Development of a High-Content High-Throughput Screening Assay for the Discovery of ATM Signaling Inhibitors

Abstract

The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.

Clinical and genomic analysis of metastatic prostate cancer progression with a background of postoperative biochemical recurrence

“…Furthermore, we found that that the metastasis signature has many genes associated with cell cycle regulation, DNA damage sensing, cytoskeleton organization and mitogen‐activated protein kinases. Kinase enrichment analysis showed that genes in the metastasis signature are highly phosphorylated by key cell cycle kinases such as ATM, CDK and MAP3K3, which play role in cell cycle checkpoint and control of G2/G1‐M transition . DNA damage sensors may be activated in early stages of prostate cancer progression through PRKDC and ATR protein kinases , which can induce RAD21 a nuclear phosphoprotein that becomes hyperphosphorylated in cell cycle M phase and has been previously described as a prognostic factor in prostate cancer .…”

Development of a Cell-Based, High-Throughput Screening Assay for ATM Kinase Inhibitors

“…Therefore, in addition to its improved ease and scale for ATM inhibitor screening, this strategy has certain advantages over a recently published high-content screening (HCS) assay for ATM kinase inhibitors that targeted ATM autophosphorylation itself. 19 Such HCS methods definitely provide in-depth information on the effects of compounds on cellular targets and cellular processes. However, our ICW assay has three advantages.…”

Implementation and Challenges of Direct Acoustic Dosing into Cell-Based Assays

“…Although intermediate plates can be used (i.e., ARPs with added buffer as described above), this requires an additional, unnecessary compound handling step to transfer the diluted compounds into the final cell plate. Direct Echo compound dosing into cell-based assays has been reported by AstraZeneca 11,[15][16][17] as well as a number of other groups [18][19][20][21] and offers the advantage of a simplified workflow without any additional steps or tip/plate consumables, reducing costs and overall assay time. Direct dosing also removes any concerns related to the long-term storage of ARPs (e.g., evaporation issues that can lead to variations in assay data quality).…”