% DMSO (control), prior to incubation for 1 h with the respective probe (50 nM final concentration). The proteins were subjected to PNGase or MilliQ (control) for 1 h to remove N-glycans. Samples were then subjected to a copper-catalyzed click reaction with Cy5-N₃ (1 μM final concentration), denatured using Laemmli buffer (4×) and resolved by SDS-PAGE. Gels were imaged using in-gel fluorescence and stained with Coomassie Brilliant Blue (CBB) as protein loading control. (B) Quantification of the lower hA₃AR band (±30 kDa). The band intensities were taken and corrected for the observed amount of protein per lane upon CBB staining. The band at 55 kDa of the PageRuler Plus ladder (not shown) was set to 100% for each gel, and band intensities were calculated relative to this band. The mean values ± SEM of three individual experiments are shown. Significance was calculated using a one-way ANOVA test using multiple comparisons (ns = not significant). (C) Control experiments with probe 9. Membrane fractions from CHO cells with or without (first lane) stable expression of the hA₃AR were pre-incubated for 30 min with antagonist (PSB-11, 1 μM final concentration) or 1% DMSO (control), prior to incubation for 1 h with 9 (50 nM final concentration) or 1% DMSO (control). Proteins were deglycosylated with PNGase for 1 h. The click mix was then added, containing CuSO₄ or MilliQ (control) and Cy5-N₃ (1 μM final concentration) or DMSO (control). The samples were then denatured with Laemmli buffer (4×) and resolved by SDS-PAGE. Gels were imaged using in-gel fluorescence and afterward stained with CBB. The image shown is a representative of three individual experiments.

Figure 5

Labeling of the hA₃AR on live CHO cells. CHO cells with or without (first lane) stable expression of the hA₃AR were pre-incubated for 1 h with antagonist (PSB-11, 1 μM final concentration) at 37 °C, prior to incubation with 9 (50 nM final concentration) for 1 h at 37 °C. After the incubation, the unbound probe was washed away with PBS. Membranes were prepared, brought to a concentration of 1 μg/μL, and subjected to the copper-catalyzed click reaction with Cy5-N₃ (1 μM final concentration). Samples were then denatured with Laemmli buffer (4×), resolved by SDS-PAGE, and imaged using in-gel fluorescence. Gels were stained by Coomassie Brilliant Blue (CBB) as loading control. (A) Labeling of glycosylated hA₃AR. (B) Labeling of deglycosylated hA₃AR. PNGase was added prior to the addition of click reagents. (C, D) Quantification of the observed signals with and without addition of antagonist (PSB-11). The band intensities were calculated using ImageLab and corrected for the amount of protein measured after CBB staining. The band at 55 kDa of the PageRuler Plus ladder (not shown) was set to 100% for each gel and band intensities were calculated relative to this band. The mean values ± SEM of three individual experiments are shown. Significance was calculated by a two-way ANOVA test using multiple comparisons (***p < 0.001; **p < 0.01).

Figure 6

Labeling of the hA₃AR observed by confocal microscopy. CHO cells with (CHO-hA₃AR) or without (CHO-K1) stable expression of the hA₃AR were pre-incubated for 30 min with PSB-11 (1 μM final concentration) or 1% DMSO (control) and incubated for 60 min with 9 or 1% DMSO (vehicle control). Cells were fixed, permeabilized, and subjected to a copper-catalyzed click reaction with TAMRA-N₃ (1 μM final concentration). The cells were then washed and kept in PBS containing 300 nM DAPI during confocal imaging. (A) Shown are DAPI staining (blue, first row), TAMRA staining (yellow, second row), and an overlay of both stains (third row). Images were acquired automatically at multiple positions in the well of interest and are representatives from two biological experiments. Scale bar = 50 μM. Figure was created using OMERO. (B) Comparison of the integrated fluorescence intensity between treatment conditions. Data was obtained from 2 × 9 fields of view, from the same experiment performed in duplicate. Each data point represents the integrated fluorescent intensity of the TAMRA signal per individual cell. Shown in the bar graphs is the average integrated fluorescence intensity of all individual cells ± SEM. Significance was calculated using a one-way ANOVA test using multiple comparisons. A significant increase in intensity is observed for the cells containing the hA₃AR and treated with 9, versus the other conditions.

Figure 7

Labeling of the hA₃AR in flow cytometry experiments. Samples were pre-incubated for 30 min with the antagonist PSB-11 and incubated for 60 min with 9 pre-clicked to a Cy5 fluorophore (9-Cy5). Samples were then washed and analyzed on Cy5 fluorescence by flow cytometry. (A) Cy5 mean fluorescence intensity (MFI) in CHO cells with (CHO-hA₃AR) and without (CHO-K1) stable expression of the hA₃AR. Values represent the mean ± SEM of three individual experiments performed in duplicate. Significance was calculated by a one-way ANOVA test using multiple comparisons (***p < 0.001; **p < 0.01; ns = not significant). (B) Representative graph showing the observed shift in MFI related to hA₃AR labeling in CHO-hA₃AR cells. (C) MFI of 9-Cy5 in neutrophils and eosinophils purified from human blood samples. Values represent the mean ± SEM (n = 4) of four donors from two individual experiments. Significance was calculated by a one-way ANOVA test using multiple comparisons (****p < 0.0001; ***p < 0.001; ns = not significant). (D) Representative graph showing the observed shift in MFI related to hA₃AR labeling in human eosinophils.