Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase Journal Article


Authors: Takagi, T.; Shum, D.; Parisi, M.; Santos, R. E.; Radu, C.; Calder, P.; Rizvi, Z.; Frattini, M. G.; Djaballah, H.
Article Title: Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase
Abstract: Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [33P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [33P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay. © 2011 Bentham Science Publishers Ltd.
Keywords: unclassified drug; cell cycle proteins; drug discovery; adenosine diphosphate; enzyme substrate; inhibitor; phosphorylation; assay; luminescence; protein-serine-threonine kinases; intermethod comparison; radiometry; adenosine triphosphate; heterodimer; false positive result; phosphate; protein kinase; technology; cdc7-dbf4 kinase; cdc7 kinase; tannin; crystal violet; protein kinase cdc7 dbf4; scintillation proximity assay; scintillation counting; gentiana
Journal Title: Combinatorial Chemistry and High Throughput Screening
Volume: 14
Issue: 8
ISSN: 1386-2073
Publisher: Bentham Science Publishers  
Date Published: 2011-05-12
Start Page: 669
End Page: 687
Language: English
DOI: 10.2174/138620711796504442
PROVIDER: scopus
PUBMED: 21564015
PMCID: PMC3643815
DOI/URL:
Notes: --- - "Export Date: 3 October 2011" - "CODEN: CCHSF" - "Source: Scopus"
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MSK Authors
  1. Toshimitsu Takagi
    6 Takagi
  2. Constantin Radu
    27 Radu
  3. David Shum
    52 Shum
  4. Paul A Calder
    9 Calder
  5. Monika Parisi
    1 Parisi
  6. Ruth E Santos
    6 Santos
  7. Zahra Hussain Rizvi
    1 Rizvi