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The Mechanics and Prediction of Small Molecule Binding to Proteins

We are presently in the process of developing a novel approach to genome-wide identification of small molecule binding proteins. Preliminary results from our group demonstrates that the similarity between the sequence of  a  protein and the sequences of affinity-selected, phage-displayed peptides can be predictive for protein binding to a small molecule ligand. Affinity-selected peptides provide information analogous to that of a consensus-binding sequence, and can be used in an analogous fashion to identify ligand binding sites. Libraries of phage-displayed peptides are being  screened for affinity to common metabolites and other small molecule ligands. The sequences of affinity-selected peptides will be determined and used as the basis of genome-wide analyses to identify proteins that have a high probability of binding to the screened ligands. Once a set of affinity-selected peptides has been validated through comparison with well-characterized proteins, it can be used for genome-wide annotation of all available genomes. Initially, affinity-selected peptide populations will be evaluated by comparison with completely sequenced microbial genomes of interest to the Department of Energy. The list of proteins identified as small molecule binders via this technique is being validated by comparison with annotated proteins to assess the false positive and false negative rates. These comparisons are then used to optimize the FORTRAN-based software written by our group members and to develop a method to calculate a confidence level for each prediction made. Web-based sequence analysis tools will provide the community with ready access to the software, the sequences of affinity-selected peptides, and the functional information generated from this project. The result will be a highly efficient means of genome annotation that will increase in utility as more ligands are screened and more genome sequences become available.

Angiogenesis

Mortality in over 90% of cancer patients is the result not of the effects of the primary lesion, but the growing out of normal cells by metastatic tumor cells at secondary sites within the body. Cellular migration of tumors is dependent upon both the successful disruption of cell-cell contacts at the primary site and the erection of proper scaffolding at the secondary site(s). A major step in scaffolding construction must include the attraction of new blood vessels (or angiogenesis) to feed and oxygenate the new tumor. These new vessels are primarily built with a class of cells called endothelial cells, which are one of the very few cell types in mammals that have the ability to migrate postembryonically.

Although a large number of antiangiogenic drugs with significant in vitro anti-cancer activity are presently in clinical trials, their mechanism of action is not well understood. Our laboratory is presently engaged in the construction of recombinant bacterial viruses which carry portions of proteins derived from endothelial cells on their surface. By studying the interaction of antiangiogenic molecules with these viral particles, we hope to identify the means by which this class of drugs inhibits the neovascularization of tumor tissue.

Early Markers for Ovarian Cancer

Ovarian cancer (OVCA) claims the lives of more women in North America each year than all other gynecologic malignancies combined. The high overall mortality from OVCA is primarily due to delays in diagnosis – although 90% of patients with early stage disease can be cured, 70% of patients at diagnosis have Stage III or higher disease, where the 5 year survival rate is only 20%. At present, there is a dearth of clinically useful antigenic markers for early stage disease. The currently used serum marker CA-125-II has limited sensitivity and specificity for detecting small volume, early stage disease, with a positive predictive value of less than 10%. We propose to use differential screening of phage displayed libraries to identify  cell surface markers specific to ovarian cancer.

The identification of an ovarian carcinoma cell-specific surface marker which is expressed early in tumorigenesis would have an immediate and substantial impact not only on diagnosis, but on development of receptor-mediated anti-cancer therapies. Phage-display derived peptides or antibodies selected for specific affinity to ovarian carcinoma cells can provide the basis for diagnostic tests for ovarian cancer and/or for targetting therapeutics to transformed cells. Analogous applications have been demonstrated for other systems (e.g., Trepel et al, 2002). For instance, successful organ targeting has been achieved with a chemically-coupled therapeutic construct (peptide-doxorubicin) (Arap et al, 1998). Peptide-bearing phage targeted to a cell surface receptor have been used successfully for gene therapy applications (Larocca et al, 1998; Poul et al, 1999) and for the induction of peptide-mediated endothelial cell apoptosis in targeted tissue (Ellerby et al, 1999 ).