In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules.

VPS34 is a class III phosphoinositide 3-kinase that acts on vesicle trafficking. This kinase has recently attracted significant attention because of the function it plays in the machinery involved in the early steps of autophagy. Moreover, because significant progress had been made in the optimization of specific kinase inhibitors, its potential to be targeted by catalytic inhibitors has been investigated by different groups. The aim of this review is to present the key in vitro assays necessary for characterizing inhibitors of the catalytic activity of VPS34. The review covers catalytic (IC50 on purified recombinant protein) and binding assays (KD, ka, kd on purified recombinant protein), and a cell-based assay (IC50 in GFP-FYVE expressing cell line). The methodology for crystallization of VPS34 protein is also presented as it can provide guidance for the design by medicinal chemistry of small molecular mass kinase inhibitor.

p53 mediated death of cells overexpressing MDM2 by an inhibitor of MDM2 interaction with p53.

The p53 tumour suppressor is frequently inactivated in human tumours. One form of inactivation results from overexpression of MDM2, that normally forms a negative auto-regulatory loop with p53 and inhibits its activity through complex formation. We have investigated whether disrupting the MDM2-p53 complex in cells that overexpress MDM2 is sufficient to trigger p53 mediated cell death. We find that expression of a peptide homologue of p53 that binds to MDM2 leads to increased p53 levels and transcriptional activity. The consequences are increased expression of the downstream effectors MDM2 and p21WAF1/CIP1, inhibition of colony formation, cell cycle arrest and cell death. There is also a decrease in E2F activity, that might have been due to the known physical and functional interactions of MDM2 with E2F1/DP1. However, this decrease is p53 dependent, as are also colony formation, cell cycle arrest and cell death. These results show that a peptide homologue of p53 is sufficient to induce p53 dependent cell death in cells overexpressing MDM2, and support the notion that disruption of the p53-MDM2 complex is a target for the development of therapeutic agents.

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments.

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 -/-human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 -/- human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

Identifying Small Molecules which Inhibit Autophagy: a Phenotypic Screen Using Image-Based High-Content Cell Analysis.

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a pro-survival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents, including acquired resistance. We aimed to identify small-molecule autophagy inhibitors using a HTS/HCA approach through a phenotypic, cell image-based assay, in order to screen multiple biological targets simultaneously and to screen compounds in a physiologically relevant environment. LC3 is a component of the autophagosome, which undergoes a cytoplasmic redistribution from diffuse to punctate dots during autophagy. We employed HeLa cells stably expressing EGFP-LC3 in a primary phenotypic screen. As a first step, a "Validation Library" of about 8,000 pre-selected compounds, about 25% of which had known biological activity and the others representing a range of chemical structures, was run in duplicate both to assess screening suitability and likely hit rate, and to give a valuable preview of possible active structures or biological targets. The primary screen of about 0.25 million compounds yielded around 10,500 positive compounds. These were tested in a suite of further cellular assays designed to eliminate unwanted positives, together with the application of chemi- and bioinformatics to pick out compounds with known biological activity. These processes enabled the selection of compounds that were the most promisingly active and specific. The screening "tree" identified, amongst others with as yet unidentified targets, chemical series active against autophagy-relevant biological targets ULK or Vsp34, validating the phenotypic screening methods selected. Finally, about 400 compounds were fully qualified after following this triage. The development of the assays, compound screening process and the compound triage is described.

Effect of DNA gyrase inhibitors pefloxacin, five other quinolones, novobiocin, and clorobiocin on Escherichia coli topoisomerase I.

Two coumarins, inhibitors of the B subunit of DNA gyrase, and six quinolones, inhibitors of the A subunit, were tested against Escherichia coli topoisomerase I-catalyzed DNA relaxation. Coumarins had no effect, whereas quinolones were inhibitors of the enzyme. This inhibition was compared with that of DNA gyrase and calf thymus topoisomerase I. The 50% inhibitory concentrations for E. coli topoisomerase I were about one order of magnitude higher than the corresponding values for E. coli DNA gyrase but were far lower than the known values for calf thymus topoisomerase I. There was a good relationship between inhibition of the two prokaryotic topoisomerases and MICs for E. coli, and the quinolones could be ranked in the same order in the three cases.

An efficient screening assay for the rapid and precise determination of affinities between leucine zipper domains.

The protein products of the jun and fos oncogenes require a functional protein-protein interaction domain, called the "leucine zipper domain", to exert their transcriptional regulatory activity. A scintillation proximity assay was developed in which the biotinylated leucine zipper domain of the Jun protein (275-315) was immobilized on streptavidin-coated microfluorospheres and in which the leucine zipper domain of the Fos protein (160-200) was used as free, labeled ligand. The Fos leucine zipper peptide specifically bound to the Jun leucine zipper peptide, and for the first time, a dissociation constant (Kd = 110 +/- 12 nM in PBS/0.1% Tween) could be determined. Optimal heterodimer formation was reached at neutral pH. Both acidic and alkaline pH decreased the association of the peptides which was, furthermore, completely abolished by 500 mM NaCl, confirming that charged residues are critical for heterodimerization. A commercially obtained recombinant Jun protein competed as efficiently as the Jun leucine zipper peptide for binding to the Fos peptide, confirming the feasibility of using the two leucine zipper peptides to study the interactions between the two transcription factors. We also injected leucine zipper peptides individually into Xenopus oocytes to study whether they would interfere with the activity of the Fos/Jun heterodimer in vivo. Both peptides blocked selectively insulin-mediated oocyte maturation with an IC50 in the range of 15 ng per oocyte. In conclusion, the scintillation proximity assay described here may be used to investigate protein-protein interactions mediated by leucine zipper structures and to identify compounds that inhibit leucine zipper association.

The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression.

Wild-type p53 is a tumor suppressor gene which can activate or repress transcription, as well as induce apoptosis. The human p53 proline-rich domain localized between amino acids 64 and 92 has been reported to be necessary for efficient growth suppression. This study shows that this property mainly results from impaired apoptotic activity. Although deletion of the proline-rich domain does not affect transactivation of several promoters, such as WAF1, MDM2 and BAX, it does alter transcriptional repression, reactive oxygen species production and sequence-specific transactivation of the PIG3 gene, and these are activities which affect apoptosis. Whereas gel retardation assays revealed that this domain did not alter in vitro the specific binding to the p53-responsive element of PIG3, this domain plays a critical role in transactivation from a synthetic promoter containing this element. To explain this discrepancy, evidence is given for a proline-rich domain-mediated cellular activation of p53 DNA binding.