We report here the development of a simple homogeneous screening protocol for identifying small-molecule inhibitors of Wee1 turnover using a luciferase reporter system. With a Z′ value greater than > 0.6 and a throughput of ~15,000 compounds per hour, the K328M-Wee1-Luc 1536-well plate assay proved to adapt very well in the context of automated uHTS and demonstrated excellent assay performance over the course of screening a small molecule collection encompassing ~218,000 distinct chemical entities.
In optimizing the assay, we found that the use of cryopreserved transiently transfected cells was beneficial to the screening process, as previously reported by others15, 23, 24
. In the case of the research presented here, it uncoupled the labor and time-consuming transfection step from the rest of the assay, greatly facilitating the organization of the screen and allowing measurement of results as fast as 20 hours after initiating the assay instead of 72 hours (i.e. if the transfection step was kept part of the process). In addition, the use of cryopreserved cells, as long as they are prepared in sufficient quantities, samples a single batch of transiently transfected cells for all the different steps involved in the lead discovery and optimization effort, hence increasing assay data consistency along the entire process.
As judged by the amount of compounds that reproduced activity in the secondary assay (), the K328M-Wee1-Luc assay yielded an unusually low confirmation rate. A retrospective analysis of the data indicated that 40 out of the 4,560 negative controls (0.87%) showed percent activation above the hit-cutoff. This suggests that ~1,900 hits out of the 2,610 found, i.e. 73%, were potentially false-positives. A primary explanation for this fact is that the calculated hit-cutoff of 8.82% is unusually low for a cell-based assay, causing compounds with marginal activity to be selected as hits.
Another explanation for the low confirmation rate lies in the fact that activation profiles observed for all active compounds, including the reference control MG132, displayed very steep dose-response curves with hill slopes >2.5. In some instances, compounds displayed a bell-shaped dose-response curve, as exemplified by compound SID14743125 (). The cell viability data for this compound indicated that this bell-shaped curve profile is likely associated with cytotoxicity at higher concentration. Taken together, this suggests that even small deviations from a compound’s expected test concentration negatively impact the confirmation rate. Nevertheless, the fact that the newly synthesized compounds confirmed their activities in our assay indicated that active compounds can be reliably identified by the K328M-Wee1-Luc assay.
The use of the N-cyclin B-Luc assay as a counterscreen proved to be an effective way of rapidly eliminating non-selective and promiscuous compounds early in the probe discovery process, allowing us to focus on a limited set of compounds that reduced Wee1 degradation. Among them, SID4243143 appeared to be the most attractive. Although this compound has also been reported as active in a non-trivial number of other cell-based assays available on PubChem (13 assays out of 359, i.e. 3.6%), our results clearly show a selective effect on the K328M-Wee1-Luc fusion protein over the N-cyclin B-Luc construct, ruling out the possibility that SID4243143 acts broadly as a proteasome inhibitor or any other mechanism that is not specific to the Wee1 degradation pathway. Moreover, SID4243143 did not affect HeLa cell viability at concentrations up to 50 μM. Results of the FACS analysis indicated that SID4243143 inhibits cell cycle progression in HeLa cells (). This preliminary insight about SID4243143’s mode of action may explain its pleiotropic effects in different cell-based assays without dismissing a selective effect on Wee1 stabilization, as indeed high levels of Wee1 prevent mitotic entry.
The confirmed robustness of the screening platform and its ability to rapidly identify and triage promising selective, non-toxic and cell-permeable compounds encourages us to further screen other chemical libraries. In addition to the possibility of identifying other potent, selective scaffolds that will be useful in cell cycle research or as possible starting points for chemotherapeutic drug development, our long-term goal is to uncover potential phospho-transferases involved in Wee1 degradation through the screen of focused-library designed at targeting kinases. Taken together, the assays and automated protocol described herein proved to be an excellent platform for interrogating large compounds libraries for inhibitors of Wee1 degradation.