Induced Proximity with Small Molecules to Explore Biologic Processes

A goal of our laboratory is to develop methods to rapidly and reversibly regulate mammalian genes that could be used like temperature-sensitive alleles in model organisms. We believe that understanding complex biologic processes will require rapid activation and inaction of proteins to allow one to remove or add the function of a protein in a living organism and then follow the orderly sequence of biochemical changes that follow. This approach allows one to define steps in biochemical pathways and distinguish causality from coincidence. As our approach we chose to use small, membrane-permeable molecules to regulate proximity of proteins, because proximity is one of the most widely used biologic control mechanisms. As seen in the illustration below we conceived of small molecules that readily pass into cells and then bring proteins into proximity by virtue of binding to small protein tags on the protein of interest (Spencer et al. Science 1993). We call these molecules CIDs or Chemical Inducers of Dimerization. A variety of CIDs of different chemical classes have been made and used to regulate nearly all levels of signaling and processes as diverse as secretion, transcription and chromosome separation. Our studies began several years ago with Stuart Schreiber in the Department of Chemical Biology at Harvard. This approach has been used at Ariad Pharmaceuticals in Cambridge to develop methods for human gene therapy and they maintain a web site for distributing materials.

Recently, Kryn Stankunas, Hank Bayle and Jason Gastwicki in the lab have developed a method to regulate the stability of proteins that appears to be applicable to many genes. They have used homologous recombination to insert a small peptide tag that destabilizes the parent protein giving the null phenotype in mice. Addition of rapamycin or a non-toxic analogue, C20-methallyl rapamycin leads to rapid stabilization of the protein and restoration of function. We have recently used this to demonstrate that GSK3b executes its functions at two different times during development and to dissect the mechanisms underlying cleft palate.