Trypsin Mutants for Structure-Based Drug Design: Expression, Refolding and Crystallisation

Abstract New techniques in drug discovery are essential for the fast and efficient development of novel innovative drugs to deal with the challenges of the future. Structure determinations of various members of serine proteinases have provided a basis for computerbased drug design within this class of enzymes. In many proteins of interest, however, this course is blocked through a lack of suitable crystals. As a strategy for circumventing such problems, we have investigated the use of surrogate proteins for studying protein ligand interactions. To test the feasibility of this approach, we have chosen bovine trypsin as a scaffold to reconstruct the ligand binding site of factor Xa. The simple modular design of trypsin, its readiness to crystallise and straightforward handling lends itself to such drug design by proxy. The expression, folding, purification, crystallographic and kinetic characterisation of bovine trypsin forms with factor Xa phenotype are presented.

[1]  W. Bode,et al.  Urethanyl-3-Amidinophenylalanine Derivatives as Inhibitors of Factor Xa. X-Ray Crystal Structure of a Trypsin/Inhibitor Complex and Modeling Studies , 2000, Biological chemistry.

[2]  J. Stürzebecher,et al.  Synthetic inhibitors of bovine factor Xa and thrombin comparison of their anticoagulant efficiency. , 1989, Thrombosis research.

[3]  K Fujikawa,et al.  The coagulation cascade: initiation, maintenance, and regulation. , 1991, Biochemistry.

[4]  A. Spada,et al.  Crystal structures of human factor Xa complexed with potent inhibitors. , 2000, Journal of medicinal chemistry.

[5]  W. Bode,et al.  A player of many parts: the spotlight falls on thrombin's structure. , 1993, Thrombosis research.

[6]  R. Huber,et al.  Structural Mapping of the Active Site Specificity Determinants of Human Tissue-type Plasminogen Activator , 1997, The Journal of Biological Chemistry.

[7]  H. Nar,et al.  Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors. , 2001, Structure.

[8]  G Klebe,et al.  Docking ligands onto binding site representations derived from proteins built by homology modelling. , 2001, Journal of molecular biology.

[9]  J. Stürzebecher,et al.  Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine. , 1997, Journal of medicinal chemistry.

[10]  Robert Huber,et al.  X-ray Structure of Active Site-inhibited Clotting Factor Xa , 1996, The Journal of Biological Chemistry.

[11]  M. Morrissey,et al.  (Z,Z)-2,7-Bis(4-amidinobenzylidene)cycloheptan-1-one: identification of a highly active inhibitor of blood coagulation factor Xa. , 1998, Journal of medicinal chemistry.

[12]  R. Huber,et al.  Structure of human des(1-45) factor Xa at 2.2 A resolution. , 1993, Journal of molecular biology.

[13]  T. Lin,et al.  Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases. , 1996, Biochemistry.

[14]  J. Hirsh,et al.  New antithrombotic agents , 1999, The Lancet.

[15]  A. Light,et al.  Refolding of serine proteinases , 1986, Journal of cellular biochemistry.

[16]  R. Huber,et al.  Structural and functional analyses of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase. , 1998, Journal of medicinal chemistry.

[17]  Horton Hr,et al.  Kinetics of papain-catalyzed hydrolysis of -N-benzoyl-L-arginine-p-nitroanilide. , 1973 .

[18]  Jean M. Severin,et al.  Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite. , 2000, Structure.

[19]  U Heinemann,et al.  High-throughput three-dimensional protein structure determination. , 2001, Current opinion in biotechnology.

[20]  G. Klebe,et al.  pH‐Dependent Binding Modes Observed in Trypsin Crystals: Lessons for Structure‐Based Drug Design , 2002 .

[21]  R. Huber,et al.  Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin , 1995, FEBS letters.

[22]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[23]  R. Huber,et al.  (4-aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[24]  N. Gordon,et al.  High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy. , 2001, Biochemistry.

[25]  A. Sali,et al.  Protein structure modeling for structural genomics , 2000, Nature Structural Biology.

[26]  J. Stürzebecher,et al.  Cyclic amides of N alpha-arylsulfonylaminoacylated 4-amidinophenylalanine--tight binding inhibitors of thrombin. , 1983, Thrombosis research.

[27]  P. Lam,et al.  Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa. , 2001, Journal of medicinal chemistry.

[28]  M. Whitlow,et al.  Discovery of N-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3, 5-difluoro-6-[3-(4, 5-dihydro-1-methyl-1H-imidazol-2-yl)phenoxy]pyridin-4-yl]-N-methylgl y cine (ZK-807834): a potent, selective, and orally active inhibitor of the blood coagulation enzyme factor Xa. , 1998, Journal of medicinal chemistry.

[29]  A. Light,et al.  Refolding of the mixed disulfide of bovine trypsinogen and glutathione. , 1979, The Journal of biological chemistry.

[30]  J. Stürzebecher,et al.  Structure-activity relationships of inhibitors derived from 3-amidinophenylalanine. , 1995, Journal of enzyme inhibition.

[31]  Wim G. J. Hol,et al.  Structural genomics for science and society , 2000, Nature Structural Biology.

[32]  U Heinemann,et al.  An integrated approach to structural genomics. , 2000, Progress in biophysics and molecular biology.

[33]  Erik Verner,et al.  Exploiting subsite S1 of trypsin-like serine proteases for selectivity: potent and selective inhibitors of urokinase-type plasminogen activator. , 2001, Journal of medicinal chemistry.

[34]  V. V. Mozhaev,et al.  The influence of effectors on the refolding (reactivation) of immobilized trypsin. , 2005, European journal of biochemistry.