50% of human cancers is related to mutations of p53. A significant number of the mutations occur at positions that do not interact DNA directly in the core domain (see mutation data base at http://www-p53.iarc.fr/index.html). This is because wild-type p53 has a low thermodynamic stability at body temperature. Such mutations can unfold the native structure and lead to the loss of function. We are interested in studying p53 and the destabilzing mutants since they are closely related to the problem of protein folding.
Specific Aim (1): Looking for small molecules that can bind to the mutant of p53 and determination of the structures of core-domain mutantsIt has been shown that some mutants of the core domain of p53 can bind target DNA but are unstable in the free form and prone for degradation. A small molecule that can bind and stabilize it can serve as a molecular chaperon for maintaining the amount of folded p53 core mutants and preventing it from degradation in cells. In collaboration with other PIs at NCI. We will try to find organic compounds that can bind to the mutants of p53 and stabilize it. We are also interested in solving the high-resolution structures of the mutants.
Specific Aim (2): Selecting short peptides that can bind to the core domain of p53 by phage-displayTo identify possible binding sites for small molecules, we will use commercially available peptide phage library to select peptides that can bind to the native state of the core domain of p53.
Specific Aim (3): Selecting scFv fragment of human antibodies that can bind to the p53 core domain using Yeast surface expression and flow-cytometryHuman single chain antibody fragment (scFv) that can bind and stabilize the p53 core mutant provides an alternative therapeutic agent to cure cancer. Recently, a library of human antibody scFv fragments has been cloned and expressed on the yeast cell surface. Flow-cytometric and magnetic bead screening methods were used to isolate scFv with nonomolar antigen-binding affinity. We would like to use this library to select the scFv that can bind the p53 core domain and stabilize it. The selected scFv alone could be the therapeutic drug.
Specific Aim (4): Delivery of WT p53 proteins into cells by using liposomeA more direct approach to rescue the mutants of p53 is to deliver WT p53 to cancer cells. It has been shown recently that folded proteins can be delivered to cells by liposome. We would like to test whether such approach can be applied to p53. Because only cancer cells can activate the function of WT p53 protein, this approach is highly selective. To do so, we have synthesized the gene of p53 and expressed and purified p53 protein in E. coli. We will use cancer cell lines with R175H mutation to test the delivery. If our test is successful, WT p53 protein alone can become a drug for curing cancer.
