Development of a novel immunoassay to detect interactions with the transactivation domain of p53: application to screening of new drugs

Tumor protein p53 acts as a trans-activator that negatively regulates cell division by controlling a set of genes required for cell cycle regulation, making it a tumor suppressor in different types of tumors. Because the transcriptional activity of p53 plays an important role in the occurrence and development of tumors, reactivation of p53 transcriptional activity has been sought as a novel cancer therapeutic strategy. There is great interest in developing high-throughput assays to identify inhibitors of molecules that bind the transcription-activation domain of p53, especially for wt p53-containing tumors. In the present study, taking MDM2 as an example, a novel amplified luminescent proximity homogeneous assay (AlphaLISA) was modified from a binding competition assay to detect the interactions between the transcription-activation domain of p53 and its ligands. This assay can be adapted as a high-throughput assay for screening new inhibitors. A panel of well-known p53-MDM2 binding inhibitors was used to validate this method, and demonstrated its utility, sensitivity and robustness. In summary, we have developed a novel protein-protein interaction detection immunoassay that can be used in a high-throughput format to screen new drug candidates for reactivation of p53. This assay has been successfully validated through a series of p53-MDM2 binding inhibitors.