Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors

Abstract

Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A(1) and A(2A) receptors (A(1)AR and A(2A)AR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A(1)AR and A(2A)AR, respectively. To proof the feasibility of MS binding on the A(1)AR and A(2A)AR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.

Keywords: Adenosine receptor; Deuteration; MS binding; Mass spectrometry; Radioligand binding.

Scheme 1

Synthesis of [²H₄]DPCPX (2). Reagents and conditions: Rh(PPh₃)₃Cl, NaBD₄, D₂O, dry THF, 60 °C, 3.5 h

Scheme 2

Synthesis of [²H₄]ZM-241,385 (5). Reagents and conditions: Et₃N, MeCN, MW, 70 °C, 3 h

Fig. 1

Standard curve of increasing concentrations of marker ligands a DPCPX with 2 nM [²H]DPCPX and b ZM-241,385 with 2 nM [²H]ZM-241,385 in matrix membrane samples. On the x-axis is plotted the concentration of marker ligand. On the y-axis is plotted the marker area TIC divided by IS area TIC (M/IS). Data shown is the average of M/IS values ± SEM from four runs in hexaplicate

Fig. 2

Typical chromatograms of a non-specific binding of DPCPX (31 pM), b total binding of DPCPX (242 pM), and c [²H]DPCPX (2 nM) in eluate containing hA₁AR membrane matrix, and of d non-specific binding of ZM-241,385 (42 pM), e total binding of ZM-241,385 (289 pM), and f [²H]ZM-241,385 (2 nM) in eluate containing hA_2AAR membrane matrix. The red lines delineate the area of peak integration

Fig. 3

Saturation of DPCPX binding to hA₁AR (a, b) and ZM-241,385 binding to hA_2AAR (c, d). Increasing concentrations of marker ligands were incubated with the respective membranes. Data shown without (a, c) and with (b, d) non-specific binding values subtracted. Graphs show mean values of one representative MS binding saturation experiment performed in duplicate

Fig. 4

Displacement of DPCPX binding to hA₁AR by CPA, 8-CPT, ZM-241,385, or NECA (a) and of ZM-241,385 binding to hA_2AAR by UK-432,097, MSX-2, DPCPX, or NECA (b). Non-specific binding is plotted at −3 on the x-axis. Graphs show mean values of one representative MS binding displacement experiment performed in duplicate

Fig. 5

Association (a, c) and dissociation (b, d) of DPCPX on hA₁AR (a, b) and ZM-241,385 on hA_2AAR (c, d). Graphs show mean values of one representative MS binding association or dissociation experiment performed in duplicate

Fig. 6

Competition association of DPCPX on hA₁AR in the presence or absence of 250 nM 8-CPT and 250 nM FSCPX (a), and of ZM-241,385 on hA_2AAR in the presence or absence of 90 nM MSX-2 and 15 nM LUF6632 (b). Graphs show mean values of one representative MS binding competition association experiment performed in duplicate

Fig. 7

Correlation plots of results obtained by MS binding and radioligand binding assays on hA₁AR and hA_2AAR. Values of marker ligands DPCPX and ZM-241,385 were measured directly on their respective binding targets hA₁AR and hA_2AAR by saturation (a, b), association (c, d), and dissociation (e, f) assays, while values of the competing ligands were measured indirectly by displacement (a, b) and competition association assays (c–f). Affinity values in pK _D and pK _i (a, b), association rates in k _on (c, d), and dissociation rates in log k _off (e, f) were compared. Correlation plots a, c, and e show MS binding results standardized with deuterium-labeled internal standard, while b, d, and f show truly label-free MS binding results without internal standard. Data points represent mean values of at least three separate experiments performed in duplicate. R ² values were calculated by linear regression performed on log-transformed values