Biomedical subjects

Philippe Roby

Publications and source records attributed to Philippe Roby.

3 recordsLinked to original sources

PubMed2005

Biochemical clustering of monomeric GTPases of the Ras superfamily.

To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen and FlashPlate, to measure nucleotide binding capacity, exchange, and hydrolysis activities of small monomeric GTPases. These two technologies have the characteristics to be very sensitive and to allow homogenous and high throughput assays. To analyze and integrate the data obtained, we developed an algorithm that allows the classification of GTPases according to their enzymatic activities. Integration and hierarchical clustering of these results revealed unexpected features of the small Rho GTPases when compared with primary sequence-based trees. Hence we propose a novel phylobiochemical classification of the Ras superfamily of GTPases.

Algorithms↗

PubMed2004

AlphaScreen kinase HTS platforms.

Kinases represents one of the most important family of targets in high throughput drug screening. Tyrosine kinases and serine/threonine kinases are known to play key roles in signal transduction as well as in cell growth and differentiation. Intense screening campaigns are underway in all major pharmaceuticals and large biotech companies to find kinase inhibitors for the treatment of inflammatory diseases, immunological disorders and cancer. The present contribution describes models that were developed to produce kinase assays amenable to HTS using AlphaScreen. Because of the flexibility allowed by AlphaScreen, kinase assays can be developed using direct or indirect approaches. Tyrosine kinase assays are usually performed with a direct format involving generic anti-phosphotyrosine antibodies while serine/threonine kinase assays are performed with an indirect format where specific antibodies are captured using protein A conjugated Acceptor beads. Streptavidin-coated Donor beads are used to capture either generic (ex. poly GT) or specific biotinylated substrates. Herein, are presented different methods to perform screening for inhibitors acting on the soluble beta-insulin receptor tyrosine kinase (IRKD), and on p38, a member of the MAP kinase family.

Drug Design↗

PubMed2003

Development of a versatile platform for nuclear receptor screening using AlphaScreen.

The interaction between nuclear receptors (NRs) and their coactivators, a key step in transcription regulation, requires a short consensus sequence called the LXXLL motif found in the coactivators' structure. Using the AlphaScreen technology, the authors have taken advantage of this receptor-coactivator interaction to develop a highly sensitive assay to identify and characterize compounds modulating NR activity. Estrogen and retinoic acid receptors were chosen as models to demonstrate the versatility of the AlphaScreen technology: (1) the assay can be designed using different antibodies to capture either full-length receptors or receptor domains that have been tagged, (2) the assay can differentiate between ligands that act as agonists or antagonists because only agonists will allow recruitment of the coactivator sequence-derived peptide, and (3) the assay gives the opportunity to screen for antagonists targeting the ligand-binding site or the dimerization interface between the receptor and the coactivator. Titration of the receptor and biotinylated peptide indicates that AlphaScreen is highly sensitive, requiring nanomolar concentration of reagents. Competition isotherms performed with known receptor antagonists demonstrate that the assay is a useful tool to rank the antagonists according to their order of potency. Overall, the results presented here indicate that the versatility, sensitivity, robustness, and ease of execution of the AlphaScreen NR assay will allow for efficient screening of NR modulators.

Antibodies↗