首页> 外文学位 >文献详情
【24h】

New advances in high-quality screening by capillary electrophoresis: A unified platform for thermodynamic and kinetic characterization of protein-small molecule interactions.

机译:毛细管电泳高质量筛选的新进展:蛋白质-小分子相互作用的热力学和动力学表征的统一平台。

获取原文
获取原文并翻译 | 示例

摘要

The development of high-quality screening assays for the identification of biologically active ligands is critical in drug discovery. This thesis is aimed at developing new advances In capillary electrophoresis (CE) for the characterization of the conformational stability and enzymatic activity of protein targets with small molecules. CE provides a convenient platform for unbiased assessment of multiple thermodynamic and kinetic parameters associated with biomolecular interactions involving regulatory protein or isomerase enzymes, where various sample pretreatment steps can be integrated directly in-capillary during analysis. The first two chapters of the thesis (Chapters II, III) outline the development of dynamic ligand exchange-affinity capillary electrophoresis (DLE-ACE) as a novel strategy for the screening of allosteric ligands based on the differential stability of urea-induced unfolding of various apo/holo-protein states of cAMP receptor protein constructs. This work introduced a label-free and multivariate approach for ligand selection based on complementary thermodynamic parameters that allowed for determination of the dissociation constant of protein-ligand interactions over a wide dynamic range (> 104, Kd ≈ nM-mM). The subsequent two chapters of the thesis (Chapters IV, V) describe the development of a novel kinetic assay for unbiased characterization of isomerase activity associated with D/L-amino acid metabolism using hydroxyproline epimerase as a model system. Stereoselective resolution of various hydroxyproline isomers was accomplished via off-line or on-line chemical derivatization with dynamic complexation using chiral selector(s) in order to screen potential inhibitors for putative epimerase and racemase activity. The integration of both thermodynamic and kinetic strategies for differentiation of mutant from wild-type enzymes was important for revealing the function of a catalytic acid/base cysteine pair in the epimerase active site. Overall, this thesis outlines an integrative framework based on CE for high-quality screening, which is relevant in reducing the high attrition rate of lead candidates in drug development.
机译:用于鉴定生物活性配体的高质量筛选测定方法的开发对于药物发现至关重要。本论文旨在开发毛细管电泳(CE)的新进展,以表征小分子蛋白质靶标的构象稳定性和酶活性。 CE提供了一个方便的平台,用于公正地评估与涉及调节蛋白或异构酶的生物分子相互作用相关的多个热力学和动力学参数,其中各种样品预处理步骤可在分析过程中直接整合到毛细管中。论文的前两章(第II章,第III章)概述了动态配体交换亲和毛细管电泳(DLE-ACE)的发展,这是一种基于尿素诱导的U折叠展开的差异稳定性筛选别构配体的新策略。 cAMP受体蛋白构建体的各种载脂蛋白/全蛋白状态。这项工作引入了基于互补热力学参数的无标记多变量配体选择方法,该方法可在较宽的动态范围内(> 104,Kd≈ nM-mM)确定蛋白质-配体相互作用的解离常数。论文的后两章(第IV,V章)描述了使用羟脯氨酸差向异构酶作为模型系统对与D / L-氨基酸代谢相关的异构酶活性进行无偏性表征的新型动力学测定方法的开发。各种羟基脯氨酸异构体的立体选择性拆分是通过使用手性选择剂通过动态络合进行离线或在线化学衍生化来完成的,目的是筛选潜在的抑制异构酶和消旋酶活性的抑制剂。对于从野生型酶中区分突变体的热力学和动力学策略的整合对于揭示差向异构酶活性位点中的催化酸/碱基半胱氨酸对的功能很重要。总体而言,本文概述了基于CE的高质量筛选的集成框架,该框架与降低药物开发中潜在候选人的高流失率有关。

著录项

  • 作者

    Gavina, Jennilee M. A.;

  • 作者单位

    McMaster University (Canada).;

  • 授予单位 McMaster University (Canada).;
  • 学科 Chemistry Analytical.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 240 p.
  • 总页数 240
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

联系方式:18141920177 (微信同号)

客服邮箱:kefu@zhangqiaokeyan.com

京公网安备:11010802029741号 ICP备案号:京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有
  • 客服微信

  • 服务号