In this grant application, we propose use of our newly developed encoded """"""""one-bead one-compound"""""""" (OBOC) combinatorial small molecule library methods to discover-peptidomimetic or small molecule substrates for p60c-src protein tyrosine kinase (PTK). The on-bead screening method will involve synthesis of small molecule libraries using the """"""""split and mix synthesis"""""""" method. Each library will have a tyrosine residue or a phenolic group plus a pfluorobeonzyl group. These will be screened against activated p60c-srcPTK with [gamma-32]ATP for phosphorylation activity. Peptidomimetic and small molecule compounds are chosen because, unlike peptides, they are more likely to be able to enter intact cells, especially when multiple aromatic and heterocyclic groups are present in the molecule. Peptidomimetic compounds synthesized on Tenta Gel or polyethylene glycol polyacrylamide bead resin are incubated with p60C-srcPTK and [gamma-32]ATP in vitro. The phosphorylated compound on beads are then identified by autoradiography and evaluated for solubility, non-specific binding and cellular uptake. Active substrates can be resynthesized with radioactive [18F]p-benzoic acid for further in vivo analysis with PET.
Specific aims of this proposal are: 1. To design and synthesize encoded """"""""one-bead one-compound"""""""" (OBOC) peptidomimetic and small molecule libraries containing fluorine and phenolic groups. 2. To screen libraries from aim 1 for peptidomimetic kinase substrates that can be phosphorylated by the p60C-src protein tyrosine kinase (PTK). 3. To decode the compounds in the positive bead, resynthesize them, and evaluate their activity as substrate for p60C-srcPTK. 4. To use autoradiography to evaluate the preferential retention of phosphorylated substrates inside cancer cell lines with high level of p60C-srcPTK. 5. To radiolabel and perform in vivo PET imaging studies in nude mice with the top three substrates with best cellular uptake and retention.
