Identification of Novel p53 Pathway Activating Small-Molecule Compounds Reveals Unexpected Similarities with Known Therapeutic Agents

Manipulation of the activity of the p53 tumor suppressor pathway has demonstrated potential benefit in preclinical mouse tumor models and has entered human clinical trials. We describe here an improved, extensive small-molecule chemical compound library screen for p53 pathway activation in a human cancer cell line devised to identify hits with potent antitumor activity. We uncover six novel small-molecule lead compounds, which activate p53 and repress the growth of human cancer cells. Two tested compounds suppress in vivo tumor growth in an orthotopic mouse model of human B-cell lymphoma. All compounds interact with DNA, and two activate p53 pathway in a DNA damage signaling-dependent manner. A further screen of a drug library of approved drugs for medicinal uses and analysis of gene-expression signatures of the novel compounds revealed similarities to known DNA intercalating and topoisomerase interfering agents and unexpected connectivities to known drugs without previously demonstrated anticancer activities. These included several neuroleptics, glycosides, antihistamines and adrenoreceptor antagonists. This unbiased screen pinpoints interference with the DNA topology as the predominant mean of pharmacological activation of the p53 pathway and identifies potential novel antitumor agents.

[1]  W. Denny,et al.  Genotoxicity of non-covalent interactions: DNA intercalators. , 2007, Mutation research.

[2]  K. Kinzler,et al.  Requirement for p53 and p21 to sustain G2 arrest after DNA damage. , 1998, Science.

[3]  Galina Selivanova,et al.  Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound , 2002, Nature Medicine.

[4]  M. R. Webb,et al.  A gel electrophoresis assay for the simultaneous determination of topoisomerase I inhibition and DNA intercalation. , 2003, Analytical biochemistry.

[5]  B. Vogelstein,et al.  Participation of p53 protein in the cellular response to DNA damage. , 1991, Cancer research.

[6]  Brad T. Sherman,et al.  DAVID: Database for Annotation, Visualization, and Integrated Discovery , 2003, Genome Biology.

[7]  E. D. Bransome,et al.  Small molecule intercalation with double stranded DNA: implications for normal gene regulation and for predicting the biological efficacy and genotoxicity of drugs and other chemicals. , 2007, Mutation research.

[8]  E. Cesarman,et al.  NF-kappaB is essential for the progression of KSHV- and EBV-infected lymphomas in vivo. , 2006, Blood.

[9]  J. Nitiss.  Targeting DNA topoisomerase II in cancer chemotherapy , 2009, Nature Reviews Cancer.

[10]  S. Kern,et al.  High-throughput measurement of the Tp53 response to anticancer drugs and random compounds using a stably integrated Tp53-responsive luciferase reporter. , 2003, Carcinogenesis.

[11]  Robert A Newman,et al.  Cardiac glycosides as novel cancer therapeutic agents. , 2008, Molecular interventions.

[12]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[13]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[14]  Paul A Clemons,et al.  The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease , 2006, Science.

[15]  Z. Weng,et al.  A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome , 2006, Cell.

[16]  Karen H. Vousden,et al.  p53 in health and disease , 2007, Nature Reviews Molecular Cell Biology.

[17]  S. Lowe,et al.  Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas , 2011, Nature.

[18]  B. Zehnbauer,et al.  Growth factor-mediated terminal differentiation of chronic myeloid leukemia. , 1994, Cancer research.

[19]  Gerard I. Evan,et al.  Modeling the Therapeutic Efficacy of p53 Restoration in Tumors , 2006, Cell.

[20]  M. Protopopova,et al.  Small molecule RITA binds to p53, blocks p53–HDM-2 interaction and activates p53 function in tumors , 2004, Nature Medicine.

[21]  E. Appella,et al.  The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX. , 2004, DNA repair.

[22]  C. Prives,et al.  Blinded by the Light: The Growing Complexity of p53 , 2009, Cell.

[23]  Alan R. Fersht,et al.  Awakening guardian angels: drugging the p53 pathway , 2009, Nature Reviews Cancer.

[24]  L. Vassilev,et al.  In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2 , 2004, Science.

[25]  Wafik S El-Deiry,et al.  Small-molecule modulators of p53 family signaling and antitumor effects in p53-deficient human colon tumor xenografts. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[26]  R. Medema,et al.  Human prostate epithelium lacks Wee1A-mediated DNA damage-induced checkpoint enforcement , 2007, Proceedings of the National Academy of Sciences.

[27]  A. Canals,et al.  DNA-binding drugs caught in action: the latest 3D pictures of drug-DNA complexes. , 2009, Dalton transactions.

[28]  Dajun Yang,et al.  Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition , 2008, Proceedings of the National Academy of Sciences.

[29]  D. Notterman,et al.  Analysis of p53-regulated gene expression patterns using oligonucleotide arrays. , 2000, Genes & development.

[30]  P. Lyu,et al.  Prazosin displays anticancer activity against human prostate cancers: targeting DNA and cell cycle. , 2007, Neoplasia.

[31]  N. Kyprianou,et al.  Effect of α1-Adrenoceptor Antagonist Exposure on Prostate Cancer Incidence: An Observational Cohort Study , 2007 .

[32]  Lee Baker,et al.  Discovery, In Vivo Activity, and Mechanism of Action of a Small-Molecule p53 Activator , 2007, Cancer cell.

[33]  G. Wahl,et al.  Regulating the p53 pathway: in vitro hypotheses, in vivo veritas , 2006, Nature Reviews Cancer.

[34]  Dale L Boger,et al.  Sequence-selective DNA recognition: natural products and nature's lessons. , 2004, Chemistry & biology.

[35]  D. Lane,et al.  Transcription — guarding the genome by sensing DNA damage , 2004, Nature Reviews Cancer.

[36]  H. Sørensen,et al.  Cancer risk among users of neuroleptic medication: a population-based cohort study , 2006, British Journal of Cancer.

[37]  F. Lombardo,et al.  Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. , 2001, Advanced drug delivery reviews.

[38]  N. Curtin,et al.  Identification and Characterization of a Novel and Specific Inhibitor of the Ataxia-Telangiectasia Mutated Kinase ATM , 2004, Cancer Research.

[39]  Laurence H. Hurley,et al.  DNA and its associated processes as targets for cancer therapy , 2002, Nature Reviews Cancer.

[40]  Paul J Hergenrother,et al.  DNA as a target for anticancer compounds: methods to determine the mode of binding and the mechanism of action. , 2007, Current opinion in biotechnology.

[41]  M. Kastan.  Wild-Type p53: Tumors Can't Stand It , 2007, Cell.

[42]  G. Semenza.  Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics , 2010, Oncogene.

[43]  S. Kurki,et al.  Reactivation of the p53 pathway as a treatment modality for KSHV-induced lymphomas. , 2007, The Journal of clinical investigation.

[44]  T. Jacks,et al.  Restoration of p53 function leads to tumour regression in vivo , 2007, Nature.

[45]  D. Malkin,et al.  Germline p53 mutations and heritable cancer. , 1994, Annual review of genetics.

[46]  K. Kinzler,et al.  Definition of a consensus binding site for p53 , 1992, Nature Genetics.

[47]  N. Kyprianou,et al.  Effect of alpha1-adrenoceptor antagonist exposure on prostate cancer incidence: an observational cohort study. , 2007, The Journal of urology.

[48]  Thierry Soussi,et al.  Assessing TP53 status in human tumours to evaluate clinical outcome , 2001, Nature Reviews Cancer.

[49]  A. Eastman,et al.  p53-based cancer therapies: Is defective p53 the Achilles heel of the tumor? , 2007, Carcinogenesis.

[50]  J. Slaton.  Small molecules that reactivate p53 in renal cell carcinoma reveal a NF-kappaB-dependent mechanism of p53 suppression in tumors , 2006 .