Evaluation of an antibody-free ADP detection assay: ADP-Glo.
Identification of kinase, especially protein kinase, modulators through high-throughput screening (HTS) has become a common strategy for drug discovery programs in both academia and the pharmaceutical industry. There are a number of platform technologies that can be used for measuring kinase activities. However, there is none that fits all criteria in terms of sensitivity, ATP tolerance, robustness, throughput, and cost-effectiveness. Therefore, development of a homogeneous and robust HTS assay for some kinase targets is still challenging. We recently evaluated the ADP-Glo assay from Promega. This is a homogeneous, signal increase assay that measures ADP production from a kinase reaction by coupled enzymes that first convert ADP to ATP and subsequently quantifies ATP using luciferase in the presence of luciferin. Since the unused ATP in the reaction is depleted prior to ADP to ATP conversion, this assay shows excellent sensitivity over a wide range of ATP concentrations. We demonstrate that ADP-Glo assay can be used for 2 kinase targets that belong to different classes, and compare the results of compound profiling with SPA and FP assay technologies.
Li
Hu
H
GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA. hu.2.li@gsk.com
Totoritis
Rachel D
RD
Lor
Leng A
LA
Schwartz
Benjamin
B
Caprioli
Peter
P
Jurewicz
Anthony J
AJ
Zhang
Guofeng
G
D000244:Adenosine Diphosphate / Q000032:analysis* / Q000737:chemistry*; D000906:Antibodies / Q000032:analysis / Q000737:chemistry; D002623:Chemistry Techniques, Analytical; D007118:Immunoassay; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000032:analysis*; D010770:Phosphotransferases / Q000032:analysis* / Q000737:chemistry*; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000906:Antibodies; D008164:Luminescent Proteins; D000244:Adenosine Diphosphate; D010770:Phosphotransferases