Jonathan J. Hollick

Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator

Summary We have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

Pilot screening programme for small molecule activators of p53

Activation of the p53 tumour suppressor is predicted to have therapeutically beneficial effects. Many current anti‐cancer therapies activate the p53 response via DNA damage. Non‐genotoxic activation of the p53 pathway would open the way to long‐term and possibly prophylactic treatments. We have established a simple protocol to screen small compound libraries for activators of p53‐dependent transcription, and to select and characterise the most interesting hits, which include non‐genotoxic activators. These compounds or their derivatives are of potential clinical interest. This approach may also lead to the identification of novel p53‐activating compound families and possibly to the description of novel molecular pathways regulating p53 activity.

Synthesis and biological characterisation of sirtuin inhibitors based on the tenovins.

The tenovins are small molecule inhibitors of the NAD(+)-dependent family of protein deacetylases known as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible applications in both cancer and neurodegenerative disease therapy. Through the synthesis of novel tenovin analogues, further insights into the structural requirements for activity against the sirtuins in vitro are provided. In addition, the activity of one of the analogues in cells led to an improved understanding of the function of SirT1 in cells.

Characterization, chemical optimization and anti-tumour activity of a tubulin poison identified by a p53-based phenotypic screen

A robust p53 cell based assay that exploits p53s function as a transcription factor was used to screen a small molecule library and identify bioactive small molecules with potential antitumor activity. Unexpectedly, the majority of the highest ranking hit compounds from this screen arrest cells in mitosis and most of them impair polymerisation of tubulin in cells and in vitro. One of these novel compounds, JJ78:1, was subjected to structure-activity relationship studies and optimised leading to the identification of JJ78:12. This molecule is significantly more potent than the original hit JJ78:1, as it is active in cells at two-digit nanomolar concentrations and shows clear antitumor activity in a mouse xenograft model as a single agent. The effects of nocodazole, a well established tubulin poison, and JJ78:12 on p53 levels are remarkably similar, supporting that tubulin depolymerisation is the main mechanism by which JJ78:12 treatment leads to p53 activation in cells. In summary, these results identify JJ78:12 as a potential cancer therapeutic, demonstrate that screening for activators of p53 in a cell-based assay is an effective way to identify inhibitors of mitosis progression and highlights p53s sensitivity to alterations during mitosis.

The discovery of nongenotoxic activators of p53: building on a cell-based high-throughput screen.

The reactivation of mutant forms of the transcriptional regulator p53 or artificially raising activated p53 levels in a controlled nongenotoxic manner are seen as two of the grand challenges in anti-cancer drug discovery. Recent reports suggest that these demanding goals are achievable. This review article focuses on the use of cell-based high-throughput screening to discover novel nongenotoxic activators of endogenous p53. This challenging approach to the early phases of drug discovery prioritises the discovery of compounds with activity in cells in the hope that the compounds discovered will ultimately be of more direct relevance to therapeutic development. However, this approach also requires that protein target identification studies are carried out. We, and others, have shown that whilst a sometimes daunting proposition, it is possible to identify the targets of compounds that activate p53.