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.

A High-Throughput Scintillation Proximity-Based Assay for Human DNA Ligase IV

“…19 The Z 0 factor constitutes a dimensionless parameter that ranges from 1 (infinite separation) to < 0. It is defined as Z 0 = 1 -(3sc + + 3sc -) / jmc + -mc -j, where sc + , sc -, mc + , and mc -are the standard deviations (s) and averages (m) of the high (c + ) and low (c -) control.…”

“…The Z 0 factor between 0 and 0.5 indicates a marginal assay and < 0 signifies a poor assay with overlap between controls. 19 …”

“…The addition of reagents was performed using the FlexDrop (Perkin Elmer) or the Multidrop 384 (Thermo) as described previously for an SPA-based assay. 19 Screening data files were obtained from the automated LEADseeker (GE Healthcare) for the SPA. The screening data files obtained were subsequently loaded into the HTS Core Screening Data Management System, a custom-built suite of modules for compound registration, plating, and data management, powered by ChemAxon Cheminformatic tools (ChemAxon).…”

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication

“…23 Instead, the study showed that a glutamic acid (Glu217) has replaced the threonine, making Cdc7 an atypical member of the serine/threonine kinase family. 23 Using a luciferase-based assay that has previously been successfully utilized to assay Cdc7 activity, 24 we compared the relative kinase activities of Cdc7 WT , Cdc7 £5A , Cdc7 £5D and Cdc7 T376A in relation to the Cdc7 1-431 negative control, an allele that is deleted for its Dbf4 interacting domain. 34 Under our experimental conditions, all three mutant alleles (Cdc7 £5A , Cdc7 £5D and Cdc7 T376A ) showed substantial kinase activity close to the level of wild type, although the difference in kinase activities between the mutants and Cdc7 WT are still significant (Fig.…”

“…21,22 Finally, other groups have successfully used recombinant human DDK in in vitro kinase assays in the absence of Cdk phosphorylation. 23,24 Thus, the significance and function of Cdk-dependent Cdc7 phosphorylation in vivo is still to be unambiguously determined.…”