The p53 family of transcription factors consists of three homologous proteins, p53, p63 and p73, encoded by three distinct genes. These proteins are involved in the regulation of many cellular functions, including cell differentiation, proliferation and death. Therefore, they have a pivotal role in tumorigenesis and significantly affect the tumor response to therapy. In tumors retaining a wild-type p53, it is well-known that the p53 activity is inhibited through interaction with major endogenous negative regulators, MDM2 and MDMX. Both proteins bind to p53 and negatively regulate the p53 activity by direct inhibition of the p53 transcriptional activity, although only MDM2 is able to induce p53 degradation. MDM2 and MDMX are also able to inhibit the p73 transcriptional activity. On the other hand, the regulation of p63 by MDM2 and MDMX is still a controversial issue in human cells. Inhibition of these interactions represents an important and appealing therapeutic strategy to reactivate p53 in these tumour cells. Additionally, the reactivation of p63 and p73, through inhibition of their interactions with MDM2 and MDMX, would represent a promising anticancer strategy to overcome the loss of p53 activity in tumor cells with null or mutated p53.

In yeast, the p53 family proteins induce growth inhibition associated with S-phase cell cycle arrest, increase of actin expression levels and depolarization, as well as autophagic cell death (Coutinho I. et al., FEBS Lett 2009; Leão M. et al., Exp Cell Res 2015). With these phenotypes, it was possible to develop high-throughput screening assays to search for modulators of these human proteins, in particular inhibitors of p53/p63/p73 interaction with MDM2 or MDMX (Leão M. et al., Biochem Pharmacol 2013; Leão M. et al., FEBS J 2013; Leão M. et al., Exp Cell Res 2015. In fact, it was shown that both MDM2 and MDMX inhibited the p53/p63/p73-induced yeast growth inhibition, S-phase cell cycle arrest and increased actin expression levels (Leão M. et al., Biochem Pharmacol 2013; Leão M. et al., FEBS J 2013; Leão M. et al., Exp Cell Res 2015).

To date, the use of the yeast p53-MDM2 interaction assay led us to the discovery of two new small molecule inhibitors of the p53-MDM2 interaction, the pyranoxanthone 1 (LEM1) and the oxazoloisoindolinone 3a (Soares J. et al., Eur J Pharm Sci 2015), as well as of a dual inhibitor of the p53-MDM2/X interaction, the compound OXAZ-1 (Soares et al., Pharmacol. Res. 2015), DIMP53-1 (Soares J. et al., Molecular Oncology, 2017), and SYNAP (Raimundo L. et al., British J. Pharmacol. 2018).