Novel, adenosine A1AR Affinity-Based Probe (AfBP) which is suitable for click conjugation for use in confocal microscopy, SDS-PAGE and chemical proteomics profiling applications. Labeling of the A1AR is more specific in live CHOhA1AR cells compared to labeling in membrane fractions.
LUF7909 acts as a partial agonist which is highly specific to the A1AR and binds covalently (apparent pKi values at A1AR are 7.8 and 9.5 (following a 4h preincubation), where a Ki shift indicates a covalent mode of action).
Applications
Live cells or membrane fractions should be incubated with LUF7909 to selectively label the desired receptor in the presence of other proteins.
The desired reporter group can subsequently be clicked onto the probe, effectively labeling the receptor.
Finally, the reporter-bound receptor is processed based on the detection method (e.g. confocal microscopy, SDS-PAGE, chemical proteomics)
Selective labeling of the A1AR in live CHO cells with LUF7909.
(A). CHO cells with or without over expression of the A1AR were pretreated with the A1 inverse agonist DPCPX (1 µM) and incubated for 1h with LUF7909 (100 nM) or 1% DMSO (control). Membranes were collected, treated with PNGases and incubated with a click mix containing AF647-N3. Samples were then subjected to SDS-PAGE and analyzed by in-gel fluorescence scanning.
(B), Volcano plot of affinity purification experiments comparing live CHOhA1AR cells treated with P30542.
(C), Confocal microscopy images. CHO cells with or without overexpression of A1AR were pretreated with FSCPX (1µM) or 1% DMSO and incubated with LUF7909 (100nM) or DMSO (1%) for 1h. Cells were then fixed and stained with TAMRA-N3 and DAPI (TAMRA = red, DAPI = blue). Arrows in figure C indicate examples of labeled membranes and labeling inside cells.
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
Diagrammatic representation of LUF7909 and applications
Diagrammatic representation of affinity-binding probe LUF7909 and workflows and applications.
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
Labeling of the A1AR in adipocyte membranes
Labeling of the A1AR in adipocyte membranes derived from mouse gonadal fat pads.
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
Specific labeling of the A1AR using LUF7909 in CHO membranes
Specific labeling of the A1AR using LUF7909 in CHO membranes overexpressing the A1AR.
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
Affinities of LUF7909
Affinities of LUF7909 and analogs for the four adenosine receptor subtypes (A1AR, A2AAR, A2BAR and A3AR)
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
LUF7909 labeling of the A1AR in live CHO cells
Selective labeling of the A1AR in live CHO cells with LUF7909.
(A). CHO cells with or without over expression of the A1AR were pretreated with the A1 inverse agonist DPCPX (1 µM) and incubated for 1h with LUF7909 (100 nM) or 1% DMSO (control). Membranes were collected, treated with PNGases and incubated with a click mix containing AF647-N3. Samples were then subjected to SDS-PAGE and analyzed by in-gel fluorescence scanning.
(B), Volcano plot of affinity purification experiments comparing live CHOhA1AR cells treated with P30542.
(C), Confocal microscopy images. CHO cells with or without overexpression of A1AR were pretreated with FSCPX (1µM) or 1% DMSO and incubated with LUF7909 (100nM) or DMSO (1%) for 1h. Cells were then fixed and stained with TAMRA-N3 and DAPI (TAMRA = red, DAPI = blue). Arrows in figure C indicate examples of labeled membranes and labeling inside cells.
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
Diagrammatic representation of LUF7909 and applications
Diagrammatic representation of affinity-binding probe LUF7909 and workflows and applications.
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
Labeling of the A1AR in adipocyte membranes
Labeling of the A1AR in adipocyte membranes derived from mouse gonadal fat pads.
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
Specific labeling of the A1AR using LUF7909 in CHO membranes
Specific labeling of the A1AR using LUF7909 in CHO membranes overexpressing the A1AR.
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
Affinities of LUF7909
Affinities of LUF7909 and analogs for the four adenosine receptor subtypes (A1AR, A2AAR, A2BAR and A3AR)
Data from Beerkens BLH et al, ACS chemical biology, 17(3131-3139)
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