Improving CPT-11 Efficacy Using Structural Biology

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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,