Reactivation of p53: from peptides to small molecules Christopher J. Brown 1 , Chit F. Cheok 1 , Chandra S. Verma 2 and David P. Lane 1 1 p53 Laboratory (p53Lab, A*STAR), 8A Biomedical Grove, #06-06, Immunos, 138648, Singapore 2 Bioinformatics Institute (A*STAR), 30 Biopolis Street, #07-01 Matrix, 138671, Singapore Approximately 27 million people are living with a tumour in which the tumour suppressing activity of p53 has been inactivated. In half of these tumours, p53 itself is not mutated but the pathway is partially abrogated. Mechan- isms include the overexpression of negative regulators of p53, such as MDM2 and MDM4, and deletion or epigenetic inactivation of the positive regulators of p53 such as ARF. In the other half of tumours, in which p53 is inactivated, p53 is mutated and 95% of these mutations lie in the core DNA-binding domain, which reflects the key role of p53 as a transcriptional activator. Reactivation of the tumour suppressive properties of p53 is a key therapeutic goal, and the use of peptides in p53 research has led directly to the development of two alternative small molecule approaches: stabilization of mutant p53 to rescue its DNA-binding activity and inhibition of MDM2 or MDM4. Introduction The tumour suppressor protein p53 is a transcription factor that guards the cell against various stress signals through the induction of cell cycle arrest, apoptosis or senescence. It consists of several domains that include the N-terminal transactivation domain, the central sequence specific DNA-binding domain, the oligomerization domain and the C-terminal regulatory domain. p53 positively and nega- tively regulates the expression of a large number and dis- parate group of genes (Figure 1). New targets of p53-induced transcription have been identified that are involved in protein translation [1,2]. These are complemented by tran- scription-independent activities and include direct effects on survival proteins in the mitochondrion [3,4], regulation of microRNA processing [5] and involvement in DNA repair pathways [6–9] (Figure 1). Through its cell cycle arrest and apoptotic activities, p53 can have a strong inhibitory effect on cell growth, making it essential to control p53 through normal development. The antiproliferative functions of p53, which are induced in response to cellular stress, are fre- quently circumvented in tumour development. Multiple mechanisms exist to negatively regulate p53, including the control of protein activity, stability and sub- cellular localization through the action of numerous other proteins that work directly or indirectly to restrain p53. These p53 regulatory proteins include ubiquitin ligases that have a role in controlling p53 stability, enzymes involved in post-translation modifications of p53 (such as kinases and acetylases), transcriptional coactivators that can modulate the transcriptional activity of p53 and many other proteins (e.g. methylation, nedylation and sumoylation of p53; reviewed in [10–12]). Other crucial proteins involved in fine-tuning these responses are phosphatases, deacetylases and deubiquitylating enzymes, which work by reversing post-translation modifications made to p53. Murine double minute 2 (MDM2) is a key E3 ubiquitin ligase that targets p53 for ubiquitin-dependent degrada- tion and that is frequently overexpressed in tumours that possess wild-type p53 (Figure 1). It is a crucial negative regulator and ensures that p53 is kept under tight regula- tion [13]. MDM2 binds to p53 and inhibits p53 transcrip- tional activity by preventing its interaction with the general transcription machinery [14]. As part of a crucial feedback loop, p53 activates transcription of the MDM2 gene [15]. Increased expression of MDM2 leads to the degradation and inactivation of p53. The resulting de- crease in p53 levels in turn causes a decrease in the rate of transcription of MDM2. MDM2 also autoubiquitinylates itself for degradation by the proteosome [16]. MDM4 is a structural homologue of MDM2 and controls p53 in a manner similar to MDM2 by blocking the transcription activation domain of p53. In addition, with no E3 ubiquitin activity of its own, MDM4 forms heterocomplexes with MDM2 and indirectly potentiates the ubiquitylation of p53. It appears that MDM2 primarily regulates p53 sta- bility and subcellular localization, whereas MDM4 can directly regulate p53 transcription [17–20]. Activation of p53 to relieve the negative regulation imposed on it is driven by several different signalling pathways. These pathways are stimulated by signals such as UV irradiation, inappropriate cell division driven by oncogenic alterations, nutrient deprivation and hypoxia [21] (Figure 1). One such signalling pathway is mediated by ADP-ribosylation factor (ARF), a small protein that binds and inhibits MDM2. This prevents p53 degradation and stabilizes p53 protein levels [22,23]. ARF has an important role in signalling to p53 in response to some oncogenes, but is not necessary for the activation of p53 in response to DNA damage [24]. Another signalling pathway utilizes the ribosomal proteins L11, L5 and L23 which mediate activa- tion of p53 in response to ribosomal stress without a requirement for ARF [23]. These proteins interact with MDM2 and activate p53 by inhibiting MDM2-mediated degradation of p53 [25–27]. A recent study of a mouse model utilizing an inducible p53 system has indicated that the response of p53 to DNA damage might not be responsible for tumour suppression Review Corresponding author: Lane, D.P. (dplane@p53lab.a-star.edu.sg). 0165-6147/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2010.11.004 Trends in Pharmacological Sciences, January 2011, Vol. 32, No. 1 53