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.
Keywords: Cdc7-Dbf4 kinase, drug discovery, inhibitor, luminescence, phosphorylation, protein kinase, ADP, detection, scintillation proximity assay, ATP, bioactives
Combinatorial Chemistry & High Throughput Screening
Title: Comparison of Luminescence ADP Production Assay and Radiometric Scintillation Proximity Assay for Cdc7 Kinase
Volume: 14 Issue: 8
Author(s): Toshimitsu Takagi, David Shum, Monika Parisi, Ruth E. Santos, Constantin Radu, Paul Calder, Zahra Rizvi, Mark G. Frattini and Hakim Djaballah
Affiliation:
Keywords: Cdc7-Dbf4 kinase, drug discovery, inhibitor, luminescence, phosphorylation, protein kinase, ADP, detection, scintillation proximity assay, ATP, bioactives
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.
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Takagi Toshimitsu, Shum David, Parisi Monika, E. Santos Ruth, Radu Constantin, Calder Paul, Rizvi Zahra, G. Frattini Mark and Djaballah Hakim, Comparison of Luminescence ADP Production Assay and Radiometric Scintillation Proximity Assay for Cdc7 Kinase, Combinatorial Chemistry & High Throughput Screening 2011; 14 (8) . https://dx.doi.org/10.2174/138620711796504442
DOI https://dx.doi.org/10.2174/138620711796504442 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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