Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library

被引:65
作者
McBride, JD
Freeman, N
Domingo, GJ
Leatherbarrow, RJ
机构
[1] UNIV LONDON IMPERIAL COLL SCI TECHNOL & MED,DEPT CHEM,LONDON SW7 2AY,ENGLAND
[2] GLAXO RES & DEV LTD,DEPT BIOMOLEC STRUCT,STEVENAGE SG1 2NY,HERTS,ENGLAND
基金
英国生物技术与生命科学研究理事会;
关键词
combinatorial chemistry; proteinase inhibitor; Bowman-Birk inhibitor; canonical loop; protein engineering;
D O I
10.1006/jmbi.1996.0360
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A synthetic library of cyclic peptides was constructed utilizing the anti-tryptic loop region of the Bowman-Birk inhibitor, D4 from Macrotyloma axillare, as a template. The loop region of this proteinase inhibitor was reproduced by an 11 residue sequence, conformationally constrained by the presence of a disulfide bridge, to act as a mimetic of the functional reactive site region of this protein. This sequence, plus a pentaglycine spacer arm, was used to create a ''one bead, one peptide'' combinatorial library after on-resin deprotection and cyclization. Randomization at three positions considered to be important for proteinase specificity (P-2, P-1 and P-2') with the genetically coded amino acids (minus cysteine) plus norleucine generated 8000 permutations. Screening this library with biotinylated alpha-chymotrypsin under appropriate conditions revealed a small number (<0.05%) of beads that selectively bound the labeled proteinase. The sequences present on these active beads were determined, and found to have a well-defined consensus. Analysis of chymotrypsin inhibition in solution using re-synthesized peptides reveals that the sequences identified are potent inhibitors with K-i values in the nanomolar range. These results show that directed randomization of the canonical loop is a powerful way of generating proteinase inhibitors with targeted specificities. Incorporation of selective random changes within a defined structural framework is found to be an effective means of generating variation in large synthetic systems. The functional basis for inhibition by the identified sequences is discussed. (C) 1996 Academic Press Limited
引用
收藏
页码:819 / 827
页数:9
相关论文
共 57 条
[1]  
Atherton E., 1989, SOLID PHASE PEPTIDE
[2]   DETERMINATION OF CONCENTRATION OF HYDROLYTIC ENZYME SOLUTIONS - ALPHA-CHYMOTRYPSIN TRYPSIN PAPAIN ELASTASE SUBTILISIN AND ACETYLCHOLINESTERASE [J].
BENDER, ML ;
BEGUECAN.ML ;
BLAKELEY, RL ;
BRUBACHER, LJ ;
FEDER, J ;
GUNTER, CR ;
KEZDY, FJ ;
KILLHEFFER, JV ;
MARSHALL, TH ;
MILLER, CG ;
ROESKE, RW ;
STOOPS, JK .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1966, 88 (24) :5890-+
[3]   A CONFORMATIONALLY HOMOGENEOUS COMBINATORIAL PEPTIDE LIBRARY [J].
BIANCHI, E ;
FOLGORI, A ;
WALLACE, A ;
NICOTRA, M ;
ACALI, S ;
PHALIPON, A ;
BARBATO, G ;
BAZZO, R ;
CORTESE, R ;
FELICI, F ;
PESSI, A .
JOURNAL OF MOLECULAR BIOLOGY, 1995, 247 (02) :154-160
[4]   NATURAL PROTEIN PROTEINASE-INHIBITORS AND THEIR INTERACTION WITH PROTEINASES [J].
BODE, W ;
HUBER, R .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1992, 204 (02) :433-451
[5]   ENCODED COMBINATORIAL CHEMISTRY [J].
BRENNER, S ;
LERNER, RA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (12) :5381-5383
[6]   A MASS-SPECTROMETRIC SOLUTION TO THE ADDRESS PROBLEM OF COMBINATORIAL LIBRARIES [J].
BRUMMEL, CL ;
LEE, INW ;
ZHOU, Y ;
BENKOVIC, SJ ;
WINOGRAD, N .
SCIENCE, 1994, 264 (5157) :399-402
[7]   STRUCTURE AND VARIATION OF HUMAN ALPHA-1-ANTITRYPSIN [J].
CARRELL, RW ;
JEPPSSON, JO ;
LAURELL, CB ;
BRENNAN, SO ;
OWEN, MC ;
VAUGHAN, L ;
BOSWELL, DR .
NATURE, 1982, 298 (5872) :329-334
[8]  
CHEN P, 1992, J BIOL CHEM, V267, P1990
[9]   SYNTHESIS IN ESCHERICHIA-COLI OF ALPHA-1-ANTITRYPSIN VARIANTS OF THERAPEUTIC POTENTIAL FOR EMPHYSEMA AND THROMBOSIS [J].
COURTNEY, M ;
JALLAT, S ;
TESSIER, LH ;
BENAVENTE, A ;
CRYSTAL, RG ;
LECOCQ, JP .
NATURE, 1985, 313 (5998) :149-151
[10]   PEPTIDES ON PHAGE - A VAST LIBRARY OF PEPTIDES FOR IDENTIFYING LIGANDS [J].
CWIRLA, SE ;
PETERS, EA ;
BARRETT, RW ;
DOWER, WJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (16) :6378-6382