Although in many tumours the loss of the tumour suppressor p53 is caused by a mutation in the gene, inactivation of wild-type p53 can also occur through failure to inactivate the ubiquitin ligase (E3) HDM2, which targets p53 for proteasomal degradation. Allan Weissman, Karen Vousden and colleagues have identified compounds that inhibit HDM2 activity, and thereby stabilize and activate wild-type p53 — these data indicate that ubiquitin ligases are viable targets for drug discovery.

High-throughput screening of small-molecule libraries identified a family of 7-nitro-5-deazaflavin compounds (HLI98s) that strongly inhibited HDM2-mediated autoubiquitylation in vitro. HLI98 compounds specifically inhibited the RING finger domain of HDM2, and not the regions that interact with p53. When primary human fibroblasts were treated with HLI98 compounds, both p53 and HDM2 levels increased. Ubiquitylated p53 was not detected, which is consistent with the compounds inhibiting ubiquitylation instead of proteasome function.

HLI98 compounds showed some selectivity for HDM2 compared with other RING finger E3s in cell lines. The compounds did not stabilize p53 in the absence of MDM2 (the mouse homologue of HDM2) in mouse embryonic fibroblasts (MEFs), indicating that they do not inhibit other E3s that target p53 for degradation, such as PIRH2 and COP1. In addition, they did not stabilize another protein, p21, which is regulated by E3 ligases other than MDM2.

So does stabilization of p53 by the HLI98 compounds also activate p53? The authors showed that the p53 that accumulated following treatment with the HLI98 compounds was transcriptionally active and induced transcription of the p53 target genes CDKN1A (encoding p21) and PUMA. An important part of the tumour-suppressor role of p53 is to induce apoptosis, and the ability of HLI98-stabilized p53 to induce apoptosis was shown by activation of caspases and an increase in cell death in treated MEFs. However, HLI98 treatment also caused p53-independent cell-cycle arrest and apoptosis, reflecting off-target activities of these compounds, such as actions against E2 enzymes of the ubiquitin system or other E3 enzymes.

These data show proof of principle for inhibitors of ubiquitin ligases and provide an alternative to inhibiting HDM2–p53 interaction as a way of reactivating p53 in tumours.