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Study of calcineurin interaction with inhibitor-1 and natural products. Towards novel calcineurin inhibitors.

机译：研究钙调神经磷酸酶与抑制剂1和天然产物的相互作用。朝向新型钙调神经磷酸酶抑制剂。

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摘要

Calcineurin (CaN) is an eukaryotic Ser/Thr protein phosphatase that plays an important role in lymphocyte activation. Inhibition of CaN leads to immune system suppression, a necessary procedure in organ transplantation. However, use of CaN inhibitors results in toxic side effects. The search for more specific immunosuppressants is therefore an important and ongoing endeavor. The focus of this thesis was to investigate methods that could lead to the discovery of novel CaN inhibitors.;CaN is closely related to protein phosphatase-1 (PP-1) but both phosphatases retain distinct substrates, regulatory proteins, and inhibitors. Two such inhibitors, okadaic acid (OA) and microcystin-LR (MCLR), potently inhibit PP-1 but are markedly less effective against CaN. Mutagenesis of CaN was undertaken to generate a form of CaN more sensitive to OA and MCLR. The optimal construct was a Y159I:F160Y:L312C:Y315L quadruple point mutant that showed 600-fold and 37-fold increased sensitivity to MCLR and OA, respectively. These studies provide the basis for chemical engineering to generate analogs of OA and MCLR that are CaN specific.;Multiple proteins use the PXIXIT amino acid sequence to interact with CaN, most notably the nuclear factor of activated T-cells (NFAT). NFAT dephosphorylation by CaN is necessary for immune system activation. The second chapter of results demonstrates that inhibitor-1 (I-1), a CaN substrate, contains the PXIXIT motif used for CaN binding. Disruption of the PXIXIT motif of I-1 reduced its dephosphorylation by CaN. I-1 therefore is a suitable substrate for identification of novel immunosuppressants that could block the interaction between CaN and the PXIXIT motif of NFAT proteins.;A new method of bioassay-guided isolation of novel CaN inhibitors from extracts of marine organisms was established in the final chapter of results. Monitoring the inhibition of CaN activity using the I-1 substrate during extract purification led to identification of three compounds: halisulfate-7, hipposulfate C, and a novel one, irregularsulfate. These compounds were identified as equipotent microM inhibitors of CaN and PP-1. The method of bioassay-guided isolation of CaN inhibitors is applicable to searching for potential drugs that could block access to the active site of CaN, or to the PXIXIT motif binding groove.
机译：钙调神经磷酸酶（CaN）是一种真核Ser / Thr蛋白磷酸酶，在淋巴细胞活化中起重要作用。抑制CaN会导致免疫系统抑制，这是器官移植的必要步骤。但是，使用CaN抑制剂会导致毒副作用。因此，寻找更特异性的免疫抑制剂是重要且持续的努力。本论文的重点是研究可能导致发现新型CaN抑制剂的方法。CaN与蛋白磷酸酶1（PP-1）密切相关，但两种磷酸酶均保留不同的底物，调节蛋白和抑制剂。冈田酸（OA）和微囊藻毒素-LR（MCLR）这两种抑制剂可以有效抑制PP-1，但对CaN的抑制作用明显较低。进行CaN诱变以生成对OA和MCLR更敏感的CaN形式。最佳构建体是Y159I：F160Y：L312C：Y315L四点突变体，其对MCLR和OA的敏感性分别提高了600倍和37倍。这些研究为化学工程产生CaN特异性的OA和MCLR类似物提供了基础。多种蛋白质使用PXIXIT氨基酸序列与CaN相互作用，最值得注意的是活化T细胞的核因子（NFAT）。 CaN对NFAT的去磷酸化对于激活免疫系统是必需的。结果的第二章证明，抑制剂1（I-1）是一种CaN底物，含有用于CaN结合的PXIXIT基序。 I-1的PXIXIT基序的破坏减少了CaN的去磷酸化作用。因此，I-1是鉴定新型免疫抑制剂的合适底物，可以阻止CaN和NFAT蛋白的PXIXIT基序之间的相互作用。;在海洋生物提取物中建立了一种生物测定指导的新型CaN抑制剂生物分离方法。结果的最后一章。在提取物纯化过程中使用I-1底物监测CaN活性的抑制作用，可鉴定出三种化合物：haliculfate-7，hipposulfate C和一种新颖的不规则硫酸盐。这些化合物被确定为CaN和PP-1的等效microM抑制剂。 CaN抑制剂的生物测定指导分离方法适用于寻找可能阻碍进入CaN活性位点或PXIXIT基序结合槽的药物。

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  • 作者

    Raszek, Mikolaj Marek.;

  • 作者单位

    University of Alberta (Canada).;

  • 授予单位 University of Alberta (Canada).;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 315 p.
  • 总页数 315
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 老年病学;
  • 关键词

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