Improving CPT-11 Efficacy Using Structural Biology

CPT-11是一种喜树碱前体药物，由酯酶激活产生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，目前正在对其他儿童和成人进行筛查 恶性肿瘤。我们最近表征了一系列羧酸酯酶(CEs)，这些酶表现出戏剧性的 它们激活CPT-11的能力存在差异。这项提案详细介绍了这些CE的结构分析，以揭开 它们在CPT-11激活中起作用。 这个项目的长期目标是利用参与CPT-11新陈代谢的酶的结构来改善 CPT-11的疗效和用途。通过结合x射线结晶学、生物化学和活体研究的工具，我们 建议解开CPT-11激活的结构基础。这些研究应该有助于小说的发展 CPT-11‘S，CE抑制剂的设计以减少药物副作用，以及CES在病毒导向的癌症联合治疗中的使用。 需要检验的假设是，羧酸酯酶之间的细微结构差异在 CPT-11在体内的激活。我们将研究三种哺乳动物的CES。第一，兔肝羧酸酯酶(RCE)， 有效地激活CPT-11。我们测定了RCE糖蛋白与一个产物的络合物的晶体结构 CPT-11激活到2.5A分辨率。这是哺乳动物CE的第一个结构。两种人类羧酸酯酶， 还将检测羧酸酯酶1(HCE1)和肠道羧酸酯酶(HICE)，hCE1的序列与 RCE(81%同一性)，但不激活CPT-11。相比之下，Hice与RCE只有47%的序列同源性，但 确实有效地激活了CPT-11。解开这些差异的结构基础是这项提议的核心焦点。 将结合X射线结晶学工具、生物化学和活体研究实现五个具体目标： 1.阐明CPT-11与兔肝内皮细胞的结合，揭示药物活化的机制。 2.确定为什么人CE1不能激活CPT-11，尽管它与RCE有很高的序列相似性。 3.检测人肠道CE的晶体结构，以解释为什么该酶有效地激活CPT-11。 4.从结构和功能上鉴定RCE、hCE1和Hice的突变体以建立分子 CPT-11激活的决定因素。 5.评估药物激活的效果和对表达RCE、hCE1和HCE1突变形式的细胞的敏感性 Hice to CPT-11. 工作人员(S)(组织、身份、状态) 北卡罗来纳大学教堂山分校 北卡罗来纳州教堂山 圣犹大儿童研究医院 田纳西州孟菲斯 KEYPERSONNEL.Seeinstructions.Usecontinuationpagesas需要按如下所示的格式提供所需信息。 从主要调查员开始。按字母顺序列出其他关键人员，姓氏在前。 名称组织角色项目 马修·R·雷丁博，北卡罗来纳大学博士，首席研究员 菲利普·M·波特，圣犹大儿童研究医院博士，联合首席研究员 Somop Bencharit，D.D.S.北卡罗来纳大学研究生助理 金杰·卡纳汉，北卡罗来纳大学学士研究生助理 施罗德·诺布尔，北卡罗来纳大学学士研究生助理 Laurie Betts，博士，北卡罗来纳大学研究技术员 克里斯托弗·莫顿，圣犹大儿童研究医院研究技术员 詹姆斯·P·莫肯，北卡罗来纳大学博士，合作者 披露许可声明。适用于SBIR/STRONNY。请参阅说明。[]是[]否 PHS398(05/01版)第2页FormPage2 在整个应用程序的底部连续编号页码。不要使用3a、3b等后缀。 首席调查员/项目主任(最后、第一、中间)：马修·罗伯特·雷丁博 必须在每张打印页和每张续页的顶部提供首席调查员/项目主任的姓名。 字体密度和大小必须符合PHS 398说明书中提供的限制和规格。 研究补助金 目录 页码 1 主页........................................................................................................................ 2-2 描述，

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,