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.

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.

Pyranone, thiopyranone, and pyridone inhibitors of phosphatidylinositol 3-kinase related kinases. Structure-activity relationships for DNA-dependent protein kinase inhibition, and identification of the first potent and selective inhibitor of the ataxia telangiectasia mutated kinase.

Structure-activity relationships have been investigated for inhibition of DNA-dependent protein kinase (DNA-PK) and ATM kinase by a series of pyran-2-ones, pyran-4-ones, thiopyran-4-ones, and pyridin-4-ones. A wide range of IC50 values were observed for pyranones and thiopyranones substituted at the 6-position, with the 3- and 5-positions proving intolerant to substitution. Related pyran-2-ones, pyran-4-ones, and thiopyran-4-ones showed similar IC50 values against DNA-PK, whereas the pyridin-4-one system proved, in general, ineffective at inhibiting DNA-PK. Extended libraries exploring the 6-position of 2-morpholino-pyran-4-ones and 2-morpholino-thiopyrano-4-ones identified the first highly potent and selective ATM inhibitor 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (151C; ATM; IC50=13 nM) and revealed constrained SARs for ATM inhibition compared with DNA-PK. One of the most potent DNA-PK inhibitors identified, 2-(4-methoxyphenyl)-6-(morpholin-4-yl)pyran-4-one (16; DNA-PK; IC50=220 nM) effectively sensitized HeLa cells to the topoisomerase II inhibitor etoposide in vitro.

Discovery, in vivo activity, and mechanism of action of a small-molecule p53 activator.

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.

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 p53's 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 polymerization of tubulin in cells and in vitro. One of these novel compounds, JJ78:1, was subjected to structure-activity relationship studies and optimized 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 depolymerization 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 p53's sensitivity to alterations during mitosis.

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.

2,6-disubstituted pyran-4-one and thiopyran-4-one inhibitors of DNA-Dependent protein kinase (DNA-PK).

6-aryl-2-morpholin-4-yl-4H-pyran-4-ones and 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones were synthesised and evaluated as potential inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). Several compounds in each series exhibited superior activity to the chromenone LY294002, and were of comparable potency to the benzochromenone NU7026 (IC(50)=0.23 microM). Importantly, members of both structural classes were found to be selective inhibitors of DNA-PK over related phosphatidylinositol 3-kinase-related kinase (PIKK) family members. A multiple-parallel synthesis approach, employing Suzuki cross-coupling methodology, was utilised to prepare libraries of thiopyran-4-ones with a range of aromatic groups at the 3'- and 4'-positions on the thiopyran-4-one 6-aryl ring. Screening of the libraries resulted in the identification of 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones bearing naphthyl or benzo[b]thienyl substituents at the 4'-position, as potent DNA-PK inhibitors with IC(50) values in the 0.2-0.4 microM range.