A New Generic Method for the Production of Protein-Based Inhibitors of Proteins Involved in Cancer Metastasis

Abstract

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this' collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 121B Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project 10704-0188), Washington, DC 20503 AGENCY USE ONLY (Leave blank)2. REPORT DATE August 1999 REPORT TYPE AND DATES COVEREDFinal (lAug 94-31 Jul 99) TITLE AND SUBTITLE A New Generic Method for the Production of Protein-Based Inhibitors of Proteins Involved in Cancer Metastasis AUTHOR(S)Marshall H. Edgell, Ph.D. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)University of North Carolina, Chapel Hill Chapel Hill, North Carolina 27599-4100 Our objective was to develop a method to make protein-based inhibitors against protein targets and as the test case to a proteinase involved in metastasis, stromelysin. The approach used phage display and a display framework which was expected to bind preferentially to the active site pocket of target proteins. While we were able to find peptides in phage display libraries that bound to active stromelysin or to cadmium inactivated stromelysin and could find binders to a test target, papain, in a constrained loop library we were unable to get the fully constrained scaffold protein to bind to its cognate target, subtilisin in the phage display system. Hence the overall objective was not obtained. As part of our efforts to characterize the binding epitope display framework, eglin c, we did develop a new method for using mutagenesis to study protein structure which we call patterned library analysis. We carried out a proof-of-principle experiment using the new method in which we were able to reproduce the values for cc-helix propensity indicating that the method does indeed work. This method should have broad utility as a method for the assessment of hypotheses concerning the determinants of protein structure. Where copyrighted material is quoted, permission has been obtained to use such material. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)U SUBJECT TERMSWhere material from documents designated for limited distribution is quoted, permission has been obtained to use the material.Citations of commercial organizations and trade names in this report do not constitute an official Department of Army endorsement or approval of the products or services of these organizations.mA&T In conducting research using animals, the investigator(s) adhered to the "Guide for the Care and Use of Laboratory Animals," prepared by the Committee on Care and use of Laboratory Animals of the Institute of Laboratory Resources, national Research Council ...