A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development.
Agonists that target the A1, A2A, A2Band A3adenosine 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 A2AR and A2BR 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 A2Aand A2Breceptors in human cells. A2Areceptor 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 A2Aand A2Breceptor 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.
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.
Karlie R
KR
Jones
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.
Uimook
U
Choi
Molecular Signaling Section, Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.
Ji-Liang
JL
Gao
Lewis and Clark Pharmaceuticals Inc., Charlottesville, VA 22901, USA.
Robert D
RD
Thompson
Lewis and Clark Pharmaceuticals Inc., Charlottesville, VA 22901, USA.
Larry E
LE
Rodman
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.
Harry L
HL
Malech
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.
Elizabeth M
EM
Kang
D000241:Adenosine / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005434:Flow Cytometry; D015870:Gene Expression; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D006801:Humans; D020014:K562 Cells; D058906:Purinergic P1 Receptor Agonists / Q000737:chemistry* / Q000494:pharmacology*; D018047:Receptors, Purinergic P1 / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D000241:Adenosine; D002118:Calcium