Control of PP1/PP2A Activity With Small Molecule Toxins

描述（由申请人提供）：结构多样的天然毒素组，包括冈田酸、花萼菌素、微囊藻毒素LR和互变霉素，通过抑制丝氨酸-苏氨酸蛋白磷酸酶PP 1和PP 2A发挥其细胞毒性作用。这种活性显著增加了细胞内多种蛋白质的磷酸化状态，这反过来又导致急性毒性作用或不受调节的细胞增殖。因此，这些化合物本身不仅是肝毒素，而且是肿瘤促进剂。 因为作为一个组，它们相对于其他已知的磷酸酶如PP 2B（钙调磷酸酶）、PP 2C和酪氨酸磷酸酶非常有效和特异地抑制PP 1和PP 2A，该组的几个成员已经成为重要的PP 1/2A结构/活性探针，并且更普遍地，成为研究细胞内信号传导途径的工具。然而，仍然需要具有增加的选择性或其他期望的性质如改善的膜渗透性的新抑制剂。最近的一份报告表明，天然毒素互变霉素抑制PP 1的数量级更有选择性（40：1）比互变霉素提供了一个重要的新的领导在这奋进。此外，大约在同一时间，冈田酸-PP 1复合物的结构被公布，首次提供了关于这种抑制剂的可靠结构信息，这将使我们能够设计比冈田酸本身更容易合成的类似物。 虽然这一领域的大多数研究都集中在PP 1和PP 2A的活性位点上，但在过去几年中，一些注意力已经转移到表征这些磷酸酶上的变构位点，该变构位点通过与各种结合（调节）蛋白相互作用来调节活性。尽管已知调节蛋白结合结构域的小肽类似物结合到PP 1的调节位点，但还没有关于调节位点的小分子配体的报道。因此，我们将准备小分子/肽杂交库，并研究它们与调控位点的相互作用，为非蛋白质PP 1激活剂的设计和合成打开大门。支持这一新领域，以及我们正在进行的抑制剂设计计划，我们将与著名的X射线晶体学家Tom Poulos教授合作进行酶学和结构研究，以获得有关我们设计所依据的假设的详细结构信息。

DESCRIPTION (provided by applicant): 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.