The structurally diverse group of natural toxins including okadaic acid, calyculin, microcystin LR, and tautomycin exert their cytotoxic effects by inhibiting the serine-threonine protein phosphatases PP1 and PP2A. This activity dramatically increases the phosphorylation state of a variety of proteins within the cell, which in turn results in acute toxic effects or unregulated cellular proliferation. The compounds themselves are therefore not only hepatotoxins, but also tumor promoters. Because as a group they inhibit PP1 and PP2A quite potently and specifically relative to other known phosphatases such as PP2B (calcineurin), PP2C, and the tyrosine phosphatases, several members of this group have become important PP1/2A structural/activity probes and, more generally, tools for studying intracellular signaling pathways. However, there is still a demand for new inhibitors with increased selectivity or other desirable properties such as improved membrane permeability. A very recent report that the naturally occurring toxin tautomycetin inhibits PP1 an order of magnitude more selectively (40:1) than tautomycin provides an important new lead in this endeavor. In addition, at about the same time, the structure of the okadaic acid-PP1 complex was published, providing for the first time solid structural information about this inhibitor that will allow us to design analogs that are much more synthetically accessible than okadaic acid itself. While most of the studies in this field have focused on the active sites of PP1 and PP2A, in the past several years some attention has shifted to characterizing an allosteric site on these phosphatases that modulates activity by interacting with a variety of binding (regulatory) proteins. Although small peptide analogues of the regulatory protein binding domains are known to bind to the regulatory site of PP1, there have been no reports of small molecule ligands for the regulatory site. We will therefore prepare libraries of small molecule/peptide hybrids and study their interactions with the regulatory site, opening the door to the design and synthesis of non-protein PP1 activators. Supporting this new area, as well as our ongoing inhibitor design program, we will conduct enzymological and structural studies in collaboration with a prominent X-ray crystallographer, Professor Tom Poulos, in order to obtain detailed structural information concerning the hypotheses upon which our designs are based.
