Departament de Bioquímica i Biologia Molecular Discovery and characterization of small molecular weight metallocarboxypeptidase inhibitors Daniel Fernández Doctoral Thesis 2009 Departament de Bioquímica i Biologia Molecular Discovery and characterization of small molecular weight metallocarboxypeptidase inhibitors Doctoral Thesis presented by Daniel Fernández to fulfill the Ph. D. degree in Biotechnology from the Universitat Autònoma de Barcelona Daniel Fernández Francesc X. Avilés Puigvert Josep Vendrell Roca Thesis co-directors Barcelona, 2009 A mis padres A Silvia Index Preface List of published material Abbreviations Introduction 1 The proteases 1 1.1 Types of proteases 1 1.2 The metallocarboxypeptidases 2 1.3 Inhibitors of CPs from natural sources 4 1.4 CPA as a model metalloprotease 5 2 Biology - carboxypeptidases 7 in health and disease 2.1 CP activities in the gastrointestinal tract 7 2.2 CP roles in acute pancreatitis and cancer 8 2.3 Extra-pancreatic carboxypeptidases 9 2.4 A CPB-like antifibrinolytic 10 3 Biotechnology - from protein 13 hydrolyzates to cancer therapy 4 Organic synthetic inhibitors 15 of caboxypeptidases 4.1 Ground-state inhibitors 16 4.2 Transition-state inhibitors 21 Objectives 23 Results and Discussion 5 Discovery of novel inhibitors 25 5.1 The arginine derivatives (family DF1) 26 5.2 Thioxophosphoranyl oxiranes (DF2) and 28 cyclobutane β-peptides (DF3) 5.3 L-alanyl derivatives (DF4) and 33 organosilicon compounds (DF5) 5.4 Peptoids (DF6) 37 5.5 Oxadiazoles (DF7) 40 6 Bioinformatics 43 6.1 The crystal structure pool 43 6.2 Interactions of the catalytic domain 44 6.3 Nature of the ODA patches 46 6.4 Predictive success of the method 47 6.5 Surface area of the ODA patches 49 6.6 Sequence-structure relationships 50 6.7 Protein-ligand relationships 54 6.8 Regionalization of the ODA patches 55 6.9 Accuracy of the predictions 57 6.10 Potential roles of the ODA patches 60 7 Crystallization and 65 X-ray diffraction studies 7.1 Structure of a new CPB crystal form 66 7.2 Assignment of residues 68 7.3 Comparison to the known tetragonal crystal 70 7.4 The aromatic cluster 72 7.5 The role of Tyr248 and its flanking region 75 7.6 CPA crystallization 80 7.7 The complex of CPA and 81 a mechanism-based inhibitor 7.8 Conformational changes 86 7.9 The zinc environment 87 7.10 The ligand structure 88 Overall discussion 91 Conclusions 97 8 Methods 99 8.1 General materials and media 99 8.2 Protein handling 101 8.3 Kinetic measurements and data treatment 103 8.4 X-ray crystallography – crystallogenesis 105 8.5 X-ray crystallography – data handling 107 8.6 X-ray crystallography – structure solution 110 8.7 Bioinformatics – the ODA method 114 8.8 Bioinformatics – virtual screening 117 8.9 Bioinformatics – resources 120 Bibliography 121 Acknowledgements 137 Publications 139 Preface When I started working at our group at the UAB, I was not aware of the impact this endeavor would be operating upon me. Peptidases are proteins that have to be carefully faced up with because of their natural job of degrading other proteins. Therefore, finding a means to modulate their activity seemed both a bewildering and challenging task. And also a top-motivating one... Five years later this feeling persists. Although some advances have been made, there seems to be no efficient means to modulate carboxypeptidase action by designed small molecular weight drugs, and therefore to shift the balance from disease to health. However, some things need time... Peptidases let me introduce into molecular and structural biology, and bioinformatics. More importantly, they allowed me to meet a fascinating new world, paradoxically the “Old world”, in a journey to a new Continent, country and nation. To a lovely city and a stimulating environment. As with peptidases, I’ve made countless people discoveries. The latter are of course not measurable by any scientific method, but constitute an important part of life. However, as this document is not intended to be a biographical account, I’ll focus on Science. Notice about the format of this Thesis At the time of writing, parts of the work done during my Ph. D. have either been published, “lost” in the review process, or remain inedit. Thus, the experimental results are organized with a general sense in Chapters 5 to 7, to cover the different approaches employed. These Chapters are not intended as a compendium of publications. Published work is included in a separate section. The same was applied to Methods. Chapter 8 covers in a general way all aspects of the techniques used, while the peculiarities of each of them can be found in the attached papers. I’ve tried to organize all parts in a well-balanced structure. Whenever possible, I’ve summarized all the information available. To begin with, Chapters 1 to 4 review background information. This is intended as a general Introduction. A brief introductory comment is included as a reference in each section of Chapters 5 to 7. Information on each specific topic can be found in the attached published material. The Discussion and Conclusions are also general. List of published material 1 Articles described in this Thesis Fernández DD, Testero S, Vendrell J, Avilés FX, Mobashery S. (2010). Chem Biol Drug Des, in press* *Our work was awarded the cover of volume 74 to appear in Feb/2010 Fernández DD, Boix E, Pallarès I, Avilés FX, Vendrell J. (2009). Biopolymers, in press Fernández DD, Avilés FX, Vendrell J. (2009). Chem Biol Drug Des 73:75-82 Fernández DD, Torres E, Avilés FX, Ortuño RM, Vendrell J. (2009). Bioorg Med Chem 17:3824-3828 Fernández DD, Avilés FX, Vendrell J. (2009). Eur J Med Chem 44:3266-3271 Fernández DD, Illa O, Avilés FX, Branchadell V, Vendrell J, Ortuño RM. (2008). Bioorg Med Chem 14:4823-4828 Fernández DD, Vendrell J, Avilés FX, Fernández-Recio J. (2007). Proteins: Structure, Function, and Bioinformatics 60:131-144 2 Inedit works Fernández DD, Montenegro M, Avilés FX, Vendrell J, García-Viloca M. (2009). In preparation Fernández DD, Avilés FX, Vendrell J, Pérez-Trujillo M, Parella T. (2009). Submitted to J Inorg Biochem 3 Crystal structures validated and deposited with major databases Cambridge Crystallographic Data Centre (CCDC) 1. deposit number: 671796 Protein Data Bank (PDB) 1. ID: 2v77 2. ID: 3glj 3. ID: 3hlp 4. ID: 3huv 5. ID: 3i1u 4 Other articles Pallarès I, Fernández DD, Comellas-Bigler M, Fernández-Recio J, Ventura S, Avilés FX, Bode W, Vendrell J. (2008). Acta Crystallogr D Biol Crystallogr 64::784-791 Niemirowicz G, Fernández DD, Solá M, Cazzulo JJ, Avilés FX, Gomis-Rüth FX. (2008). Mol Microbiol 70:853-866 Bayés A, Fernández DD, Solá M, Marrero A, García-Piqué S, Avilés FX, Vendrell J, Gomis-Rüth FX. (2007). Biochemistry 46:6921-6930 Abbreviations ACE, angiotensin-converting enzyme ADEPT, antibody-directed enzyme prodrug therapy Ala, alanine Arg, arginine Asn, asparagine Asp, aspartic acid bCPA, bovine carboxypeptidase A (= CPA) bCPA1, bovine carboxypeptidase A1 (= bCPA) bPCPA1, bovine procarboxypeptidase A1 BzlSA, 2-benzylsuccinic acid CBP, cyclobutane β-peptide CCP, cytosolic carboxypeptidase CP, carboxypeptidase CPA, carboxypeptidase A CPB, pancreas carboxypeptidase B ( = CPB1) CPD, carboxypeptidase D CPE, carboxypeptidase E CPM, carboxypeptidase M CPN, carboxypeptidase N CPU, human plasma CPB ( = TAFI/CPR/CPB2) CPZ, carboxypeptidase Z CTGC, chymotrypsinogen C Cys, cysteine Da, dalton dCPD, domain II of duck carboxypeptidase D ( = CPD) DMSO, dimethylsulfoxide ECI, endogenous carboxypeptidase inhibitor (= latexin) GEMSA, 2-(2-guanidinoethylthio)succinic acid Gln, glutamine Glu, glutamic acid Gly, glycine HaCPA, CPA from cotton bollworm (Helicoverpa armigera) hCPA1, human carboxypeptidase A1 hCPA2, human carboxypeptidase A2 hCPA4, human carboxypeptidase A4 hCPB, human pancreas CPB ( = CPB/CPB1) hCPU, human plasma CPB (= TAFI/CPR/CPB2) hCPM, human carboxypeptidase M ( = CPM) His, histidine hPCPA1, human procarboxypeptidase A1 HzCPB, CPB from corn earworm (Helicoverpa zea) Ile, isoleucine iPr, iso-propyl LCI, leech carboxypeptidase inhibitor Leu, leucine Lys, lysine MD, molecular dynamics MES, 2-(N-morpholino)ethanesulfonic acid Met, metionine MMP, matrix metalloproteinase MR, molecular replacement MW, molecular weight ODA, Optimal Docking Area PCI, potato carboxypeptidase inhibitor PCP, procarboxypeptidase pCPB, porcine pancreas carboxypeptidase B PDB, Protein Data Bank Phe, phenylalanine PPE, proproteinase E Pro, proline SDS, sodium dodecyl sulfate Ser, serine Tris, 2-amino-2-(hydroxymethyl)propane-1,3-diol TAFI, thrombin-activable fibrinolysis inhibitor (=CPU/CPR/plasma CPB/CPB2) TCI, tick carboxypeptidase inhibitor TcMCP-1, metallocarboxypeptidase 1 from Trypanosoma cruzi TcMCP-2, metallocarboxypeptidase 2 from Trypanosoma cruzi Thr, threonine TvCPT, carboxypeptidase T fom Thermoactynomices vulgaris Trp, tryptophan Tyr, tyrosine Val, valine Z , carbobenzoxy Introduction 1 The proteases Proteases are proteins that catalyze the hydrolysis of the C-N bond, or peptide bond, of protein and peptide substrates and are therefore called peptidases as well. Proteases were recognized early in the history of biochemistry. In the nineteenth century, one primary focus of research was on digestive proteases like pepsin and trypsin. Another important digestive protease named carboxypeptidase was isolated and characterized in the last years of the second decade of the twentieth century. Digestive proteases are located in the gastro-intestinal tract and are mainly responsible for the digestion of food proteins (Erickson & Kim, 1990). Peptidases located extracellularly in the blood or other extracellular compartments of the body often play regulatory roles in carefully controlled processes, such as blood clotting, fibrinolysis, complement activation, fertilization, and hormone production. Intracellularly located proteases exhibit a wide range of roles. They are found in the Endoplasmic Reticulum, the Golgi apparatus, or the lysosomes, among others. Their functions include the processing of peptide hormones, cytokines, and ubiquitin mediated proteolysis, among others (Fricker, 1988; Skidgel & Erdös, 1998; Fujiwara et al., 1999). 1.1 Types of proteases Several protease classification schemes have emerged.