Selective Photoaffinity Probe That Enables Assessment of Cannabinoid CB2 Receptor Expression and Ligand Engagement in Human Cells

Abstract

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB₂R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB₂R-selective bifunctional probe that covalently captures CB₂R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB₂R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.

Figure 1

Design, synthesis, and two-step photoaffinity labeling of LEI121. (A) LEI121 was designed by replacing the hydantoin group of LEI101 by a central amide that carries both the photoreactive diazirine moiety (blue) and the ligation handle (red). (B) LEI101 was docked into a CB₂R homology model based on a recently published CB₁R crystal structure. (C) Synthesis of LEI121, reagents and conditions: (a) NaBH₄, DCM:MeOH (2:1), rt, 99%; (b) (4-formylphenyl)boronic acid, Pd(PPh₃)₄, K₂CO₃, toluene:EtOH (4:1), 80 °C, 89%; (c) DiPEA, Ms-Cl, DCM, 0 °C, quant.; (d) thiomorpholine 1,1-dioxide, K₂CO₃, ACN, 60 °C, 95%; (e) N-methylmorpholine, HOBt, EDC, DCM, quant.; (f) HCl, dioxane, 60%; (g) K₂CO₃, NaBH(OAc)₃, MeOH:DCM (1:1), 28%; and (h) diazirine 13, EDC, HOBt, DCM, 14%. (D) Two-step photoaffinity labeling. After incubation of proteome or whole cells with the probe, UV-irradiation causes the formation of a carbene, which is able to insert itself into a C–H, O–H, or N–H bond of the targeted protein. The resulting protein–probe complex is tagged using a copper-catalyzed azide–alkyne cycloaddition (CuAAC) with the following conditions: NaAsc, CuSO₄, THPTA, and tag 14, 15, or 16 to enable SDS-PAGE, mass spectrometry, or flow cytometry analysis, respectively.

Figure 2

Molecular pharmacology of LEI101 versus LEI121. (A) Reduced [³H]CP55940 binding to CB₂R in LEI121-treated membranes from CB₂R-overexpressing CHO cells was observed after UV-irradiation and washout, but not in THC- or nonirradiated LEI121-treated membranes. Data presented are the mean ± SEM of three (two in case of THC) independent experiments performed in duplicate. Statistics performed is a two-tailed t-test (****p-value < 0.0001). (B,C) β-Arrestin recruitment and G protein activation were measured as described previously,^, showing inverse agonistic activity of LEI121 (■) (β-arrestin recruitment pEC₅₀ (E_max), 7.3 ± 0.3 (−12 ± 4); G protein activation pEC₅₀ (E_max), 6.6 ± 0.2 (−50 ± 7)), which is in contrast to the agonism activity of LEI101 (●) (β-arrestin recruitment pEC₅₀ (E_max), 7.0 ± 0.3 (41 ± 6); G protein activation pEC₅₀ (E_max), 6.6 ± 0.2 (65 ± 8)). Efficacy (E_max) is normalized to the effect of 10 μM CP55940. Data are presented as the mean ± SEM of three independent experiments performed in duplicate, except for β-arrestin recruitment of LEI121 (4 experiments in duplicate).

Figure 3

Validation of CB₂R labeling by LEI121 in CB₂R-overexpressing CHO cells. (A) LEI121 labels displaceable bands (▶ = CB₂R) specifically in CB₂R-overexpressing membranes, not in wild-type CHO membranes, and only in the presence of all necessary components (active protein, probe, UV-irradiation, ligation components). (B) Addition of PNGase F shows a decrease of higher MW glycosylated CB₂R bands, and an increase in full-length, probe-bound CB₂R signal (∼41 kDa). The gel shown is a representative result of three independent experiments. (C) Isolation of CB₂R was achieved in live CB₂R-overexpressing cells by ligation with biotin-N₃ (15), followed by avidin enrichment, trypsinization, and proteomics. CB₂R peptides identified from three independent experiments are highlighted in magenta. (D) Quantification of dose-dependent displacement of LEI121-labeled bands in SDS-PAGE experiments by different cannabinoid ligands. Results shown are the mean ± SEM of 2 (CP55940 and 2-AG) or 3 (SR144528 and HU910) independent experiments. The inlay shown is a representative gel of concentration-dependent displacement of LEI121 labeling by 2-AG (see also Figure S8).

Figure 4

Visualization of endogenous CB₂R expression in live HL-60 cells by LEI121. (A) Representative dot plot of the selected HL-60 cell population for each individual experiment. (B,C) Representative histograms showing fluorescence intensity differences between the untreated sample (AF647-fluorophore only), the LEI121-treated sample, and the sample with CP55940 (CP, B) or SR144528 (SR, C). (D) Pretreatment with CP55940 (10 μM) and SR144528 (20 μM) induced ∼50% and ∼25% displacement, respectively, of the labeling induced by LEI121. Statistics performed was a two-tailed t-test, and the results shown are the mean ± SEM of the background-corrected, normalized mean fluorescence intensity (MFI) values of three independent experiments performed in duplicate (**p-value < 0.01, ****p-value < 0.0001).

Figure 5

Visualization of endogenous CB₂R expression in PBMCs by LEI121. (A) Representative dot plot of the selected PBMC population (debris, doublets, and dead cells excluded). (B) From left to right: representative dot plots of selected positive populations, representative histograms of these populations to show fluorescence intensity differences, and scatter plots showing % labeling by LEI121 ∓ CP55940 of four donors, in triplicate (n = 12), normalized to the average maximum LEI121 signal per donor. The line represents the mean. Statistics was performed using a two-tailed t-test (*p-value < 0.05, ****p-value < 0.0001, ns = not significant) on the mean background-corrected, normalized MFI values of LEI121 ∓ CP55940 (N = 4). Significant displacement with CP55940 was observed in CD3⁺ T cells, CD14⁺ monocytes, and CD19⁺ B cells. (C) The specific fluorescent signal of LEI121 indicates the level of CB₂R expression in these cell types. The highest signal was found in CD19⁺ B cells, which is also the case for the level of CB₂R mRNA expression measured for these cell types. The β₂ microglobulin mRNA expression per cell type was determined as control. Bar graph shows the mean ± SEM of background-corrected specific fluorescence of LEI121 (for details of the calculations in (B) and (C), see Table S3 and Data Analysis).