The lethal factor of Bacillus anthracis is a Zn-endoproteinase that targets the mitogen activated protein kinase kinase (MEK) family of signaling proteins. The toxin recognizes particular members of this family and cleaves a motif that is required for the interaction of MEKs with their downstream effectors--mitogen-activated protein kinases (MAPKs). As a consequence, multiple MAPK pathways are inhibited, and this impairs the viability of affected cells. The goal of this project is to develop an in vivo screening system that allows for the rapid identification of potent and highly specific therapeutics. Rather than screening for compounds that affect particular aspects of lethal factor function in vitro (e.g., its proteolytic activity of the toxin or substrate affinity), we will select for molecules that protect whole cells from lethal factor toxicity using specially engineered strains of the budding yeast Saccharomyces cerevisiae. The yeast strains will be designed to conditionally express anthrax lethal factor and to require the function of a human MEK for survival. When expression of the lethal factor is turned on, the MEK will be inactivated and the cells will die. By selecting for the viability of yeast cells, we can screen hundreds of thousands of compounds and identify potent anti-toxins in a matter of weeks. An important advantage of this approach is that it requires maintenance of MEK function. Agents that interfere with MEK signaling will not pass through the screen. Another attractive feature of this system is that in comparison to mammalian cell-based screens, yeast allow for a much faster response to new mutant strains of the pathogen due to its relatively rapid rate of growth. In addition to providing proof of concept, we will optimize the yeast-based system for high-throughput screening of peptide and cDNA libraries, as well as combinatorial libraries of membrane-permeable small molecules.
