STRUCTURAL STUDIES OF HUMAN TOPOISOMERASE I AND DRUG PROCESSING ESTERASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 建议对两种在人类疾病中具有重要意义的蛋白质进行检测。首先，拓扑异构酶I通过松弛超螺旋张力在DNA代谢中发挥关键作用，也是抗癌药物喜树碱的唯一靶点。该应用程序的P.I.使用SSRL的设备确定了DNA与DNA络合物中的人拓扑异构酶I的晶体结构。我们现在建议检测喜树碱耐药和过敏形式的酶与DNA的复合体，以揭示这些药物的作用模式。这种蛋白质-DNA复合体的晶体是手部的，但内部的X射线衍射很弱(~4.5？)，因此需要同步辐射。其次，喜树碱衍生物伊立替康在体内通过肝脏羧酸酯酶加工成活性形式。我们已经结晶出一种能有效激活伊立替康的兔肝羧酸酯酶。这些研究的目标是了解这种关键的抗癌药物是如何在人体内加工的。这种酶的六方晶体(空间群P6322)在内部将X射线衍射到3.8？，并包含一个长的c轴(275？)。需要同步辐射来提高衍射分辨率，并分离由于我们本源的几何约束而重叠的反射。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The examination of two proteins of importance in human disease is proposed. First, topoisomerase I plays a critical role in DNA metabolism by relaxation superhelical tension and is also the sole target of the anti-cancer drug camptothecin. The P.I. of this application determined crystal structures of human topoisomerase I in complexes with DNA using the facilities at SSRL. We now propose to examine camptothecin resistant and hypersensitive forms of the enzyme in complex with DNA to unravel the mode of action of these drugs. Crystals of this protein-DNA complex are in-hand but diffract x-ray weakly in-house (~4.5 ¿); thus synchrotron radiation is required. Second, the camptothecin derivative Irinotecan is processed in vivo to its active form by liver carboxylesterases. We have crystallized a rabbit liver carboxylesterase that efficiently activates Irinotecan. The goal of these studies is to understand how this critical cancer drug is processed in humans. Hexagonal crystals of this enzyme (space group P6322) diffract x-rays to 3.8 ¿ in-house and contain a long c-axis (275 ¿). Synchrotron radiation is required to improve diffraction resolution and to separate reflections that overlap due to geometric constraints at our home source.