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Structural analysis of the cancer promoting matrix metalloproteinase-9 in complexes with novel pharmacological inhibitors [Elektronische Ressource] : molecular structure of the hemopexin domain of MT1-MMP, including novel dimerization models / Anna Maria Tochowicz

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Published 01 January 2006
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Structural analysis of the cancer promoting
Matrix Metalloproteinase–9 in complexes with novel
pharmacological inhibitors



Molecular structure of the hemopexin domain of
MT1-MMP, including novel dimerization models




Anna Maria Tochowicz
Max-Planck-Institute für Biochemie
Abteilung Strukturforschung
D-82152 Martinsried, München
Max-Planck-Institute für Biochemie
Abteilung Strukturforschung


Structural analysis of the cancer promoting
Matrix Metalloproteinase–9 in complexes with novel
pharmacological inhibitors

Molecular structure of the hemopexin domain of
MT1-MMP, including novel dimerization models

Anna Maria Tochowicz

Vollständiger Abdruck der von der Fakultät für Chemie
der Technischen Universität München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigen Dissertation.


Vorsitzender: Univ. - Prof. Dr. Hans Jürgen Neusser

Prüfer der Dissertation: 1. apl. Prof. Dr. Dr. h.c. Robert Huber, i.R.
2. Univ. - Prof. Dr. Johannes Buchner



Die Dissertation wurde am 27.09.2006 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 10.11.2006 angenommen.
Acknowledgements


Acknowledgments

The present work was performed from March 2003 to September 2006 in the Abteilung
für Strukturforschung at the Max-Planck-Institut für Biochemie in Martinsried, under the
supervision of Prof. Dr. Robert Huber and Prof. Dr. Wolfram Bode.
I am deeply grateful to Prof. Dr. Robert Huber for giving me the opportunity to work in
his group, for his generous support and for excellent conditions in the department. I am
especially thankful to Prof. Dr. Wolfram Bode for accepting me in his group, the
continuous support and guidance during all these years, and the critical review of my
work.
Many thanks to Prof. Luis Moroder and Dr. Alessandra Barazza for providing us with the
bivalent inhibitors and for a successful collaboration. To Dr. Mateo Zanda for funding
and encouraging the “MMP-9 project”, many thanks.
I am especially grateful to Dr. Peter Göttig for introducing me to the crystallography
field, for providing criticism, valuable advice, suggestions, and finally for careful reading
of my manuscript. I am also very thankful to Dr. Klaus Maskos for introducing me to the
metalloproteinase field and enzyme kinetics, for the critical discussions of some results
and for encouragement any time I needed it.
A special thank to my best friend here in Munich, Mekdes Debela for this wonderful and
good time we have had together, and for fruitful discussions not only about science.
During this time I had a chance to work with and enjoy many great lab- and office (never
to be forgotten “Kinderzimmer”) mates and I would like to thank all of them for having
been good colleagues, in the present and in the past, for a long and a short time: Dr.
Martin Augustin, Dr. Irena Bonin, Dr. Michael Engel, Dr. Rainer Friedrich, Dr. Pablo
Fuentes-Prior, Dr. Michael Groll, Dr. Stefan Henrich, Dr. Daniela Jozic, Dr. Dorota
Ksiazek, Cora Keil, Sandra Lepthien, Dr. Martin Locher, Dr. Sofia Maciera, Vesna Mors,
Dr. Kerstin Rohr, Elizabeth Ruge, Dr. Peter Sondermann, Rasso Willkomm, Dr.
Magdalena Wisniewska. A special thank to Charlotte Ungewickell for her german
teaching lessons. Acknowledgements


I would like to acknowledge the secretaries Renate Rüller, Monika Schneider and
Monika Bumann for their help during these years.
A deep thank to those friends that have tried to be always with me, in good and bad
times, with calls or writing or just visiting me, for their long-term, certainly intense
friendship: Klaudia Kosowska-Shick, Izabella Noll, Joanna Nowakowska, Mariola
Majewska and Magdalena Broniatowska. I really hope that no matter where we are, we
always will stay in touch.
I am especially indebted to my lovely sister, for our outstanding friendship, for
motivating me, and for being with me always when I need it. Thanks, my dear! Finally, a
special thank to my Mother for her patience, encouragement and support.
Table of Contents

Acknowledgements

Table of Contents 1

Chapter 1 Summary 4

Zusammenfassung 6

Chapter 2 Introduction 8

2.1 Proteases 8
2.1.1 Types of proteases 9

2.1.1.1 Metalloproteinases 10 .1 Structural features of Matrix metalloproteinases (MMPs) 13

2.2 Gelatinase B (MMP-9) 15
2.2.1 Physiological and pathological implications of gelatinases 15
2.2.2 Gelatinase B in cancer 16
2.2.3 Principal features of the human MMP-9 17
2.2.4 Substrate specificity of gelatinase 19
2.2.5 Regulation of gelatinase B activity 21
2.2.6 Activation of pro-MMP-9 in vivo by Trypsin, APMA and Stromelysin 21
2.2.7 Inhibition of MMP-9 24
2.2.7.1 Tissue Inhibitors of Metalloproteinases, TIMPs 24
2.2.7.2 Synthetic MMPs Inhibitors (MMPIs) 28 .1 First-generation MMPIs 28
2.2.7.2.2 Next-generation MMPIs 29 .3 Bivalent Inhibitor 31

2.3 Membrane-type 1 matrix metalloproteinase (MT1-MMP) 33
2.3.1 General properties of MT1-MMP 33
2.3.2 MT1-MMP as an important pericellular modifier 34
2.3.2.1 Cell surface localization of MT1-MMP 34
2.3.2.2 Functions of MT1-MMP 35
2.3.3 Regulation of MT1-MMP; controlling cell function 37
2.3.3.1 Inhibition of MT1-MMP activity 37
2.3.3.2 Proteolytic processing 37
2.3.3.3 Regulation of MMP-2 activation 38

Chapter 3 Experimental Procedures 41

3.1 Molecular biology methods 41
3.1.1 Cloning 41
3.1.1.1 Mini-catalytic domain of MMP-9 (Mini-MMP-9) 41
3.1.1.2 Pro-catalytic MMP-9 (ProMMP-9; ProMMP-9 ΔcollV Δpex) 42
3.1.2 Plasmid Preparation and restriction analysis 42
1 Table of Contents

3.1.3 Agarose Gel Electrophoresis 42
3.1.4 E. coli Transformation by Electroporation 43
3.1.5 Cell cultures; plate, liquid and glycerol cultures 43
3.2 Protein chemical and biochemical methods 44

3.2.1 Mini-MMP-9 44
3.2.1.1 Expression and refolding 44
3.2.1.2 Purification and Protein concentration 44
3.2.1.3 Determination of the protein concentration 45
3.2.1.4 Electrophoresis of proteins on SDS-polyacrylamide gels (SDS) 45
3.2.1.5 Protein Transfer and Western Blots 47
3.2.1.6 Protein analytical work 48 .1 N - terminal amino acid sequencing 48
3.2.1.6.2 Mass spectroscopy 48 .3 Circular dichroism 49
3.2.1.7 Kinetic measurement .1 Theoretical aspects of protease-inhibitor interactions 49
3.2.1.7.2 Activity assay of Mini-MMP-9 in vitro 50

3.2.2 ProMMP-9 51
3.2.2.1 Expression 51
3.2.2.2 Purification and refolding 51
3.2.2.3 Functional characterization 52 .1 Kinetic measurement 53
3.2.2.3.1.1 Trypsin and APMA activation assay 53 .1.2 Inhibition assays 53

3.2.3 MT1-MMP Hpex (hemopexin-like) domain 54
3.2.3.1 Analysis of the oligomeric state in solution 54 .1 Gel filtration chromatography 54
3.2.3.1.2 Molecular weight determination by analytical ultracentrifuge analysis;
Sedimentation Velocity and Sedimentation Equilibrium 55

3.3 Protein crystallography methods 57

3.3.1 Mini-MMP-9 57
3.3.1.1 Crystallization 57
3.3.1.2 Co-crystallization of complexes of Mini-MMP-9 with different
inhibitors 58
3.3.1.3 Data collection and structure determination 59
3.3.2 Molecular replacement; Introduction 59
3.3.2.1 The Translation Function 60
3.3.2.2 The Rotation Function 62

3.3.3 MT1-MMP PEX domain 64
3.3.3.1 Crystallization 64
3.3.3.2 Data collection and structure determination 65


2 Table of Contents

Chapter 4 Results 66

4.1 Wild type mini-MMP-9 and the mutant E402Q 66
4.1.1 Protein purification and characterization 66
4.1.1.1 Kinetic measurement 68
4.1.2 Protein crystallization and data collection 69
4.1.3 Phasing and Refinement 71
4.1.4 Description of the structures; Mini-catalytic domain with
different inhibitors 73
4.1.4.1 The principal features of the MMP-9 catalytic domain 73
4.1.4.2 MMP-9-barbiturate inhibitor interaction (RO206-0222) 75
4.1.4.3 MMP-9-phosphinate inhibitor interaction (AM409) 78
4.1.4.4 MMP-9-carboxylate inhibitor interaction (An1) 80
4.1.4.5 MMP-9-hydroxamate inhibitor interaction (MS560) 82
4.1.4.6 MMP-9-carboxylate inhibitor interaction (MJ24) 84

4.2 Pro-catalytic MMP-9 (ProMMP-9 ΔcollV Δpex) 89
4.2.1 Protein purification and characterization 89
4.2.2 Activation by Trypsin, APMA and by Stromelysin-1 89
4.2.3 Inhibition of ProMMP-9 by bivalent inhibitors 91
4.2.3.1 Kinetic measurements 93

4.3 MT1-MMP Hpex domain 97
4.3.1 Protein purification and characterization 97
4.3.2 Protein crystallization and data collection 100
4.3.3 Structure determination 101
4.3.4 Description of the structure 102
4.3.4.1 New ideas for dimerization of MT1-MMP Hpex domain 103

Charter 5 Discussion 106

5.1 Slow binding inhibitors of gelatinases 107
5.2 The barbiturate ring as a chelator of the catalytic zinc 109
5.3 The phosphinic peptide compounds as highly potent inhibitors 111
5.4 The function and influence of halogens substituted to the inhibitors 113
5.5 Comparison and contribution of the flexible Arg424 side chain
to the selectivity 116
5.6 MT1-MMP Hpex domain novel dimerization models 119

Chapter 6 Literature 123

Charter 7 Appendix 142

7.1 Abbreviations 142
7.2 Index of Figures 144
7.3 Index of Tables 148
3 Summary

Chapter 1 Summary

Cancer is a life-threatening disorder and one of the major prevailing health problems.
The Matrix Metalloproteinase-9, also called gelatinase B, is a critical component of the
angiogenic switch driving metastasis in various cancers. Additionally it promotes
osteoarthritis, atherosclerosis or heart failure. Under healthy conditions, the MMP-9
proteolytic activity is strictly regulated by the endogenous tissue inhibitors of matrix
metalloproteinases (TIMPs), while disruption of this balance leads to a detrimental
excess of active MMP-9. Specific inhibition of MMP-9 may prevent tumor growth
therefore, it is essential to design potent inhibitors in order to compensate for the loss at
physiological regulation. Even though many inhibitors have been studied, none was
sufficiently specific and efficient for any individual MMP. In an attempt to clarify
important determinants for the specific inhibition of MMP-9, the inactive E402Q mutant
of the truncated catalytic domain of human MMP-9 was prepared, and the X-ray crystal
structures of this recombinant miniMMP-9 were determined with a number of catalytic
zinc-directed synthetic inhibitors of different binding type. The complex structures of
MMP-9 with relatively selective, tight binding inhibitors, namely a pyrimidine-2,4,6-
trione (RO206-0222), a phosphinic acid (AM409), a carboxylate (An1), a trifluoromethyl
hydroxamic acid inhibitor (MS560), and a difluoro carboxylate inhibitor (MJ24) were
solved. The crystal structures revealed that these five inhibitors bind in a similar manner,
compromising between optimal coordination of the catalytic zinc, favorable hydrogen
bond formation in the active-site cleft, and accommodation of their large P1’ groups in
the overall rigid S1’ cavity. Furthermore, three MMP inhibitors were designed which are
capable of differentiating the gelatinases MMP-2 and -9 from the other members of the
MMPs family, by applying the concept of bivalent inhibitors: Al134 (Peg ), Al134 4
(Peg ), and Al134 (Peg ). The latter one proved that the bivalent inhibition concept for 6 8
MMP-9 is working in principle, but requires additional fine tuning of the compound.
Dimerization is an important mechanism to regulate activity of MMPs, in particular for
the cancer related MMP-2. MT1-MMP Hpex fixes, through a bound TIMP-2, a
proMMP-2 molecule presenting a scissile peptide bond toward a second non-inhibited
MT1-MMP molecule, which facilitates proMMP-2 activation. For a structural analysis of
4 Summary

the MT1-MMP dimerization the recombinant MT1-MMP hemopexin domain was
crystallized. The crystals contain a monomer with two different, symmetrical and
asymmetrical binding sites. The symmetrical MT1-MMP-Hpex dimer interface is mixed
polar and hydrophobic, and served as a model for MT1-MMP dimerization. In contrast to
the corresponding asymmetrical MT1-MMP dimer, the symmetrical complex is in
accordance with the known dimerization of the transmembrane segments including the
cytoplasmic tail. Furthermore, the complex formation of Hpex MT1-MMP and one
TIMP-2 molecule via its C-terminal part is also possible.

5 Zusammenfassung


Zusammenfassung

Lebensbedrohliche Krebserkrankungen sind eines der größten medizinischen Probleme
unserer Zeit. Matrixmetalloproteinase 9 (MMP-9), oder Gelatinase B, ist eine der
entscheidenden Komponenten bei der Gefäßneubildung, welche die Metastasierung
diverser Krebsarten vorantreibt, sowie Osteoarthritis, Atherosklerose und Herzversagen
fördert. Im gesunden Zustand wird die proteolytische Aktivität von MMP-9 durch die
endogenen Gewebsinhibitoren der Matrixmetalloproteinasen (TIMP) streng reguliert,
während die Störung dieses Gleichgewichts zu einem schädlichen Überschuss aktiver
MMP-9 führt. Da die gezielte Inhibition von MMP-9 das Tumorwachstum verhindern
könnte, ist es erforderlich, wirksame Inhibitoren herzustellen, die den Verlust der
physiologischen Regulation kompensieren. Obwohl eine Vielzahl von Inhibitoren
untersucht wurde, besaß bisher keiner genügend spezifische Wirksamkeit für die
einzelnen MMP. Zur Bestimmung der molekularen Grundlagen der MMP-9-Inhibition
wurden die inaktive E402Q-Mutante der verkürzten katalytischen Domäne von humaner
MMP-9 gereinigt und die Röntgenkristallstrukturen dieser rekombinanten Mini-MMP-9
mit synthetischen Inhibitoren bestimmt, die das katalytische Zink in unterschiedlicher
Weise binden. Die MMP-9-Komplexe mit folgenden selektiven, stark bindenden
Inhibitoren wurden strukturell aufgeklärt: ein Pyrimidin-2,4,6-trion (RO206-222), eine
Phosphinsäure (AM409), ein Carboxylat (An1), eine Trifluoromethylhydroxamsäure
(MS560) und ein Difluorocarboxylat (MJ24). Die Kristallstrukturen zeigten, dass die fünf
Inhibitoren ähnlich binden, wobei ein Ausgleich zwischen optimaler Koordination des
katalytischen Zink, günstigen Wasserstoffbrücken in der Spalte des aktiven Zentrums und
der Aufnahme großer P1´-Reste in der insgesamt rigiden S1´-Tasche gefunden wird. Des
Weiteren wurden drei MMP-Inhibitoren synthetisiert, die aufgrund bivalenter
Funktionalität die Gelatinasen MMP-2 und -9 von den anderen MMPs unterscheiden
sollten: Al134(Peg ), Al134(Peg ) und Al134(Peg ). Letzterer bestätigte prinzipiell die 4 6 8
Anwendbarkeit des Konzepts der bivalenten Inhibition für MMP-9, wenngleich eine
weitere Optimierung der Verbindung notwendig ist.
Die Dimerisierung ist ein wichtiger Mechanismus bei der Steuerung der MMP-Aktivität,
insbesondere für die an Krebs beteiligte MMP-2. Die MT1-MMP (MMP-14) bindet im
Komplex mit dem Inhibitor TIMP-2 ein Pro-MMP-2-Molekül, das einem zweiten, nicht-
6