Development of a high-throughput robotic fluorescence-based assay for HsEg5 inhibitor screening.
HsEg5 has microtubule-activated ATPase activity and plays essential roles in bipolar spindle formation. Because HsEg5 is validated as an attractive cancer target, in vitro biochemical assays have been developed for identifying compounds with high inhibitory activity. Several compounds, including quinazoline ring-containing compounds, have been identified and are currently in clinical trials. Although considerable progress has been made during recent years, limitations of HsEg5 in vitro screening assays still reside in two main aspects. First, colorimetric-based assays exhibit relatively low sensitivity and limited dynamic range that are unable to accurately measure compounds with nanomolar potencies. Second, current fluorescence assays are relatively low throughput without "mix and read" homogeneous features. In this study, we describe a sensitive fluorescence-based assay for HsEg5-specific inhibitors. By coupling several enzymes' activities, the release of ADP was measured quantitatively through red fluorescent resorufin. The Km for ATP hydrolysis in this assay was calculated as 23 microM. The known HsEg5 inhibitors CK0106023 and CK0238273 gave IC50 values of 9.8 and 30.6 nM, respectively. Our fluorescence assay has a 20-fold increase in sensitivity with broader dynamic range when compared with a colorimetric assay. We further automated this assay for high-throughput screening with a Z' factor of 0.8.
Zhang
Bin
B
Department of Chemical Genomics, ArQule Inc., Woburn, MA 01801, USA. bzhang@arqule.com
Senator
David
D
Wilson
Christopher J
CJ
Ng
Shi-Chung
SC
D000244:Adenosine Diphosphate / Q000032:analysis / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D045744:Cell Line, Tumor; D003124:Colorimetry / Q000379:methods; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D007211:Indoles; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008175:Lung Neoplasms / Q000473:pathology; D009930:Organic Chemicals / Q000494:pharmacology; D010078:Oxazines; D011743:Pyrimidines / Q000494:pharmacology; D012371:Robotics; D012680:Sensitivity and Specificity; D012997:Solvents / Q000494:pharmacology
C487201:CK0106023; C506942:CK0238273; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D009930:Organic Chemicals; D010078:Oxazines; D011743:Pyrimidines; D012997:Solvents; C007293:DAPI; D000244:Adenosine Diphosphate; C014180:resorufin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004121:Dimethyl Sulfoxide