p53 transcriptional activity as a tool to uncover novel and diverse druggable targets in cancer

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that