Understanding p53 structure and targeting mutant p53 for improved cancer therapy

Sammanfattning: The p53 gene family consists of p53, p63 and p73. The three proteins share a high degree of structural similarity in their DNA-binding domains but have rather different functions. The tumor suppressor p53 acts as the guardian of the genome and is activated in response to cellular stress. p53 is a transcription factor that activates downstream target genes to induce cell cycle arrest, senescence or apoptosis. However, the TP53 gene is inactivated by missense mutation in about half of human tumors. Therefore, reactivation of mutant p53 is an attractive strategy for novel cancer treatment. The mutant p53-reactivating compound PRIMA-1, identified in a cellular screen of the NCI Diversity set, suppresses tumor cell growth in a mutant p53-dependent manner. The methylated analog PRIMA-1Met, now named APR-246, is even more potent. APR-246 has been successfully tested in a phase I/IIa clinical trial. A phase II clinical trial in ovarian cancer is ongoing. Both PRIMA-1 and APR-246 are prodrugs that are converted to the active compound methylene quinuclidinone (MQ), a Michael acceptor that binds covalently to cysteines in p53's DNA-binding domain. In paper I, we tested the effect of APR-246 on primary adult skin keratinocytes with p63 mutations from patients with the EEC developmental syndrome. We showed that APR-246 can partially rescue morphological defects of the mutant p63-carrying EEC keratinocytes, and expression of differentiation and stratification markers. Furthermore, we found that APR-246 restored the expression of p63 target genes. Our findings demonstrate that APR-246 can also target mutant forms of p63. In paper II, we found that the Michael acceptor activity of MQ is critical for MQ binding, thermostabilization and refolding of mutant p53. We identified Cys277 as a prime binding site for MQ in p53. Cysteine to alanine substitution at this position abolished both MQ binding and thermostabilization. Moreover, we found that both Cys124 and Cys277 are required for APR-246 mediated mutant p53 reactivation in His175 mutant p53-carrying tumor cells. In paper III, we selected potential thiol-reactive compounds including Michael acceptors, primary alcohols, imines and aldehydes, with top p53 selectivity based on datamining of the NCI database. Multivariable analysis identified different functional groups associated with various features of mutant p53 reactivation. Michael acceptors are more prone to high toxicity and thiol reactivity. Alcohols and imines are more associated with p53 refolding. Aldehydes are more likely to stabilize p53. These results may facilitate the design of novel mutant p53-targeting compounds. Paper IV describes a p53-like gene in the hydrothermal annelid Alvinella pompejana. Sequence alignment and structure modeling indicated that this p53 homolog is more similar to p63 and p73. Consistent with this finding, the DNA-binding domain of Alvinella has high thermostability. We identified repacking features in the hydrophobic core of Alvinella p53 that are associated with its high thermostability. Understanding structural features of p53 family proteins that govern stability may provide insights for development of mutant p53-reactivating drugs.