A YEAST GENETIC SYSTEM FOR SELECTING SMALL-MOLECULE INHIBITORS OF PROTEIN-PROTEIN INTERACTIONS IN NANODROPLETS

Cellular processes are mediated by complex networks of molecular inter actions. Dissection of their role most: commonly is achieved by using genetic mutations that alter, for example, protein-protein interaction s, Small molecules that accomplish the same result would provide a pow erful camplement to the genetic approach, but it generally is believed that: such molecules are rare, There art several natural products, ho wever, that illustrate the feasibility of this approach. Split-pool sy nthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attache d to cell-sized polymer beads. Here, we describe a genetic system comp atible with split-pool synthesis that allows the detection of cell-per meable, small molecule inhibitors of protein-protein interactions in 1 00- to ZOO-nr cell culture droplets, prepared by a recently described technique that: arrays large numbers df such droplets. These ''nanodro plets'' contain defined media, cells, and. one or more beads containin g approximate to 100 pmol of a photoreleasable small molecule and a co ntrolled number of cells. The engineered Saccharomyces cerevisiae cell s used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disrupti on of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction, We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKB12 can be disrupt ed by the small molecule FK506 at nanomolar concentrations in nanodrop lets. This system should provide a general method for selecting cell-p ermeable ligands that can ire used to study the relevance of protein-p rotein interactions in living cells or organisms.