Structural and functional basis for therapeutic modulation of p53 signaling.

Effective modulation of structural features and/or functional properties of the major tumor suppressor p53 as a wild-type or cancer-associated mutant protein represents a major challenge in drug development for cancer. p53 is an attractive target for therapeutic design because of its involvement as a mediator of growth arrest and apoptosis after exposure to chemoradiotherapy and/or radiotherapy. Although most clinically used cytotoxic agents target stabilization of wild-type p53, there are a number of approaches that hold promise for reactivation of mutant p53. On the other hand, brief blockade of p53 may reduce toxicity from systemic cytotoxic therapy. Screens for restoration of p53 transcriptional responses in p53-deficient cells may provide a functional means to develop anticancer therapeutics. Structure-based modulation continues to hold promise for development of peptides or small molecules capable of modulation of either wild-type or mutant p53 proteins.

Pharmacological characterization of CP-547,632, a novel vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for cancer therapy.

Signaling through vascular endothelial growth factor (VEGF) receptors (VEGFRs) is a key pathway initiating endothelial cell proliferation and migration resulting in angiogenesis, a requirement for human tumor growth and metastasis. Abrogation of signaling through VEGFR by a variety of approaches has been demonstrated to inhibit angiogenesis and tumor growth. Small molecule inhibitors of VEGFR tyrosine kinase have been shown to inhibit angiogenesis, inhibit tumor growth, and prevent metastases. Our goal was to discover and characterize an p.o. active VEGFR-2 small molecule inhibitor. A novel isothiazole, CP-547,632, was identified as a potent inhibitor of the VEGFR-2 and basic fibroblast growth factor (FGF) kinases (IC(50) = 11 and 9 nM, respectively). It is selective relative to epidermal growth factor receptor, platelet-derived growth factor beta, and other related TKs. It also inhibits VEGF-stimulated autophosphorylation of VEGFR-2 in a whole cell assay with an IC(50) value of 6 nM. After oral administration of CP-547,632 to mice bearing NIH3T3/H-ras tumors, VEGFR-2 phosphorylation in tumors was inhibited in a dose-dependent fashion (EC(50) = 590 ng/ml). These plasma concentrations correlated well with the observed concentrations of the compound necessary to inhibit VEGF-induced corneal angiogenesis in BALB/c mice. A sponge angiogenesis assay was used to directly compare the inhibitory activities of CP-547,632 against FGF receptor 2 or VEGFR-2; this compound potently inhibits both basic FGF and VEGF-induced angiogenesis in vivo. The antitumor efficacy of this agent was evaluated after once daily p.o. administration to athymic mice bearing human xenografts and resulted in as much as 85% tumor growth inhibition. CP-547,632 is a well-tolerated, orally-bioavailable inhibitor presently under clinical investigation for the treatment of human malignancies.

Restoring p53-dependent tumor suppression.

p53 represents an ideal target for anti-cancer drug design, because p53 is mutated in more than half of human tumors. Most of the remaining tumors, although carrying wild-type p53, have defects in the p53-mediated apoptotic pathway. Activation of p53 activity by either chemotherapy or radiotherapy induces p53-dependent apoptosis in tumor cells with wild-type p53. Supplying exogenous wild-type p53 in cancer cells by gene delivery is effective in suppressing tumor growth of both mutant and wild-type p53-containing tumors. Blockage of p53 degradation pathways either by overexpression of ARF or interruption of MDM2:p53 interaction is effective in inducing p53 triggered tumor cell death. Since unlike most other tumor suppressor genes, mutant p53 is over expressed in tumor cells, a promising approach involves restoring tumor-suppressing function to mutant p53. The activity of the mutant p53 in tumor cells is restorable based on the fact that PAb241 antibody against the carboxy-terminus of p53 and peptides corresponding to the p53 carboxy-terminus can restore specific DNA-binding ability to some mutant p53 proteins. High throughout screening of chemical libraries has led to the identification of a group of small synthetic molecules such as CP-31398, which can restore p53 function to mutant p53 by stabilizing the active conformation of the protein that is destabilized in many mutants. Subsequent identification of PRIMA-1 provides further evidence to the possibility of developing anti-cancer drugs that may rescue mutant p53. Further understanding of the mechanisms by which CP-31398 and PRIMA-1 restore p53 activity may not only lead to discovery of more potent analogs but may also suggest new strategies for p53-targeting in tumor therapy.

Stabilization of p53 by CP-31398 inhibits ubiquitination without altering phosphorylation at serine 15 or 20 or MDM2 binding.

CP-31398, a styrylquinazoline, emerged from a high throughput screen for therapeutic agents that restore a wild-type-associated epitope (monoclonal antibody 1620) on the DNA-binding domain of the p53 protein. We found that CP-31398 can not only restore p53 function in mutant p53-expressing cells but also significantly increase the protein level and promote the activity of wild-type p53 in multiple human cell lines, including ATM-null cells. Cells treated with CP-31398 undergo either cell cycle arrest or apoptosis. Further investigation showed that CP-31398 blocks the ubiquitination and degradation of p53 but not in human papillomavirus E6-expressing cells. Of note, CP-31398 does not block the physical association between p53 and MDM2 in vivo. Moreover, unlike the DNA-damaging agent adriamycin, which induces strong phosphorylation of p53 on serines 15 and 20, CP-31398 exposure leads to no measurable phosphorylation on these sites. We found that CP-31398 could also stabilize exogenous p53 in p53 mutant, wild-type, and p53-null human cells, even in MDM2-null p53(-/-) mouse embryonic fibroblasts. Our results suggest a model wherein CP-31398-mediated stabilization of p53 may result from reduced ubiquitination, leading to high levels of transcriptionally active p53. Further understanding of this mechanism may lead to novel strategies for p53 stabilization and tumor suppression in cancers, even those with absent ARF or high MDM2 expression.

The mutant p53-conformation modifying drug, CP-31398, can induce apoptosis of human cancer cells and can stabilize wild-type p53 protein.

CP-31398, a styrylquinazoline, emerged from a screen for therapeutic agents that restore a wild-type DNA-binding conformation of mutant p53 to suppress tumors in-vivo (Science 286, 2507, 1999). We investigated the growth inhibitory mechanism of CP-31398 using nine human cancer cell lines containing wild-type, mutant or no p53 expression. Six of nine cell lines underwent apoptosis after exposure to CP-31398, while two cell lines, DLD1 colon cancer and H460 lung cancer, underwent exclusively cell cycle arrest. Cell cycle arrest preceded the apoptosis in some cases. CP-31398 did not inhibit growth of the p53 non-expressing ovarian cancer cell line SKOV3. Interestingly, we found that wild-type p53 protein is stabilized upon CP-31398 exposure. p53 target genes such as p21WAF1/Cip1, and KILLER/DR5 were upregulated by CP-31398, but their expression did not correlate with arrest or apoptosis induction. Combination of CP-31398 and TRAIL or chemotherapeutic agents enhanced cancer cell killing effect possibly through upregulation of p53-regulated genes such as KILLER/DR5. Bax-/-, wild-type p53-expressing cells displayed reduced susceptibility to killing by CP-31398. An Affymetrix GeneChip Array screen revealed that CP-31398 alters expression of non-p53 target genes in addition to p53-responsive genes. Although our preliminary data suggest that CP-31398 does not alter wild-type p53:MDM2 interaction, further efforts are required to elucidate the mechanism of wild-type p53 stabilization by CP-31398. The results increase our understanding of CP-31398 action, and suggest strategies for improving its specificity, possibly through use of microarrays to screen related compounds with higher mutant p53-specificity.