A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

Abstract

Agonists that target the A₁, A_2A, A_2B and A₃ adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A_2AR and A_2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A_2A and A_2B receptors in human cells. A_2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A_2A and A_2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds.

Figure 1. Sequence homology between the four adenosine receptors.

The A_2A and A_2B receptors show the most sequence homology among the adenosine receptors, with these two receptors having a 61% identity.

Figure 2. CRISPR vector design for knockout of A_2AR in K562 Cells.

(a) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A_2AR gene. Additional arrowheads indicate genes with similar regions to the A_2AR gene, including the A_2BR gene on chromosome 17. (b) Left, guide-RNAs found for the A_2AR gene. Right, guide sequence #1 was selected for insertion into the plentiCRISPR loxP vector plasmid DNA. BsmBI cut sites were added to gRNA #1 to facilitate insertion. (c) Top, Cas9 cut site in the A_2AR gene. Bottom, map of the plentiCRISPR construct. (d) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.

Figure 3. Development of a stable A_2AR knockout cell line in K562 cells using CRISPR.

(a) Flow cytometry for Cas9 expression in K562 cells before and after transduction with the plentiCRISPR loxP A2AR. After puromycin selection to remove non-transduced cells, 96% of cells were successfully transduced. (b) Flow cytometry for removal of the loxP cassette using the pcDNA3.1 ZEO T2A CRE-GFP plasmid. 32% of cells express GFP, indicating successful removal of the proviral vector cassette. These cells were further selected using zeocin for isolation of cassette free cells. (c) Left, RT-PCR of clones selected from loxP cassette-free A_2AR knockout K562 cells. Right, sequencing of clone 12 reveals a one base (T) insertion on one allele and an 18 base deletion on the other for homologous knockout of the A_2AR gene.

Figure 4. Calcium flux assay for the BAY60 A_2BR agonist compound shows significant specificity to the A_2BR receptor.

A2AR KO, naïve and A2BR OE cells were treated with BAY60 and release of calcium was measured as relative fluorescence units (RFU). Cells with vehicle addition were used for baseline reads.

Figure 5. Flow cytometry analysis of engineered A_2A and A_2B receptor overexpressing cell lines.

(a) Representative flow cytometry for naïve and A_2AR overexpressing (A2AR90) K562 cells. (b) Representative flow cytometry for A_2AR knockout (A2AR KO) and A_2BR overexpressing (A2BR OE) K562 cells (stained for expression of YFP).

Figure 6. Calcium flux assays for LNC-3047 and LNC-3015, two newly developed A_2AR agonist compounds, show specificity for LNC-3047 but not for LNC-3015.

(a,b) Top, A2AR KO, naïve and A2AR90 cells were treated with LNC-3047 or LNC-3015 and release of calcium was measured as RFU. Cells with vehicle addition were used for baseline reads. Bottom, comparison of A2AR KO and A2BR OE lines treated with LNC-3047 or LNC-3015.

Figure 7. Treatment of naïve CD4 T-cells with LNC agonist compounds induces FoxP3 expression.

Representative flow cytometry for treated human CD4 T-cells. Cells were initially gated on CD4⁺CD25⁺, followed by gating for FoxP3⁺ cells. Fold change in FoxP3 expression in cells treated with LNC compounds is shown. Error bars are defined as mean + SEM, n = 5 for all points. Student’s T-test with Mann-Whitney was used to determine significance. *p = 0.0318, **p = 0.0079.