Molecular characterization of the hdm2-p53 interaction.

A number of viral oncogenes target the tumour suppressor protein p53 and inactivate its function. This is an important step in tumourogenesis. The cellular oncogene hdm2 acts through a similar mechanism. It binds the N terminus of p53, thereby interfering with the ability of p53 transcriptionally to activate genes responsible for growth arrest or apoptosis after genotoxic insults. The disruption of the interaction of the two proteins therefore comprises a promising therapeutic target for treatment of the subset of human cancers in which this pathway is active. In this paper we attempt to characterize the p53-hdm2 interaction biochemically. We analyse the potential of a series of peptide inhibitors, derived from previously described mdm2 binding peptide display phage, to disrupt this interaction in ELISA assays. We conclude that F19, W23 and L26 of p53 are critical contact points for p53 binding to hdm2. Furthermore, we show the potential of the monoclonal antibody 3G5 to interfere with binding of p53 to hdm2 in ELISA assays. Consequently, we define the binding site of 3G5 on hdm2 using overlapping peptides derived from the N terminus of hdm2 and phage display libraries. The result indicates L66, Y67 and E69 on hdm2 as critical binding points for 3G5. In electrophoretic mobility shift assay we demonstrate the formation of hdm2-p53 complexes that can be disrupted in the presence of 3G5 or inhibitory peptides. Finally, we describe the effects of NEM and DTT on the interaction between the two molecules in ELISA assays. All our results are discussed in the light of the recently published crystal structure of the mdm2-p53 complex. A striking correspondence between our findings and the crystal structure is revealed.

Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo.

BACKGROUND: The transcriptional activation function of the p53 tumour suppressor protein is induced by DNA damage and results in growth arrest and/or apoptotic responses. A key component of this response is the dramatic rise in p53 protein concentration resulting from an increase in the protein's stability. Very recently, it has been suggested that interaction with the Mdm2 protein may target p53 for rapid degradation. We have designed a gene encoding a small protein that binds tightly to the p53-binding pocket on the Mdm2 protein. We have constructed the gene by cloning a phage display optimised Mdm2-binding peptide into the active-site loop of thioredoxin. RESULTS: When introduced into cells containing low levels of wild-type p53, this protein causes a striking accumulation of the endogenous p53 protein, activation of a p53-responsive reporter gene, and cell cycle arrest mimicking the effects seen in these cells after exposure to UV or ionising radiation. Microinjection of a monoclonal antibody to the p53-binding site on Mdm2 achieves a similar effect, establishing its specificity. CONCLUSIONS: These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein-protein interactions in cells.

Identification of novel mdm2 binding peptides by phage display.

The oncogene mdm2 and its human homologue hdm2 bind to the tumour suppressor protein p53 and inactivate its function as a transcription factor. This has been implied as a possible mechanism for cancer development in several tumours including human sarcomas. The mdm2-p53 interaction is therefore a much persued target for the development of anti-cancer drugs. In order to find novel high affinity ligands for hdm2 which would interfere with its binding to p53 we screened phage display peptide libraries for mdm2 binding phage. We found a series of 12 and 15mer peptides which interact strongly with hdm2. The peptide sequences show striking homology with the previously established mdm2 binding site on p53, confirming that the peptide defined 18TFSDLW23 region is crucial for the interaction but that contact between the two molecules extends to position L26 on p53. Free synthetic peptides derived from the phage selected sequences proved to be up to 100 times stronger inhibitors of the p53-mdm2 interaction than the p53 derived wt-peptide in several ELISA-assays. This illustrates the potency of phage display libraries in the search for new peptide based lead structures designed to mimic or inhibit therapeutically important protein-protein interactions.

An evaluation of the anxiolytic SC 48,274 in generalized anxiety disorder (GAD)

1. The present study evaluated the safety and efficacy of two dosages of SC 48,274 (1mg and 25mg) as compared to placebo in subjects with Generalized Anxiety Disorder (GAD). 2. This was a randomized, double-blind, placebo-controlled, parallel-group study which was part of one of three large multicenter trials which evaluated a total of 5 doses of SC 48,274 (.25, 1, 5, 25, and 100mg bid). Following a 7-day placebo baseline period, patients entered 4 weeks of double-blind treatment and a 7-day placebo follow-up period. 3. Efficacy was assessed weekly throughout the study with the Hamilton Anxiety Rating Scale (HAM-A), and Clinical Global Impression (CGI), and at treatment endpoint with the Covi Anxiety Scale, Raskin Depression Scale and Hamilton Depression Rating Scale (HAM-D). A diagnosis of GAD according to DSM-III-R criteria (with the exception that a GAD minimum duration of 3 months was allowed), a HAM-A score > or = 20 (anxious mood and tension items > or = 2), HAM-D less than HAM-A, Covi Anxiety Score > or = 8, Raskin Depression Scale less than the Covi Anxiety, and age of 18 to 65 years were necessary for inclusion in the study. 4. Patients received one of two dosages of SC 48,274, either 1mg (n = 28), 25mg (n = 9), or placebo (n = 28) bid, during the 4-week randomized portion of the trial. 5. Mean changes from baseline in HAM-A scores for the 1mg, 25mg, and placebo groups after 4 weeks treatment were -5.1, -4.2, and -1.9, respectively. Changes were significant for the 1mg group vs. placebo (F = 8.93, p = 0.004), but not for the 25mg group (F = 2.26, p = 0.138). 6. CGI severity of illness scores were also significant for the 1mg group versus placebo at the end of treatment (X2 = 3.8, p = 0.05), but not for the 25mg group (X2 = 0.90, p = 0.343). Neither group showed significant CGI improvement scores by end of treatment. 7. The most frequent adverse events associated with the study drug (n = 37) were headache (n = 7), nausea (n = 3), palpitations (n = 4) and chest pains (n = 2). There was, however, no apparent pattern of adverse events distinguishing SC 48,274 from placebo.