Abstract
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.
Highlights
The p53 protein, the guardian of the genome, plays an essential role in the regulation of cell cycle, apoptosis and DNA repair by defending cells against various cellular stresses, such as hypoxia and DNA damage[1,2,3]
It is well known that MDM2 is representative of a p53-negative regulator in which the N-terminal domain directly binds the TAD1 of p53 via a putative helix formed by residues 18–2612
If proteins like MDM2, which compete with anti-p53 antibody to interact with p53 transactivation domain (TAD) domain, are present in the solution, as shown in Fig. 1a lower panel, there are no acceptor beads in close proximity to the donor beads, and no signal
Summary
The p53 protein, the guardian of the genome, plays an essential role in the regulation of cell cycle, apoptosis and DNA repair by defending cells against various cellular stresses, such as hypoxia and DNA damage[1,2,3]. There are approximately 22 million patients suffering from different kinds of cancer that are affected by p535 Half of these patients bear wild-type p53 in tumor cells, but its function is impaired by negative regulators through degradation or inhibition[6]. Among these negative regulation motifs, binding of the transactivation domain (TAD) of p53, blocking its transcriptional activity, is crucial[7, 8]. The purpose of this study is to develop a homogenous immunoassay, termed an AlphaLISA, for monitoring total free p53 TAD, which can be widely used to detect the TAD binding to a variety of regulators via competition assay This detection method could be applied to screen new inhibitors that disrupt the binding and reactivate p53. We used MDM2 as an example to develop an AlphaLISA assay to measure interactions with p53 and further validated its ability to screen potential inhibitors by successfully identifying known p53-MDM2 binding inhibitors, such as Nutlin-3a
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