Control of PP1/PP2A Activity With Small Molecule Toxins

描述(由申请人提供)：结构多样化的天然毒素组包括冈田酸、帽藻毒素、微囊藻毒素LR和交托霉素，通过抑制丝氨酸-苏氨酸蛋白磷酸酶PP1和PP2A发挥细胞毒作用。这种活性极大地增加了细胞内各种蛋白质的磷酸化状态，进而导致急性毒性效应或不受控制的细胞增殖。因此，这些化合物本身不仅是肝毒素，而且还是肿瘤促进剂。 因为作为一个组，它们对PP1和PP2A的抑制非常有效，并且相对于其他已知的磷酸酶，如PP2B(钙调神经磷酸酶)、PP2C和酪氨酸磷酸酶，这一组中的一些成员已经成为重要的PP1/2A结构/活性探针，更广泛地说，是研究细胞内信号通路的工具。然而，仍然需要具有更高选择性或其他所需性能的新的缓蚀剂，例如改善膜的通透性。最近的一份报告称，自然产生的毒素交托霉素比交托霉素更有选择性地抑制PP1一个数量级(40：1)，为这一努力提供了重要的新线索。此外，几乎在同一时间，冈田酸-PP1复合体的结构被公布，首次提供了关于这种抑制剂的固体结构信息，这将使我们能够设计出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PP1和PP2A的活性部位上，但在过去的几年里，一些注意力转移到了这些磷酸酶上的变构部位，它通过与各种结合(调节)蛋白的相互作用来调节活性。虽然已知调节蛋白结合域的小肽类似物与PP1的调节位点结合，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/肽杂交物的文库，并研究它们与调控位点的相互作用，从而为设计和合成非蛋白PP1激活剂打开大门。为了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射线结晶学家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基于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.