Staurosporine-based binding assay for testing the affinity of compounds to protein kinases.
Staurosporine is a broad-spectrum inhibitor of both tyrosine and serine/threonine protein kinases. Excitation of staurosporine and its analogues at 296 nm results in major emission bands centered at 378 and 396 nm. The intensity of the emission bands is enhanced on binding to the adenosine triphosphate (ATP) site of many protein kinases. This property was used to develop a competitive displacement assay for evaluating the binding affinity of small molecules to protein kinases. The assay was validated in both cuvette and plate formats for several phosphorylated and non-phosphorylated protein kinases. The throughput of the assay is high enough to be used in drug discovery for screening as well as lead optimization.
Vertex Pharmaceuticals Incorporated, Cambridge, MA 02139, USA.
Ganesh H
GH
Iyer
Paul
P
Taslimi
S
S
Pazhanisamy
D001665:Binding Sites; D001667:Binding, Competitive; D002227:Carbazoles / Q000494:pharmacology; D026121:Indole Alkaloids / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D019311:Staurosporine / Q000378:metabolism* / Q000494:pharmacology; D000099190:MAP-Kinase-Activated Kinase 2
D002227:Carbazoles; D026121:Indole Alkaloids; D047908:Intracellular Signaling Peptides and Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D019311:Staurosporine; D000099190:MAP-Kinase-Activated Kinase 2; C049985:staurosporine aglycone