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Improving CPT-11 Efficacy Using Structural Biology

利用结构生物学提高 CPT-11 功效

基本信息

批准号：
7169845
负责人：
Matthew R Redinbo
金额：
$ 24.45万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-02-01 至 2009-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7169845
关键词：
Acute Adult Adverse drug effect Antineoplastic Agents Authorization documentation Binding Biochemical Biochemistry Biological Assay Camptothecin Camptothecin-11 Carboxylic Ester Hydrolases Cells Chemotherapy-Oncologic Procedure Child Childhood Clinical Cocaine Colon Carcinoma Complex Crystallography Development Disclosure Doctor of Dental Surgery Doctor of Philosophy Drug Metabolic Detoxication Engineering Enzymes Face Ginger Glycoproteins Goals Heroin Human Instruction Intestines Knowledge Liver Malignant Neoplasms Mammalian Cell Metabolism Molecular Names Narcotics North Carolina Numbers Oryctolagus cuniculus Overdose Play Poison Principal Investigator Printing Process Prodrugs Protein Glycosylation Range Research Research Project Grants Resolution Role Role playing therapy SN-38 Saint Jude Children&apos s Research Hospital Series Solid Neoplasm Structure Testing Type I DNA Topoisomerases Universities Viral analog base carboxylesterase density design drug efficacy drug mechanism enzyme structure esterase human TOP1 protein human carboxylesterase 1 improved in vivo inhibitor/antagonist mutant novel programs protein structure size structural biology tool

项目摘要

CPT-11 is a camptothecin prodrug that is activated by esterases to yield SN-38, a potent topoisomerase I poison. CPT-11 has been approved for use against colon cancer and is currently being screened for other childhood and adult malignancies. We have recently characterized a series of carboxylesterases (CEs) that demonstrate dramatic differences in their ability to activate CPT-11. This proposal details the structural analyses of these CEs to unravel the role they play in CPT-11 activation. The long-range goal of this project is to use the structures of the enzymes involved in CPT-11 metabolism to improve the efficacy and use of CPT-11. By combining the tools of x-ray crystallography, biochemistry and in vivo studies, we propose to unravel the structural basis of CPT-11 activation. These studies should facilitate the development of novel CPT-11's, the design of CE inhibitors to reduce drug side effects, and the use of CEs in viral-directed cancer co-therapies. The hypothesis to be tested is that subtle structural differences between carboxylesterases play central roles in the activation of CPT-11 in vivo. We will examine three mammalian CEs. The first, a rabbit liver carboxylesterase (rCE), efficiently activates CPT-11. We have determined the crystal structure of the rCE glycoprotein in complex with a product of CPT-11 activation to 2.5 A resolution. This is the first structure of a mammalian CE. Two human carboxylesterases, carboxylesterase 1 (hCE1) and intestinal carboxylesterase (hiCE), will also be examined, hCE1 is similar in sequence to rCE (81% identity) but does not activate CPT-11. hiCE, in contrast, shares only 47% sequence identity with rCE but does efficiently activate CPT-11. Unraveling the structural basis of these differences is a central focus of this proposal. Five specific aims will be pursued combining the tools of x-ray crystallography with biochemical and in vivo studies: 1. Elucidate how CPT-11 binds to the rabbit liver CE and unravel the mechanism of drug activation. 2. Determine why human CE1 is unable to activate CPT-11 despite its high sequence similarity with rCE. 3. Examine the crystal structure of human intestinal CE to illuminate why this enzyme efficiently activates CPT-11. 4. Characterize, both structurally and functionally, mutants of rCE, hCE1 and hiCE designed to establish the molecular determinants of CPT-11 activation. 5. Assess the efficacy of drug activation and the ability to sensitize cells expressing mutant forms of rCE, hCE1 and hiCE to CPT-11. PERFORMANCESITE(S) (organizationc,ity, state) University of North Carolina at Chapel Hill Chapel Hill, NC St. Jude Children's Research Hospital Memphis, TN KEYPERSONNEL.Seeinstructions.Usecontinuationpagesas neededto providetherequiredinformationinthe formatshownbelow. StartwithPrincipalInvestigator.List allotherkeypersonnelin alphabeticalorder,last namefirst. Name Organization RoleonProject Matthew R. Redinbo, Ph.D. University of North Carolina Principal Investigator Philip M. Potter, Ph.D. St. Jude Children's Research Hospital Co-Principal Investigator Sompop Bencharit, D.D.S. University of North Carolina Graduate Assistant Ginger Carnahan, B.S. University of North Carolina Graduate Assistant Schroeder Noble, B.S. University of North Carolina Graduate Assistant Laurie Betts, Ph.D. University of North Carolina Research Technician Christopher L. Morton, B.S. St. Jude Children's Research Hospital Research Technician James P. Morken, Ph.D. University of North Carolina Collaborator Disclosure Permission Statement. Applicableto SBIR/STTROnly. Seeinstructions. [] Yes [] No PHS398(Rev.05/01) Page 2 FormPage2 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. ¿ Principal Investigator/Program Director (Last, first, middle): REDINBO, Matthew Robert The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. Type density and size must conform to limits and specifications provided in the PHS 398 Instructions. RESEARCH GRANT TABLE OF CONTENTS Page Numbers 1 Face Page .................................................................................................................................................. 2- 2 Description,

CPT-11是一种喜树碱前体药物，可被酯酶激活产生SN-38，一种有效的拓扑异构酶I毒物。 CPT-11已被批准用于治疗结肠癌，目前正在筛查其他儿童和成人癌症。恶性肿瘤。最近，我们发现了一系列羧酸酯酶（CE），它们激活CPT-11的能力存在差异。本建议书详细介绍了这些CE的结构分析，它们在CPT-11激活中发挥的作用。该项目的长期目标是利用CPT-11代谢中涉及的酶的结构来改善CPT-11代谢。 CPT-11的功效和用途。通过结合X射线晶体学，生物化学和体内研究的工具，我们提出解开CPT-11激活的结构基础。这些研究应该对小说的发展起到促进作用 CPT-11，CE抑制剂的设计以减少药物副作用，以及CE在病毒导向的癌症联合治疗中的应用。待检验的假设是羧酸酯酶之间的细微结构差异在羧酸酯酶的合成中起核心作用。 CPT-11在体内的活化。我们将研究三种哺乳动物的CE。第一种是兔肝羧酸酯酶（rCE），能有效激活CPT-11我们已经确定了与产物复合的rCE糖蛋白的晶体结构 CPT-11激活至2.5 A分辨率。这是哺乳动物CE的第一个结构。两种人类羧酸酯酶，羧酸酯酶1（hCE 1）和肠羧酸酯酶（hCE），hCE 1在序列上与 rCE（81%同一性），但不激活CPT-11。相比之下，hiCE与rCE只有47%的序列同一性，能有效激活CPT-11揭示这些差异的结构基础是本提案的核心重点。将结合X射线晶体学与生物化学和体内研究的工具，实现五个具体目标： 1.阐明CPT-11如何与兔肝CE结合，并阐明药物活化的机制。 2.确定尽管人CE 1与rCE具有高度序列相似性，但其不能激活CPT-11的原因。 3.检查人肠道CE的晶体结构，以阐明为什么这种酶有效地激活CPT-11。 4.对rCE、hCE 1和hiCE突变体进行结构和功能表征，以建立分子生物学模型。 CPT-11激活的决定因素。 5.评估药物活化的功效和致敏表达rCE、hCE 1和 hiCE到CPT-11。地点（组织、城市、州）北卡罗来纳州查佩尔山大学查佩尔山圣裘德儿童研究医院孟菲斯，田纳西州关键人员。参见说明。需要使用续页以下面所示的格式提供所需信息。 Starí首席调查员，按顺序列出所有关键人员，姓在前。名称组织机构RoleonProject 马修河Redinbo博士北卡罗来纳州大学首席研究员菲利普·M Potter博士St. Jude Children's Research Hospital联合首席研究员 Sompop Bencharit，D.D.S.北卡罗来纳州研究生助理 Ginger Carnahan，理学士北卡罗来纳州研究生助理 Schroeder Noble，理学学士北卡罗来纳州研究生助理劳里贝茨博士北卡罗来纳州大学研究技术员 Christopher L. Morton，BS圣裘德儿童研究医院研究技术员 James P. Morken博士北卡罗来纳州大学合作者披露许可声明。仅适用于SBIR/STTR。参见说明。[]是[]否 PHS 398（修订版05/01）第2页表格第2页在整个应用程序的底部连续编号页面。不要使用后缀，如3a，3b。首席研究员/项目主任（最后，第一，中间）：雷丁博，马修罗伯特主要研究者/项目负责人的姓名必须在打印页和续页的顶部提供。字体密度和尺寸必须符合PHS 398说明中提供的限制和规格。研究资助目录页码 1 首页...... 2- 2 产品描述：