CompleteThesis_ChristianeRiedinger.pdf

Tumour Suppressors and Oncogenes:
Structure, Function and Drug Design

Christiane Riedinger

Laboratory of Molecular Biophysics and
Christ Church College, University of Oxford

Michaelmas Term 2007/2008

A thesis submitted in partial fulfilment of the requirements
for the degree of Doctor of Philosophy at the University of Oxford.

Abstract
The work towards this thesis comprises two independent projects that are linked through the
process of carcinogenesis. In cancer cells, it is often the case that tumour suppressors,
proteins that prevent uncontrolled cell division are down-regulated, while proto-oncogenes
promoting cell growth are up-regulated. By applying structural biology in combination with
biophysical methods, we have tried to provide the basis for further development of anti-
cancer drugs inhibiting the interaction between the tumour suppressor p53 and the oncogene
MDM2. Furthermore, this work has engaged in structural and functional characterisation of
the potential tumour suppressor protein Doc1.

Inhibiting the MDM2-p53 Interaction

The transcription factor p53 is the cell’s major tumour suppressor, often described as the
“guardian of the genome”. Due to its crucial role in preventing uncontrolled cell
proliferation, the disruption of p53-function is one of the major steps during carcinogenesis.
In many cancers, the function of p53 is disrupted through elevated levels of its main
antagonist, the ubiquitin ligase MDM2. Activation of wild type p53 in these cancer types has
therefore become a focus of cancer drug discovery. The MDM2-p53 interaction has been
localised to a relatively small hydrophobic pocket within the MDM2 N-terminal domain, to
which p53 makes contact through only three conserved amino acids. This renders the
MDM2-p53 interaction one of the few protein-protein interactions amenable to structure-
based drug design of small molecule inhibitors. A relatively novel class of MDM2-
antagonists are the Isoindolinones, developed by our collaborators at the Northern Institute
for Cancer Research at the University of Newcastle. In this study, we have attempted to
elucidate the molecular details of Isoindolinone binding to MDM2, in order to provide a
structural basis for further rational inhibitor-design, with the ultimate aim of developing
more potent candidates for clinical trials.
This goal was pursued applying two major methods: Crystallography and NMR
Spectroscopy. Since MDM2 is a challenging target for protein crystallisation, we have
attempted to alter the surface-properties of MDM2 through protein-engineering in order to
improve its crystallisability. While standard NMR methods of structure determination cannot
be employed in this system, we have obtained a molecular model of Isoindolinone binding to
MDM2 through detailed analysis of inhibitor-induced chemical shifts.
While our efforts to obtain a high-resolution X-ray structure of MDM2 complexed to an
Isoindolinone have been unsuccessful, the chemical shift analysis has provided insights into
the binding modes of Isoindolinone inhibitors.

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Structure and Function of Doc1, a Potential Tumour Suppressor

Deleted in Oral Cancer 1 (Doc1) is a potential tumour suppressor first identified in 1998. Its
name stems from the fact that the protein could not be detected in malignant human oral
keratinocytes, suggesting a potential involvement in cellular growth-control. Not much
information is available about the structure and function of Doc1 to date: The protein has
been shown to suppress DNA replication through association with DNA polymerase α
primase, and was later identified as a CDK2-binding protein. Furthermore, Doc1 is a 12kDa
protein consisting of an unstructured N-terminus and a helical C-terminal domain. The aim
of this study was therefore to further characterise the interaction of Doc1 with CDK2 and to
determine its structure by NMR spectroscopy.
Employing a variety of methods, we have not been able to demonstrate a direct interaction
between Doc1 with CDK2. Furthermore, we have not been able to reproduce one of the
initial experiments establishing a Doc1/CDK2 interaction. We must therefore suspect that a
third component not present in out assays mediates the interaction or that there is indeed no
interaction between Doc1 and CDK2 in vivo. Using a proteomics approach, we have not
been able to identify a protein Doc1 ligand, but present preliminary evidence for a potential
DNA interaction. The structural characterisation of Doc1 has progressed to the initial stages
of NMR structure calculations. Combining all structural evidence available at this point, we
propose a structural model for Doc1.

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To Prof Justus Henatsch ().

iv

Acknowledgements
Abstract
I would like to thank my supervisors Prof Jane Endicott, Dr Jim McDonnell and Prof
Martin Noble for supervision and guidance throughout the four years of my DPhil.

Introduction
Many people deserve to be mentioned here, and I will try my best to point out the
special contribution that each individual person has made.

Methods
For precious NMR advice on many occasions, I would like to thank Dr Christina
Redfield, Dr Jonathan Boyd, Mr Nick Soffe, Dr to-be Charlie Taylor and Dr Ioannis
Vakonakis. Each of them has always been very generous with their help and support.
Another important person to be mentioned here is Dr Emma Boswell. Emma
supervised me during my Diploma Thesis in 2003. Very patiently, she turned a crude
chemist into a potential structural biologist. She taught me the daily business of
structural biology (yes, it’s cloning and protein expression!) and I would have been
much less successful without such great supervision at the beginning of my scientific
career.
I would also like to thank the members of the LMB, in particular Dr Aude Echalier
and Dr Nick Brown for providing me with samples of various cell-cycle proteins.
For literally millions of HeLa cells, I would like to thank Dr Ildem Akerman and Dr
Anette Medhurst, both working in Dr Nick Lakin’s lab at the Oxford Biochemistry
Department.

Results
I would like to thank Prof Douglas McAbee for making Biochemistry sound cool.
In times of motivational break-downs (and happy times), there was the infamous trio
of Harefield’s housemates to pick me up: Dr Tee, Dr Why and Dr Kay. Doctors, I
owe you a great deal… you mean a lot to me! Doctors Loco, Pea and π are also have
to be included in this round of thanks.
Non-scientists (Claudilaudi, Lisa, my family, Katrin et al.) must have had a hard time
coping with endless discussions about mutants, clones, magnets and dead proteins
over the years. I thank you for not rejecting the geek-ness and for even showing real
interest in my work.
Finally: Charlie (most special), Duc, Fiona, Franziska and Loukas deserve the most
gratitude of all. I have the most non-scientific, irrational, non-structural-biology-
related, yet robust and well-justified feelings for you und ich danke Euch von
ganzem Herzen – Ihr seid echt super!

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Abbreviations
AUC Analytical Ultracentrifugation

BSA Bovine serum albumin

CDK Cyclin dependent kinase

C-terminal Carboxy terminal

CyclinA Human CyclinA

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

Doc1 Deleted in Oral Cancer

DSS 2,2-Dimethyl-2-silapentane-5-sulfonate
sodium salt

DTT Dithio-threitol

E.coli Escherichia Coli

EDTA Ethylene diamine tetra-acetic acid

ESI-MS Electrospray Ionization MS

FPLC Fast protein liquid chromatography

GST Glutathione S-transferase

HBS HEPES-buffered saline

HEPES N-[2-Hydroxyethyl]piperazine-N-[2-
ethanesulphonic acid]

HPLC High performance liquid chromatography

HSQC Heteronuclear Single Quantum
Correlation

IPTG Iso-propyl–D-thiogalactoside

ITC Isothermal titration calorimetry

kDa Kilo Dalton

LB Luria Bertani Broth

MALDI-TOF Matrix Assisted Laser ionisation

MAPK Mitogen activated protein kinase

MDM2 Mouse Double Minute 2

MTG Mono-thioglycerol

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MS Mass Spectrometry

NMR Nuclear Magnetic Resonance

NOE Nuclear Overhauser Effect

N-terminal Amino terminal

MS Mass spectrometry

OD Optical density

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PDB Protein data bank

PEG Polyethylene glycol

ppm Parts per million

Prozac Fluoxetine hydrochloride

Rmsd Root mean square deviation

RNA Ribonucleic acid

rpm Revolutions per minute

SAR Structure-Activity-Relationships

SDS-PAGE Sodium dodecyl sulphate-polyacrylamide
gel electrophoresis

SPR Surface Plasmon Resonance

Sw Sweep width

t1dw t1dwell time

TFA Triflouroacetic acid

Tm Melting temperature

Tricine N-tris[hydroxymethyl]methylglycine

Tris Tris[hydroxymethyl]aminomethane

UV Ultraviolet radiation

WT Wild type

X. laevis Xenopus laevis

vii

Foreword – Tumour Suppressors: Structure, Function and Drug Design

Foreword

Tumour Suppressors and Oncogenes:

Structure, Function and Drug Design

viii


Cancer is a highly heterogeneous disease and a major cause of death in the Western World

{U.K., 2005 #131}. Even though some cancers can be cured, for many types there is a great

need for improvement in cancer therapies. This will require the development of new

methods, as well as more detailed study of the cellular causes of cancer.

Cancer cells are advantaged over normal cells in that they carry DNA defects that promote

cell growth. Therefore, it is essential for the understanding of cancer development to

elucidate underlying mechanisms of cell growth and division. Tight regulation of the

activities of proteins that regulate cell proliferation, and the presence of pathways that detect

damage are essential to prevent cells from dividing uncontrollably, and to minimise the risk

of genetic defects being passed on to the daughter cells. The loss of these growth control

mechanisms leads to uncontrolled cell proliferation, which is the main characteristic of

cancer.

In cancer cells, it is often observed that negative regulators of the cell cycle (tumour

suppressors) are inhibited in their function, either through mutations or deregulation of the

associated pathways {Sherr, 2004 #71}. On the other hand, growth-stimulating agents

(oncogenes) are often found to be up-regulated, either through amplification of the wild-type

protein or mutations that result in increased activity {Felsher, 2004 #85}. The aim of this

thesis is to further our understanding of the structural mechanisms underlying tumour-

suppressor/oncogene interactions, with the view of correcting their imbalance through

rational structure-based drug development.

One focus of my D.Phil. is the inhibition of the interaction between the tumour suppressor

p53 and its antagonist MDM2 using small molecules. P53 is the cell’s major tumour

suppressor and is tightly controlled by its main antagonist, the proto-oncogene MDM2.

During carcinogenesis, the disruption of p53’s function is a significant event, which can be

achieved through elevated levels of MDM2. In these cancer types, wild-type p53 fails to

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carry out its function as a tumour suppressor, which is the essential step towards the

development of malignant cells. Thus, inhibiting the MDM2/p53 interaction is a promising

target for cancer drug development in these cases, as releasing wild-type p53 would enable

the tumour suppressor to resume its native function, triggering apoptosis in the affected cells.

We are currently investigating a new class of small molecules designed to disrupt the

MDM2-p53 interaction. By gaining insights into the binding of these inhibitors to MDM2,

we are aiming to provide the basis for improving the specificity and affinity of inhibition,

with the ultimate aim of delivering a candidate for clinical trials.

In a second project, I have been investigating the structure and function of a relatively novel

tumour suppressor protein Deleted-in-Oral-Cancer 1 (Doc1), a potential CDK2 inhibitor.

Cyclin-dependent kinases (CDKs) are the main coordinators of cell-cycle progression and

are therefore often affected during cancer development. The name Deleted-in-Oral-Cancer

results from the failure to detect the Doc1 in human oral cancer cells. The fact that the

protein is absent in the examined tumour tissues indicates that it may play an important role

in the healthy cell and makes it a very interesting target for basic characterisation. The main

aims of this project were therefore to characterise the structural properties of Doc1, as well

as to study its interaction with CDK2. This project, if successful, will lead to a more detailed

understanding of the causes of oral cancer in particular, and can hopefully serve as a starting

point for the development of new treatments against this disease.

As these two projects are independent of each other, this thesis has been divided into two

major parts: The first one describes my studies of the MDM2-p53 interaction, the second

part describes the structural characterisation of Doc1.

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Part I

Inhibiting the MDM2-p53 Interaction

Table of Contents – Part I
Table of Contents.............................................................................................................. 1
Table of Figures ................................................................................................................ 4
Table of Tables ................................................................................................................. 6
Table of Equations............................................................................................................ 7
Section 1................................................................................................................... 8
Introduction to the MDM2-p53 Interaction .................................................................... 8
1.1 The MDM2-p53 Interaction is an Important Target in Drug Design .................. 9
1.2 The p53 Tumour Suppressor ................................................................................ 10
1.2.1 The History of p53 Discovery.......................................................................... 10
1.2.2 The p53 Network .............................................................................................. 11
1.2.2.1 P53 Activation .......................................................................................... 13
1.2.2.2 P53 Function............................................................................................. 13
1.3 The Oncogene MDM2 .......................................................................................... 14
1.4 Regulation of p53 by MDM2 ............................................................................... 15
1.5 Structural Details of MDM2 and p53 .................................................................. 18
1.5.1 Overall Structure of p53 ................................................................................... 18
1.5.2 Overall Structure of MDM2............................................................................. 19
1.5.3 The MDM2 N-terminal Domain ...................................................................... 20
1.6 The MDM2-p53 Interaction ................................................................................. 23
1.7 Inhibitors of the MDM2-p53 Interaction............................................................. 25
1.7.1 Peptidic Inhibitors............................................................................................. 26
1.7.2 Natural Inhibitors .............................................................................................. 29
1.7.3 Small Molecule Inhibitors ................................................................................ 30
1.7.4 Nutlin-Inhibitors ............................................................................................... 32
1.7.5 Benzodiazepinedione Inhibitors....................................................................... 33
1.7.6 Isoindolinone Inhibitors of the MDM2-p53 Interaction................................. 35
Section 2................................................................................................................. 38
Towards a Crystallisable Form of MDM2 .................................................................... 38
2.1 Crystallising MDM2 ............................................................................................. 39
2.2 MDM2 Surface Engineering ................................................................................ 41
2.2.1 Decreasing the Entropic Penalty of Crystallisation ........................................ 41
2.2.2 Generating MDM2 Surface Mutants by Gene Synthesis ............................... 43
2.2.2.1 Cloning by Assembly PCR or Gene Synthesis ...................................... 43

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2.2.2.2 Primer Design ........................................................................................... 44
2.2.2.3 Materials and Methods............................................................................. 45
2.2.2.4 Results....................................................................................................... 46
2.2.3 Generating MDM2 Surface Mutants using Site-directed Mutagenesis......... 49
2.2.4 Generating MDM2 Surface Mutants by Overlap-Extension PCR ................ 50
2.2.4.1 Cloning by Overlap-Extension PCR ....................................................... 50
2.2.4.2 Primer Design ........................................................................................... 51
2.2.4.4 Results....................................................................................................... 52
2.2.5 P53-Binding of MDM2 Surface Mutants........................................................ 55
2.2.5.2 Results....................................................................................................... 56
2.3 Generation of a Truncated MDM2 Point Mutant................................................ 59
2.3.1 MDM225-108 L33E ............................................................................................. 59
2.3.1.2 Results....................................................................................................... 63
2.4 Methylation of MDM2 ......................................................................................... 64
2.4.1 Reductive Methylation of Lysine Residues .................................................... 64
2.4.1.2 Results....................................................................................................... 65
2.5 Conclusions ........................................................................................................... 67
2.5.1 Summary of Constructs and Crystallisation Conditions ................................ 67
2.5.2 Effects of the Ligand on Crystallisation.......................................................... 68
2.5.3 Effects of the Protein on Crystallisation.......................................................... 69
Section 3................................................................................................................. 71
Insights into Isoindolinone Binding Modes from Chemical Shift Perturbations........ 71
3.1 Structural Insights into Isoindolinone-Binding ................................................... 72
3.2 Protein Production................................................................................................. 74
3.2.1 Materials and Methods ..................................................................................... 74
3.2.2 Results ............................................................................................................... 75
3.3 MDM2 Backbone Assignments ........................................................................... 77
3.3.1 Introduction ....................................................................................................... 77
3.3.2 Optimising Protein Solubility .......................................................................... 78
3.3.2.1 Introduction .............................................................................................. 78
3.3.2.3 Results....................................................................................................... 79
3.3.3 Sample Preparation and Data Acquisition ...................................................... 80
3.3.4 Results ............................................................................................................... 80

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3.3.4.1 Backbone Assignments............................................................................ 80
3.3.4.2 Interpretation of p53-induced Chemical Shift Changes ........................ 84
3.4 NMR Titrations with Isoindolinones ................................................................... 91
3.4.1 Introduction ....................................................................................................... 91
3.4.1.1 The 1H/15N Heteronuclear Single Quantum Correlation (HSQC)
Experiment .............................................................................................. 91
3.4.1.2 NMR Titrations ........................................................................................ 93
3.4.2 Materials and Methods ..................................................................................... 96
3.4.2.1 Sample Preparation .................................................................................. 96
3.4.2.2 Data Processing ........................................................................................ 97
3.4.2.3 Resonance Assignments .......................................................................... 98
3.4.2.4 Data Analysis............................................................................................ 98
3.4.3 Results ............................................................................................................. 100
3.4.3.1 General Effects ....................................................................................... 100
3.4.3.2 Broadening Effects................................................................................. 106
3.4.3.3 Comparison to p53 ................................................................................. 108
3.4.3.4 Comparison to Nutlin-3 ......................................................................... 111
3.5 Determining Binding Modes using Chemical Shifts ........................................ 116
3.5.1 Introduction to Chemical Shift....................................................................... 116
3.5.2 Materials and Methods ................................................................................... 118
3.5.2.1 Analysis of Combined Chemical Shift Changes by Magnitude.......... 119
3.5.2.2 Analysis of the Direction of Chemical Shift Changes ......................... 119
3.5.3 Results from Chemical Shift Analysis .......................................................... 121
3.5.3.1 Chemical Structure of Isoindolinone Inhibitors ................................... 121
3.5.3.2 Analysis of Isoindolinone-induced Chemical Shifts by Magnitude ... 123
3.5.3.3 Analysis of the Direction of Chemical Shift Change........................... 126
3.5.3.4 Relating the Difference in Chemical Shift Change to Structural
Differences of Isoindolinone Inhibitors............................................... 130
3.5.4 Manual Docking.............................................................................................. 135
3.5.4.1 Structural Details of Isoindolinone Binding to MDM2 ....................... 135
3.5.4.2 Choice of the Acceptor Structure for Manual Docking ....................... 136
3.5.4.3 Nomenclature of Enantiomers............................................................... 139
3.5.4.4 Results..................................................................................................... 141
3.5.5 Ab initio Computational Docking .................................................................. 144
3.5.5.1 Introduction ............................................................................................ 144
3.5.5.2 Materials and Methods........................................................................... 144
3.5.5.3 Results..................................................................................................... 145
3.5.5.4 Isoindolinone Binding Modes ............................................................... 148
3.6 Summary of Results............................................................................................ 151

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3.7 Conclusions ......................................................................................................... 154
3.8 Future Work......................................................................................................... 158
Section 4............................................................................................................... 161
Final Discussion............................................................................................................ 161
Section 5............................................................................................................... 163
Bibliography.................................................................................................................. 163

Table of Figures
Figure 1-1. Cellular responses induced by p53 as a result of cytotoxic stress.. ...................... 12
Figure 1-2. The MDM2-p53 auto-regulatory feedback loop.................................................... 17
Figure 1-3. Schematic representation of the domain organisation of p53. .............................. 18
Figure 1-4. Schematic representation of the domain organisation of MDM2......................... 19
Figure 1-5. Pseudo-symmetrical arrangement of MDM2’s N-terminal domain, forming a
hydrophobic pocket at their interface................................................................................ 21
Figure 1-6. The NMR structure of MDM2’s N-terminal domain (1Z1M). ............................. 22
Figure 1-7. Crystal structure of a p53 peptide bound to MDM2 at 2.6Å resolution (1YCR). 23
Figure 1-8. Triad of p53 amino acids forming the main contacts to MDM2. ......................... 24
Figure 1-9. Inhibitory peptides of the MDM2-p53 interaction ................................................ 26
Figure 1-10. Crystal structure of peptide 8 (an 8-mer p53 peptide analogue) bound to MDM2
............................................................................................................................................. 28
Figure 1-11. Natural inhibitors of the MDM2-p53 interaction. ............................................... 29
Figure 1-12. Small-molecule inhibitors of the MDM2-p53 interaction. ................................. 31
Figure 1-13. Inhibitors of the Nutlin-series.. ............................................................................. 32
Figure 1-14. Binding modes of Nutlin-inhibitors atomic resolution........................................ 33
Figure 1-15. Benzodiazepine-inhibitors of the MDM2-p53 interaction. ................................. 34
Figure 1-16. Benzodiadepinedione inhibitor in complex with MDM2. .................................. 35
Figure 1-17. Initial Isoindolinone inhibitors of the MDM2-p53 interaction. .......................... 36
Figure 1-18. SAR of the Isoindolinone Series........................................................................... 37
Figure 2-1. Crystal contacts from MDM2 crystal structure 1YCR, 1T4E and 1RV1. ........... 40
Figure 2-2. Clusters of Glutamate-Lysine pairs within MDM217-125 amenable to surface
engineering by site-directed mutagenesis. ........................................................................ 42
Figure 2-3. PCR 1 and 2 of Gene Synthesis. ............................................................................. 43
Figure 2-4. Assembly PCR products for gene assembly PCRs 1 and 2................................... 47

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Figure 2-5. Example of an expression trial of MDM2 K94E95A surface mutant produced by
gene synthesis.. ................................................................................................................... 48
Figure 2-6: Overlap Extension PCR........................................................................................... 50
Figure 2-7. Example of overlap extension PCRs to generate MDM2 surface mutants. ......... 53
Figure 2-8. Preparative scale S200 gel filtration profiles of MDM2 surface mutants. ........... 54
Figure 2-9. 1 H/15N-HSQC spectra of MDM2 surface mutants and WT. ................................. 56
Figure 2-10. 1 H/15N-HSQC spectra of MDM2 surface mutants and WT bound to the p53-
peptide................................................................................................................................. 57
Figure 2-11. Crystal structure of the MDM2 L33E mutant (1RV1). ....................................... 59
Figure 2-12. Introducing the L33E mutation into pGEX6P1. .................................................. 60
Figure 2-13. Expression of MDM225-108-L33E. ......................................................................... 63
Figure 2-14. Gel filtration profile of 1mg WT and methylated MDM2 on an S75 K16
column................................................................................................................................. 66
Figure 2-15. Ensemble of 20 NMR structures of apo-NMR (1Z1M)...................................... 69
Figure 3-1. Expression and purification of MDM217-125. .......................................................... 76
Figure 3-2. Previous backbone assignments of MDM2 and application to the MDM217-125
construct.............................................................................................................................. 77
Figure 3-3. Backbone assignment of apo- and holo-MDM2.................................................... 82
Figure 3-4. Assigned 1 H/15N HSQC of apo-MDM2. ................................................................ 83
Figure 3-5. Assigned 1 H/15N HSQC of holo-MDM2 bound to a p53-peptide. ....................... 84
Figure 3-6. Chemical shift changes induced by p53. ................................................................ 86
Figure 3-7. Chemical shift changes induced by p53 mapped onto the structure of MDM2
(1YCR)................................................................................................................................ 87
Figure 3-8. Overlay of the apo and holo-structure of MDM2 (1Z1M and 1YCR)................. 89
Figure 3-9. The 1H/15N HSQC pulse sequence.......................................................................... 92
Figure 3-10. Exchange phenomena in NMR spectroscopy. ..................................................... 94
Figure 3-11. Residues excluded from analysis, highlighted in blue. ....................................... 99
Figure 3-12. Isoindolinone-induced chemical shift changes in MDM217-125......................... 101
Figure 3-13. Weighted chemical shift changes (1 H and 15
N) of all Isoindolinone inhibitors
tested in this thesis mapped onto the primary sequence of MDM217-125....................... 103
Figure 3-14. Average per-residue chemical shift change for all Isoindolinones tested plotted
onto structure of MDM217-125 (1YCR). ........................................................................... 104
Figure 3-15. Degree of line broadening / intermediate exchange per residue caused by
Isoindolinone binding. ..................................................................................................... 106
Figure 3-16. Movement of K93 and H96 side-chain upon ligand binding to MDM2. ......... 107

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Figure 3-17. Difference in Isoindolinone and p53 induced chemical shift changes. ............ 110
Figure 3-18. Original titration spectra and chemical shift changes mapped onto MDM2’s
primary structure for Nutlin-3.. ....................................................................................... 111
Figure 3-19. Chemical shift changes induced by Nutlin-3. .................................................... 113
Figure 3-20. Difference in chemical shift changes induced by Nutlin-3 and Isoindolinones
........................................................................................................................................... 115
Figure 3-21. Structural relationships between the Isoindolinone inhibitors tested in this
thesis.................................................................................................................................. 123
Figure 3-22. Chemical shift perturbations induced by Isoindolinone inhibitors mapped onto
the MDM2 structure (1YCR). ......................................................................................... 125
Figure 3-23. Magnitude of chemical shift change for inhibitors 8247 and 8248. ................. 125
Figure 3-24. “Per-residue” plots for amino acids V75, L82, K93 and L103......................... 129
Figure 3-25. Comparison of chemical shift perturbations by all inhibitors on each residue of
MDM2’s hydrophobic pocket. ........................................................................................ 131
Figure 3-26. Comparison of chemical shift change within group C of isodindolinone
inhibitors. .......................................................................................................................... 133
Figure 3-27. Per-residue plot for residue Y100 of MDM2..................................................... 134
Figure 3-28. Possible orientations of Isoindolinones in MDM2’s hydrophobic pocket....... 135
Figure 3-29. Selection of a structure for docking of Isoindolinones...................................... 138
Figure 3-30. Nomenclature of Isoindolinone enantiomers using inhibitor 8231 as the
example. ............................................................................................................................ 141
Figure 3-31. Possible binding modes derived from the NMR data and manual docking..... 143
Figure 3-32. Binding models of Isoindolinone inhibitors derived from NMR data and
molecular docking with the program GOLD.................................................................. 150
Figure 3-33. Structural relationship of Isoindolinone inhibitors............................................ 152
Figure 3-34. Summary of Isoindolinone binding models derived from chemical shift data and
molecular docking. ........................................................................................................... 154
Figure 3-35. Different inhibitors bound to MDM2. ................................................................ 155
Figure 3-36. Binding model for inhibitors of group B obtained from manual docking.. ..... 156
Figure 3-37. SAR of Isoindolinones similar to group B. ........................................................ 158

Table of Tables
Table 2-1. Primers for gene synthesis of the MDM2 gene.. ..................................................... 45
Table 2-2. Primer combination for gene synthesis.................................................................... 45

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Table 2-3. Primers for side-directed mutagenesis. .................................................................... 49
Table 2-4. Primers for overlap-extension PCR. . ...................................................................... 52
Table 2-5. Primers for the MDM225-108-L33E mutant............................................................... 61
Table 2-6. ESI-MS results for methylated versus WT MDM2. ............................................... 65
Table 2-7. Summary of all MDM2 constructs and crystallisation conditions......................... 67
Table 3-1. Dialysis test to improve MDM2 stability. ............................................................... 79
Table 3-2. Inhibitors used for NMR titrations........................................................................... 97
Table 3-3. Average chemical shift changes observed in inhibitor titrations. ........................ 102
Table 3-4. Criteria for the analysis of magnitude of chemical shift....................................... 123
Table 3-5. Analysis of per-residue plots. ................................................................................. 127
Table 3-6. Structural restrains obtained from the NMR data.. ............................................... 136
Table 3-7. Selection of MDM2 Structures available in the protein data bank. ..................... 136
Table 3-8. Docking results from manual docking. .................................................................. 142
Table 3-9. Comparison of GOLD docking results to manual docking results. ..................... 148

Table of Equations
Equation 2-1. Reductive methylation of amides. ...................................................................... 64
Equation 3-1. Calculation of chemical shift differences........................................................... 99
Equation 3-2. Calculation of combined chemical shifts ........................................................... 99
Equation 3-3. Definition of chemical shift. ............................................................................. 116

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Part I: Section 1 - Introduction to the MDM2-p53 Interaction

Section 1

Introduction to the MDM2-p53 Interaction

8


1.1 The MDM2-p53 Interaction is an Important
Target in Drug Design

The transcription factor p53 is the cell’s major tumor suppressor (Levine 1997) and found to

be mutated in more than 50% of human cancers (Hainaut and Hollstein 2000). Targeting

tumour cells with altered p53 is therefore an important strategy in drug discovery, both in

academia and industry (Blagosklonny 2002). Since overcoming p53 function is one of the

major steps in carcinogenesis, cancer cells with wild-type p53 have found other means of

inhibiting its activity. In many cancer cells, disruption of p53 function is achieved through

over-expression of its main antagonist, the ubiquitin ligase MDM2 (Momand, Jung et al.

1998). These tumour cells require a different approach in cancer treatment, but offer a major

advantage for therapeutic intervention: once p53 inhibition is alleviated, its tumour

suppressor activity is unleashed, triggering apoptosis in such cells. In fact, three very recent

publications have stated that restoration of p53 function alone is sufficient to cause tumour

regression (Martins, Brown-Swigart et al. 2006; Ventura, Kirsch et al. 2007; Xue, Zender et

al. 2007). Disruption of the MDM2-p53 interaction in MDM2-amplified cells with WT-p53

has therefore become an important target in drug discovery. The molecular details of

MDM2-p53 binding present one of the few cases in which protein-protein interactions are

amenable to inhibition by small molecules (Kussie, Gorina et al. 1996).

During the course of this thesis, I have studied Isoindolinone-inhibitors of the MDM2-p53

interaction, gathering structural information about Isoindolinone binding to MDM2 by

crystallography and NMR. These structural insights were required to provide the basis for

further development of compounds with increased specificity and affinity, with the ultimate

aim of generating more suitable candidates for clinical trials.

9


1.2 The p53 Tumour Suppressor

Activation of the p53 protein protects the organism against the propagation of cells that carry

damaged DNA with potentially oncogenic mutations. Because of its essential function in

growth control, the p53 tumour suppressor is sometimes termed the “Guardian of the

Genome” (Lane 1992). Currently, over 19,000 publications discussing p53 are available on

Pubmed; a result of nearly 30 years of dedicated research into the subject. Unfortunately, it

will be impossible to cover such a large body of work in this context. Instead, this section

will give a brief general overview about p53 function, and then focus on the aspects of the

MDM2-p53 interaction relevant to this work.

1.2.1 The History of p53 Discovery

Even though p53 was first discovered in 1979, it took another 10 years for its true function to

be revealed. P53 was first identified in complex with the SV40 tumour antigen, a protein

encoded by the SV40 DNA virus that causes malignant transformation in mice (Lane and

Crawford 1979), (Linzer and Levine 1979). Subsequently, the p53 gene product was reported

to cause immortalisation upon over-expression and transformation in conjunction with the

Ras oncogene (Eliyahu, Raz et al. 1984; Jenkins, Rudge et al. 1984; Parada, Land et al.

1984). P53 was therefore initially classified as an oncogene, due to the fact that the cDNA

used in these early experiments carried mutations in the p53 gene, resulting in aberrant p53-

function. In the late 1980’s, it became apparent that p53’s transforming activity might be a

result of mutations causing a loss of function in the wild-type (WT) protein. Furthermore,

frequent observations of p53 inactivation in malignant cell lines suggested that the loss of

p53 function might offer an advantage in tumour development. In 1989, WT-p53 was shown

to prevent transformation (Finlay, Hinds et al. 1989), leading the way for a reinterpretation

of earlier results and subsequent classification of p53 as a tumour suppressor.

10


Soon after, p53 was found to activate transcription (Fields and Jang 1990), (Raycroft, Wu et

al. 1990), and was identified as a sequence-specific DNA-binding protein (Kern, Kinzler et

al. 1991), performing cell-cycle “checkpoint” functions (Kuerbitz, Plunkett et al. 1992).

Today, p53 is recognised as the cell’s major tumour suppressor and the most frequently

inactivated gene in human cancers (Vousden and Lu 2002).

1.2.2 The p53 Network

P53 has so far been best understood in its function as a tumour suppressor, responding to

signals of DNA damage and aberrant growth, initiating DNA repair, cell cycle arrest and, if

necessary, apoptosis. These functions are mainly provided through p53’s activity as a

transcription factor, controlling the expression of dozens of genes (Vogelstein, Lane et al.

2000). Even though it has not been the focus of this work, it is important to note that non-

transcriptional activities of p53 have also recently been reported (Vousden 2005).

Furthermore, more recent studies have investigated the function of p53 beyond its

involvement in cancer prevention (Vousden and Lane 2007), suggesting an important

contribution of p53 to other aspects of cellular life. The various pathways of p53 control and

function form a highly complex network of cellular events, in which p53 plays a central role.

Figure 1-1 attempts to summarise this network, by illustrating the most important causes of

p53 activation and the resulting down-stream events.

11


Figure 1-1. Cellular responses induced by p53 as a result of cytotoxic stress. This figure has been
extended from Vousden and Lane (Vousden and Lane 2007). Different kinds of cytotoxic stress
(grey), most classically DNA damage and oncogene activation, lead to activation of p53 through
different pathways. P53 activation occurs according to three major principles: Activation of p53
through post-translational modifications, sub-cellular localisation of the protein and stabilisation of
p53 due to reduced proteasomal degradation. Depending on the type of stress, the activation pathway
and the cell type, p53 then initiates the cellular response (yellow). Initiation of cell cycle arrest, DNA
repair and apoptosis represent its most classical functions providing tumour suppression. Other
functions, such as maintenance of genomic stability, inhibition of angiogenesis, involvement in
senescence and cell survival are also part of the repertoire of p53-induced down-stream events.

12


1.2.2.1 P53 Activation

Due to the potentially suicidal response that can be triggered by p53, a low cellular

concentration is maintained during normal cell development. Tight regulation of p53 levels

is achieved in three different ways addressing the protein’s activity, stability and sub-cellular

localisation. P53’s activity can be controlled through various post-translational modifications

such as phosphorylation, acetylation or glycosylation. The stability of p53 levels is mainly

dependent on the rate of ubiquitin-mediated degradation, while localisation of p53 in

different sub-sections of the cell can be regulated through shuttling the transcription factor in

and out of the nucleus. Furthermore, there are several pathways leading to p53 activation,

also affecting the type of response triggered by the tumour suppressor.

P53 levels are highly sensitive to DNA damage: Even a single break within a double-

stranded DNA molecule can lead to elevation of p53 levels (Vogelstein, Lane et al. 2000). If

DNA damage is caused by ionising radiation, then the central signalling protein in DNA

damage control, the kinase ATM (Ataxia telangiectasia mutated) activates p53 either through

direct phosphorylation or indirectly or via the transducer kinase Chk2 (Lukas, Lukas et al.

2004). P53 activation can also be induced via the kinase ATR (Ataxia telangiectasia and

Rad3 related), which is activated by the presence of single stranded regions of DNA

(Munger 2002).

Oncogene activation, such as expression of the Ras or Myc gene products (Munger 2002)

results in activation of p53 via the p14/p19ARF tumour suppressor, a negative regulator of

MDM2. The function of p14/p19ARF is described further in Section 1.3.

1.2.2.2 P53 Function

Keeping in mind additional functions mentioned in Figure 1-1, the major effects of p53

activation are the initiation of cell cycle arrest, DNA repair and apoptosis, accomplished

through regulation of DNA transcription. One important transcriptional target of p53 is the

13


cyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1, which serves to prevent entry from G1

to S phase, as well as G2 to mitosis transitions. In terms of triggering apoptosis, the protein

Bax and the tumour necrosis factor (TNF) are amongst the most important proteins activated

through the transcription factor p53. For a more complete discussion of transcriptional

regulation by p53, the reader is referred to Laptenko et al. (Laptenko and Prives 2006).

Interestingly, amongst the genes controlled by p53, there are some targets which themselves

modulate the activity of p53. One of these genes is MDM2, p53’s main antagonist, which is

able to return p53 levels back to normality at the end of a successful stress response.

1.3 The Oncogene MDM2

The Murine Double Minute Clone 2 (MDM2) is the main antagonist of p53, and was first

identified in 1987 as one of three genes amplified in a spontaneously transformed mouse cell

line (3T3DM) (Cahilly-Snyder, Yang-Feng et al. 1987). Amplification of the MDM2 gene in

rodent cells was shown to result in high tumourigenic potential, suggesting MDM2 is an

oncogene (Fakharzadeh, Trusko et al. 1991). A 90kDa protein found to co-

immunoprecipitate with the p53 tumour suppressor was later identified as the MDM2 gene

product and MDM2 was suggested to down-regulate p53 activity (Momand, Zambetti et al.

1992). The human MDM2 orthologue was mapped onto chromosome 12q13-14. When the

MDM2 gene was found amplified in 30% of osteosarcomas and soft tissue tumours,

MDM2’s function as an oncoprotein was confirmed (Oliner, Kinzler et al. 1992). Overall,

MDM2 is amplified in 7% of all human tumours (Momand, Jung et al. 1998). In 1997,

MDM2’s function as an E3 ubiquitin ligase was revealed, transferring a ubiquitin moiety

onto p53 from via cysteine residue in MDM2’s C-terminus (Honda, Tanaka et al. 1997).

Furthermore, MDM2 contains a nuclear localisation and export signal similar to that found

in several viral proteins (Roth, Dobbelstein et al. 1998). By shuttling in an out of the nucleus,

14


MDM2 transports p53 to the cytoplasm where ubiquitin-mediated degradation can occur and

where it can no longer carry out its function as a transcription factor (Tao and Levine 1999).

Many in vivo experiments have demonstrated that MDM2’s main function in untransformed

cells is the regulation of p53. MDM2-/- mice are not viable and die within the first days of

gestation (Jones, Roe et al. 1995). Deletion of the p53 gene in addition to MDM2 provides a

rescue from cell death (Montes de Oca Luna, Wagner et al. 1995), indicating that an

increased level of p53 in the absence of MDM2 drives the cells into apoptosis. p53-- mice

develop normally, but acquire tumours within the first three months after birth (Donehower,

Harvey et al. 1992). This is also the case for mice deficient in both MDM2 and p53

(McMasters, Montes de Oca Luna et al. 1996).

Apart from its main function as a suppressor of p53 activity, MDM2 interacts with a range of

other proteins, which either regulate MDM2 activity in upstream events, or are affected by

MDM2 in downstream events. These interactions are beyond the scope of this thesis; so only

one other MDM2-binding protein in addition to p53 will be mentioned here. This protein is

the p14/p19ARF tumour suppressor, which was one of the first proteins shown to interact with

MDM2 (Quelle, Zindy et al. 1995; Zhang, Xiong et al. 1998). P14/p19ARF is expressed from

the INK4a locus in an alternate reading frame (Quelle, Zindy et al. 1995) and indirectly

activates p53 in response to oncogene activation by inhibiting MDM2’s ubiquitin ligase

activity (Silva, Silva et al. 2003). Furthermore, p14/p19ARF blocks MDM2-dependent nuclear

export of p53 through localisation of MDM2 to the nucleolus (Weber, Taylor et al. 1999),

and thereby allows nuclear p53 to function as a transcription factor.

1.4 Regulation of p53 by MDM2

MDM2 and p53 are mutually regulated through an auto-regulatory feedback-loop (Picksley

and Lane 1993; Wu, Bayle et al. 1993), first described in 1993. In normal cells, the

15


concentration of p53 is maintained at low levels due to a short half-life. Upon DNA damage,

MDM2-mediated degradation is prevented by phosphorylation of conserved p53 Serine and

Threonine residues (Oren, Maltzman et al. 1981; Reich, Oren et al. 1983; Ashcroft, Taya et

al. 2000), increasing p53’s half-life from several minutes to hours (Harris and Levine 2005).

Alternatively, p53 levels are increased in response to oncogene activation via the p14/p19ARF

pathway, releasing MDM2’s tight control of p53 function. Elevated levels of p53 promote

transcription of MDM2 (Barak, Juven et al. 1993) and the MDM2 protein inhibits p53

activity through three independent mechanisms (Figure 1-2):

(i) MDM2 binds to the transactivation domain of p53 and thereby inhibits the

interaction of p53 with the transcription machinery, preventing transcription of p53-

regulated genes (Momand, Zambetti et al. 1992; Chen, Marechal et al. 1993).

(ii) Secondly, MDM2 contains a nuclear export signal able to induce transport of p53

into the cytoplasm, where p53 can no longer act as a transcription factor (Tao and

Levine 1999).

(iii) Finally, the E3 ubiquitin ligase MDM2 (Honda, Tanaka et al. 1997) targets p53 for

degradation by the proteasome (Haupt, Maya et al. 1997), through addition of a

ubiquitin moiety to the C-terminal region of p53.

16


Figure 1-2. The MDM2-p53 auto-regulatory feedback loop. In response to cytotoxic stress, p53 is
activated and transported to the nucleus, where it acts as a transcription factor of genes implicated in
cell cycle control. MDM2 is one of the genes controlled by p53 and is expressed upon activation of
p53. MDM2 inhibits p53 function by (1.) blocking p53’s transcriptional activity through direct
binding to the transactivation domain, (2.) through promotion of nuclear export of p53 and (3.)
through induction of ubiquitin-mediated degradation. This Figure has been modified from P.Chène
(Chene 2003).

In summary, elevation of p53 levels results in increased MDM2 levels, which in turn inhibit

the function of p53 and decrease p53 levels through proteasomal degradation. These out-of-

sync oscillations in the levels of both proteins (Lev Bar-Or, Maya et al. 2000) open a

window for p53-activity, but also offer the possibility of aborting the p53-response after

successful recovery from DNA damage.

In tumour cells where increased activity of MDM2 creates an imbalance in the complex

equilibrium between MDM2 and p53, a compound designed to bind MDM2 in order to

17


dissociate p53 could rescue p53 function and induce cell cycle arrest or apoptosis in the

affected cells.

1.5 Structural Details of MDM2 and p53

The sites of interaction between MDM2 and p53 were first identified in 1993 by yeast-two

hybrid screen and immuno-precipitation (Chen, Marechal et al. 1993; Oliner, Pietenpol et al.

1993). Both sites of interaction were found to lie in the N-termini of the proteins. The

following section is giving a brief overview of the overall domain organisation of MDM2

and p53, in order to illustrate the context in which the two interacting N-termini exist in vivo.

1.5.1 Overall Structure of p53

P53 is a tetrameric 393 amino acid protein (Stenger, Mayr et al. 1992), composed of five

major sub-sections connected by flexible linkers (Levine 1997).

Figure 1-3. Schematic representation of the domain organisation of p53.

The transactivation domain is responsible for MDM2-binding (Fields and Jang 1990;

Raycroft, Wu et al. 1990) and is located at the N-terminus (residues 1-42). NMR and circular

diochroism studies of a fragment comprising the transactivation domain and the adjacent

Proline rich region showed that this region is natively unfolded, lacking tertiary and

secondary structure elements (Dawson, Muller et al. 2003). However, upon binding to

MDM2, the transactivation domain adopts a helical conformation (Kussie, Gorina et al.

18


1996). The DNA binding domain comprises residues 102-292 (Cho, Gorina et al. 1994) and

is the region of p53 most affected by mutations in cancer cells (Vousden and Lane 2007).

The NMR structure of the human p53 DNA-binding domain was solved in 2006 (Canadillas,

Tidow et al. 2006), and is available under the PDB code 2FEJ (Berman, Westbrook et al.

2000). The oligomerisation domain (residues 324-355) is responsible for the formation of a

tetramer in vivo. The NMR structure of this domain has been deposited under the PDB code

2J0Z. The C-terminal domain (residues 367-393) is capable of binding to single-stranded

DNA and RNA (Figure 1-3) (Lee, Elenbaas et al. 1995).

1.5.2 Overall Structure of MDM2

MDM2 is a 491 amino acid protein composed of several functional domains interlinked by

less structured regions, not all of which are well characterised in terms of their structure and

function.

Figure 1-4. Schematic representation of the domain organisation of MDM2.

The p53 binding site is located in MDM2’s N-terminus (residues 19-102), followed by

nuclear localisation and export signals (residues 179-185 and 190-202). Structurally, the N-

terminal domain is the most well characterised part of MDM2: There are several NMR and

X-ray structures available, both in the apo-form and complexed to various peptide or small-

molecule ligands. A subset of these structures will be described in more detail in the

remainder of this introduction. The central region of the protein, often referred to as the

acidic domain (residues 222-272), is rich in negatively charged residues. This region is

19


followed by a Zinc finger domain (residues 290-335), for which the NMR structure has been

determined (PDB code 2C6B) (Yu, Allen et al. 2006). The C-terminal region (residues 399-

489) contains a RING-finger motif responsible for MDM2’s ubiquitin ligase activity. The

NMR structure of this domain was revealed by NMR spectroscopy (2HDP) (Kostic, Matt et

al. 2006).

1.5.3 The MDM2 N-terminal Domain

The first structure of MDM2’s p53-binding domain was solved by X-ray crystallography in

1996, in complex with a 15-residue p53 peptide (Kussie, Gorina et al. 1996). In addition to

the details of MDM2-p53 binding (described in Section 1.5.4), this structure revealed that

MDM2’s N-terminal section is composed of two major repeats of secondary structure. These

repeats are arranged pseudo-symmetrically along an approximate dyad axis, as shown in

Figure 1-5a. The interface of the repeats on either side of the symmetry axis is lined with

hydrophobic residues that form a deep cleft at the surface of MDM2 (Figure 1-5b), to which

the p53-peptide binds. To describe the elements of secondary structure within MDM2,

Kussie et al. have introduced a useful nomenclature, which is described in Figure 1-5 and

will be applied throughout the remainder of this thesis.

The hydrophobic pocket arising at the interface of the two repeats is composed of the

following structural elements: The bottom of the cleft is composed of two helices, α1 and

α1’. Helices α2 and α2’ form the sides of the pocket, and two β-sheets seal the ends at either

side of the pocket. Even though the molecule is built from symmetrically arranged elements

of secondary structure, the cleft itself is asymmetrical, as a result of different lengths of the

helices forming the walls of the pocket.

20


Figure 1-5. Pseudo-symmetrical arrangement of MDM2’s N-terminal domain, forming a
hydrophobic pocket at their interface. Elements of secondary structure have been labelled
according to Kussie et al.: repeat one (cyan), β1 - 26-30, α1 - 32-42, β2 - 48-49, α2 - 51-63, β3 - 66-
68, repeat two (blue), β1’ - 73-77, α1’ - 81-87, β2’ - 89-93, α2’ - 95-104, β3’ - 106-108. (a) Schematic
representation of the secondary structure highlighting the axis of symmetry. (b) Three-dimensional
structure highlighting the hydrophobic pocket formed at the interface of the two repeats (grey).

21


In 2005, Uhrinova et al. solved the NMR structure of the MDM2 N-terminal in the absence

of a ligand (Uhrinova, Uhrin et al. 2005). This structure revealed that the p53-binding

domain is less structured in the holo-state, consisting of shorter elements of secondary

structure than observed for the p53-bound form. The decrease in structural definition was

also reflected in the restraint-density, as the structure is based on only 8 inter-residue NOEs

per residue. Furthermore, the heteronuclear NOE ratio (for more information, see Part II of

this thesis) for the well-structured residues was relatively low (0.7±0.1), indicating that even

the structured parts of the protein show increased flexibility. Overall, the decreased structural

definition of the apo-domain, as well as problems due to protein instability, signal overlap

and conformational exchange resulted in a final structural bundle of rather poor convergence

(RMSD ~ 1Å, for residues 28-104).

Figure 1-6. The NMR structure of MDM2’s N-terminal domain (1Z1M). Colour coding of the
elements of secondary structure is identical to Figure 1-5. N and C-termini of MDM2 were found to
be unstructured.

When comparing p53-bound MDM2 to the holo-structure (Figures 1-5b and 1-6), it becomes

evident that the hydrophobic pocket of MDM2 is smaller in the absence of a ligand.

Furthermore, the holo-binding pocket is partly occluded by the otherwise unstructured N-

terminal tail of MDM2 (Figure 1-6). It had previously been assumed that the N-terminus of

MDM2 forms a “flexible lid” folding over the hydrophobic pocket in the absence of a ligand

22


(McCoy, Gesell et al. 2003), which is replaced upon p53 binding. This was confirmed here,

as Uhrinova et al. were able to observe through-space correlations between the N-terminal

tail of MDM2 and the hydrophobic cleft (Uhrinova, Uhrin et al. 2005).

1.6 The MDM2-p53 Interaction

The molecular details of p53-binding to MDM2 were revealed in the crystal structure by

Kussie et al. (Kussie, Gorina et al. 1996), which formed the basis for structure-based design

of MDM2-antagonists. Upon binding to MDM2, the p53-peptide forms an amphipathic helix

highly complementary to the hydrophobic pocket at the surface of MDM2. This helix

extends over approximately 2.5 turns, with three extended residues at the C-terminal side

(Figure 1-7).

Figure 1-7. Crystal structure of a p53 peptide bound to MDM2 at 2.6Å resolution (1YCR),
depicting the helical conformation of the p53 peptide.

The main contacts of the interaction between MDM2 and p53 are made by three highly

conserved p53 residues, namely Phe19, Trp23 and Leu26, which insert deeply into the

23


hydrophobic pocket of MDM2 (Figure 1-8). These three residues occupy three sub-pockets

at the surface of MDM2, from now on referred to as the Phe19, Trp23 and Leu26 sub-pockets.

For more information on this nomenclature, the reader is referred to Figure 3-28 of Section

3.5.4. The interface between the two proteins consists mainly of van-der-Waals contacts,

with only two hydrogen bonds contributing to the interaction. One hydrogen bond is formed

between the NH-group of the Trp23-sidechain of p53 and the carbonyl group of Leu54 of

MDM2. The second hydrogen bond is formed between the backbone amide of p53’s Phe19

and the side-chain amide of Gln72 of MDM2 (Figure 1-8).

Figure 1-8. Triad of p53 amino acids forming the main contacts to MDM2. (a) Binding of the
three hydrophobic acids Phe19, Trp23 and Leu26 to the hydrophobic pocket of MDM2. (b) The
tryptophan-side chain of p53 is inserted into the deepest sub-pocket of MDM2. Stars indicate the
location of the two hydrogen bonds contributing to the interaction.

The importance of the three side-chains of residues Phe19, Trp23 and Leu26 as main

contributors to the interaction was later confirmed in studies with a retro-inverso p53 peptide

(an all-D peptide with reversed sequence), which did not display significant loss in affinity

compared to the WT-peptide (Sakurai, Chung et al. 2004). This supports the hypothesis that

binding is merely driven by the side-chain contacts, and the backbone and helix of the p53

peptide functions mainly as a scaffold to hold the side-chains in the correct orientation.

The MDM2-p53 interface presents a special case in protein-protein interactions in that most

contacts are hydrophobic as opposed to polar. Furthermore, the interface is fairly small,

spanning only 600-800Å2 (Klein and Vassilev 2004). The three side-chains of p53 forming

24


the main contacts to MDM2 have a molecular weight of approximately 300 Daltons, which

renders this protein-protein interaction one of the few targets amenable to inhibition by

small-molecules.

1.7 Inhibitors of the MDM2-p53 Interaction

Since the publication of the crystal structure of the MDM2-p53 complex (Kussie, Gorina et

al. 1996), many resources have been invested in the development of a pharmacophore model

for this protein-protein interaction. A pharmacophore is a three-dimensional substructure of a

molecule that carries the essential features responsible for a drug’s biological activity. It does

not represent a real molecule or a real association of functional groups, but a purely abstract

concept that accounts for the common molecular interaction capacities of a group of

compounds towards their target structure. Detailed exploration of the 1YCR crystal structure

was an obvious starting point for the development of a pharmacophore:

Comparing the binding epitopes of MDM2 and p53, MDM2 presents a more well-defined

binding site, to which the p53 peptide, normally unstructured in solution, binds in a

complementary shape. Inhibitors should aim to mimic the less structured binding partner, in

this case the p53-peptide. Since the interaction is mainly hydrophobic, MDM2-antagonists

have to be lipophilic. This is favourable, on the one hand, since burial of lipophilic groups in

the binding pocket is accompanied by partial desolvation of the inhibitor, which makes the

binding-event entropically favourable. On the other hand, hydrophobicity is accompanied by

a decrease in bioavailability of the inhibitor through decreased solubility. Finally, it is

desirable for the inhibitor to mimic at least one of the hydrogen bonds involved in p53-

binding.

In the late 1990’s, the Novartis group in Switzerland as well as a research group at the

University of Dundee engaged in the development of a more detailed pharmacophore model,

25


determining amino acid preferences of MDM2’s hydrophobic pocket. This work resulted in

the identification of highly potent peptide inhibitors (Picksley, Vojtesek et al. 1994; Bottger,

Bottger et al. 1996; Bottger, Bottger et al. 1997; Garcia-Echeverria, Chene et al. 2000), much

more active than initial small-molecule inhibitors (Duncan, Gruschow et al. 2001; Stoll,

Renner et al. 2001; Zhao, Wang et al. 2002).

1.7.1 Peptidic Inhibitors

In the year 2000, the Novartis group published the most potent MDM2 inhibitor to date, an

8-residue p53-peptide analogue, named peptide 8 (Figure 1-9) (Garcia-Echeverria, Chene et

al. 2000). This peptide was developed from a phage display peptide library (Bottger, Bottger

et al. 1996), based on synthetic peptides derived from p53’s MDM2-binding region

(Picksley, Vojtesek et al. 1994), and subsequent structure-based design. Phage display

resulted in the identification of peptide 2, displaying 28-fold increased potency compared to

the WT peptide 1 (Figure 1-9).

Figure 1-9. Inhibitory peptides of the MDM2-p53 interaction (Garcia-Echeverria, Chene et al.
2000). Peptide 1 corresponds to the WT p53-peptide. The positions of the three conserved residues
forming the main contacts with MDM2 are indicated in bold. Residues represented in blue are
derivatives of natural amino acids: α-aminoisobutyric acid (Aib), phosphonomethylphenylalanine
(Pmp), 6-chlorotryptophane (6-Cl-Trp) and 1-amino-cyclopropanecarboxylic acid (Ac3C). IC50 values
are stated as nanomolar concentrations.

Assuming that it is entropically favourable for the peptide to adopt a helical conformation

prior to MDM2-binding, non-natural amino acids which are known to stabilise helices, such

as α-aminoisobutyric acid and 1-amino-cyclopropanecarboxylic acid (Figure 1-9), were

26


introduced, resulting in a further increase of affinity. In the next step, attempts to improve

binding of the Tyr22 and Trp23 side-chains were made. Tyr22 was replaced by

phosphonomethylphenylalanine (Figure 1-9), in order to allow salt-bridge formation between

this side-chain and Lys94 of MDM2. This substitution was able to increase binding-affinity 7-

fold. The most significant increase in affinity was achieved through the introduction of a

chloro-moiety at position 6 of the Trp23 side-chain. The chloro-substituent is able to exploit

the Trp23-pocket of MDM2 more fully, resulting in a 60-fold increase in affinity. In total, a

1700-fold increase in binding capacity compared to the initial WT-peptide was achieved by

this rational approach (Garcia-Echeverria, Chene et al. 2000).

When talking about binding-affinity in this context, it is important to note that the peptides’

capacity to bind MDM2 was determined by an in vitro competition assay. In this assay,

ELISA plates covered with GST-MDM2 were incubated with the inhibitory peptides. After

addition of full-length p53, the amount of full-length p53 bound to MDM2 in the presence of

the inhibitory peptide was determined by Western Blotting (Garcia-Echeverria, Chene et al.

2000). By using different concentrations of inhibitors, the concentration of inhibitor required

to replace 50% of the reporter ligand, in this case full-length p53, can be determined. This

value is called the median inhibitory concentration, or IC50. It is a useful parameter to

compare potencies of inhibitor potencies when absolute affinities are not available.

In 2006, the crystal structure of peptide 8 bound to MDM2 was solved (2GV2) (Sakurai,

Schubert et al. 2006). This structure verified that the main contacts of binding are still made

by the three conserved residues, Phe19, Trp23 and Leu26 (Figure 1-8), but that peptide 8 shows

increased steric complementarity compared to the WT peptide. The chloro-substituent of the

Trp23-sidechain is able to explore a void in the deep pocket of MDM2, making additional

contacts with residues Phe86 and Ile99 of MDM2. The Pmp-substitution for Tyr22, however,

did not have the predicted effect of forming a salt-bridge with Lys94 of MDM2. It is instead

projecting into the solvent.

27


Figure 1-10. Crystal structure of peptide 8 (an 8-mer p53 peptide analogue) bound to MDM2
(2GV2). Upper panel: The entire peptide complexed to MDM2, showing Phe19, 6-Cl-Trp23 and Leu26
side-chains in blue. The side chains of non-natural amino acids such as α-aminoisobutyric acid (Aib),
phosphonomethylphenylalanine (Pmp) and 1-amino-cyclopropanecarboxylic acid (Ac3C) are shown
in grey. Lower panel: On the left, the Trp23 side-chain of the WT peptide is shown in the same
orientation as the 6-chlorotryptophane of the synthetic peptide (right hand Figure). The chloro-
substitution (green) enables full exploration of the binding pocket, increasing the affinity several-fold.

28


1.7.2 Natural Inhibitors

In 2001, the first natural inhibitors of the MDM2-p53 interaction were published (Stoll,

Renner et al. 2001). These inhibitors were chalcone derivatives, compounds derived from

1,3-diphenyl-2-propen-1-one, and had previously been known as substances with tumour

suppressing activity (Dore and Viel 1974). Now, these compounds could be identified as

MDM2 antagonists, binding in the same location as p53, as established by chemical shift

mapping (Stoll, Renner et al. 2001). It is assumed that the dicholorophenyl-moiety resides in

the Trp23-pocket of MDM2. The IC50’s of these naturally occurring compounds lay in the

micromolar range, with the potent compound (compound B) having an IC50 of approximately

50-70µM (Figure 1-11).

Figure 1-11. Natural inhibitors of the MDM2-p53 interaction.

29


In the same year, Chlorofusin, a natural substance identified from over 53000 microbial

extracts, was identified as an MDM2-antagonist (Duncan, Gruschow et al. 2001).

Chlorofusin is a fungal metabolite, inhibiting the MDM2-p53 interaction with an IC50 of

4.6µM (Figure 1-11). To date, there is no information about the binding-mode of

Chlorofusin.

In 2006, a third natural inhibitor was reported (Tsukamoto, Yoshida et al. 2006). The

compound hexylitaconic acid was isolated from a culture of marine-derived fungus,

Arthrinium sp. (Figure 1-11), and was shown to inhibit MDM2-p53 binding with an IC50 of

50µM.

1.7.3 Small Molecule Inhibitors

The first non-natural MDM2-antagonist, named Syc (non-peptidic small-molecule

synthesised compound), was reported in 2002 (Zhao, Wang et al. 2002). Following

computer-aided design and subsequent synthesis of 23 compounds, five compounds were

found to exert an inhibitory effect on the MDM2-p53 interaction in cellular assays. The main

scaffold of these compounds is bicyclic with two varying aromatic substituents, hoped to

mimic the interaction of Phe19 and Trp23 with MDM2 (Figure 1-12).

In 2004, Galatin et al. reported a sulfonamide-based compound (Galatin and Abraham 2004)

(Figure 1-2), identified by 3D database searches of the National Cancer Institute database as

a lead compound for further drug design. This compound had an IC50 of 32µM.

In 2006, Spiro-oxindoles were reported as inhibitors of the MDM2-p53 interaction,

following structure-based design. It was decided to base the database search on compounds

with an oxindole ring, in order to mimic the Trp23 side-chain of p53. The compound

spiro(oxindole-3,3’-pyrrolidine) was chosen as a starting point for further compound design,

since the spiropyrrolidine ring is able to provide a suitable scaffold for the attachment of R-

30


groups to mimic the interactions of Phe19 and Leu26 with MDM2. The most potent compound

(compound 1, Figure 1-12) identified had a Ki value of 86nM based on a fluorescence-

polarisation binding assay. Compared to the most potent peptide inhibitor tested in the same

assay (Garcia-Echeverria, Chene et al. 2000), this compound was still 100-times less potent.

In 2006, a Spiro-oxindole with a Ki of 3nM was identified (compound 8, Figure 1-12) (Ding,

Lu et al. 2006), based on further rational design.

Figure 1-12. Small-molecule inhibitors of the MDM2-p53 interaction.

31


1.7.4 Nutlin-Inhibitors

The most potent class of small-molecule inhibitors to date are the “Nutlins” (for Nutley-

inhibitor), a cis-imidazoline based series of inhibitors identified by screening a library of

synthetic chemicals (Vassilev, Vu et al. 2004). The compounds were synthesised as racemic

mixtures and separated with the use of chiral colums. For the compound Nutlin-3 (Figure 1-

13), only one enantiomer was found to be active.

Figure 1-13. Inhibitors of the Nutlin-series. The IC50 values of the three published inhibitors were
260nM, 140nM and 90nM for Nutlin-1, Nutlin-2 and Nutlin-3, respectively. These values were
determined by SPR, in a competition assay measuring the binding of MDM2 in the presence of
inhibitors to an immobilised p53 peptide (Vassilev, Vu et al. 2004).

In order to investigate the molecular details of Nutlin-binding to MDM2, the crystal structure

of the Nutlin-2/MDM2 complex was solved at 2.3Å resolution (1RV1) (Vassilev, Vu et al.

2004). In the same year, an NMR structure of a Nutlin-like compound bound to humanised

Xenopus MDM2 was also published (1TTV) (Fry, Emerson et al. 2004). Both structures

show that the hydrophobic pocket of MDM2 adopts a similar shape to the p53-bound pocket,

and that both inhibitors bind in a p53-like fashion: One chloro/bromo-phenyl moiety points

32


in to the Trp23-pocket of MDM2, while the other one projects into the sub-pocket

accommodating the Leu26-sidechain of p53. Both halogeno-phenyl groups are held in

position by the imidazoline scaffold, replacing the helical backbone of p53. The 2-

isopropoxy- and 2-ethoxy-moieties of the 4-methoxy-substituent sit in the position of the

Phe19 side-chain of p53. The N-piperazinyl moiety points into the solvent. Its position could

not be defined in the NMR-structure (Figure 1-14).

Figure 1-14. Binding modes of Nutlin-inhibitors atomic resolution. (a) Crystal structure of Nutlin-
2 bound to human MDM2 (1RV1). (b) NMR structure of a Nutlin-like compound bound to Xenopus
MDM2 (1TTV). Note that the N-piperazinyl moiety of the inhibitor is not displayed due to a lack of
NOEs observed to define its position, and due to undefined stereochemistry of this R-group.

The effects of Nutlin-inhibitors have been well characterised in vivo and the most potent

compounds have entered clinical trials. However, the successful development of an anti-

cancer drug is not guaranteed, hence there is still a need for alternative compounds.

1.7.5 Benzodiazepinedione Inhibitors

In 2005, a second high-resolution inhibitor-MDM2 structure was reported (Grasberger, Lu et

al. 2005). A Benzodiazepinedione antagonist of MDM2 was identified through screening of

over 338,000 compounds in an affinity-based fluorescence assay named ThermoFluor,

measuring ligand-induced stabilisation of the target-protein (Pantoliano, Petrella et al. 2001).

Interestingly, this was not the first time a benzodiazepine derivative was mentioned as a

33


potential inhibitor of the MDM2-p53 interaction. In 2001, a benzodiazepine-2-one had been

identified by Majeux et al., as part of the establishment of a computational method to

evaluate electrostatic desolvation energies of receptor-ligand complexes (Figure 1-15)

(Majeux, Scarsi et al. 2001).

Figure 1-15. Benzodiazepine-inhibitors of the MDM2-p53 interaction.

From the initial high-throughput assay reported by Grasberger et al., 1216 compounds,

amongst those 116 Benzodiazepinediones, were selected for further characterisation using a

fluorescent peptide displacement assay. This assay was able to show that

Benzodiazeponediones bind MDM2 in the p53-binding region (Parks, Lafrance et al. 2005).

The most potent compounds according to this assay were selected for further optimisation,

ultimately leading to identification of compound 1, binding to MDM2 with a KD of 80nM

(Figure 1-15). Compound 1 has been co-crystallised with MDM2 in a 2.7Å resolution crystal

structure (PDB code 1TAE). The two para-chlorophenyl moieties point into the Trp23 and

Leu26 sub-pockets, held in place by the benzodiazepine heterocycle (Figure 1-16). A similar

orientation of chlorophenyl-groups was also observed for Nutlin-binding (Vassilev, Vu et al.

2004). The iodobenzo-component of the inhibitor is located in the Phe19 sub-pocket,

similarly to the p53-peptide. An interesting feature of this structure is the location of the N-

terminal portion of MDM2, a helix comprising residues 17-25, which folds over the

34


hydrophobic pocket and extends the Leu26 sub-pocket, intensifying contacts with the para-

chlorobenzoyl-moiety of the inhibitor.

Figure 1-16. Benzodiadepinedione inhibitor in complex with MDM2. (a) Binding mode of the
inhibitor. (b) The N-terminus of MDM2 (residues 17-25, shown in blue) folds over the binding-site
and extends the Leu26 sub-pocket accommodating the para-chlorobenzoyl-moiety.

Due to the close superposition of elements of the Benzodiazepinediole-substituents with the

three crucial side-chains of p53, it was suggested that the Benzodiazepinediole-scaffold

could serve as an α-helix mimetic in a more general sense (Cummings, Schubert et al. 2006).

This might have applications in the identification of drug leads targeting other protein-

protein interactions.

1.7.6 Isoindolinone Inhibitors of the MDM2-p53 Interaction

This thesis focuses on the elucidation of the binding modes of Isoindolinone inhibitors of the

MDM2-p53 interaction, developed by our collaborators at the Northern Institute for Cancer

Research at the University of Newcastle upon Tyne. Hardcastle et al. first reported inhibitors

with an Isoindolinone scaffold in 2005, identified from in silico screening (Hardcastle,

Ahmed et al. 2005). Initial compounds displayed modest affinity for MDM2 with an IC50 of

approximately 200µM (Figure 1-17). These compounds were further developed in a program

of focused library synthesis guided by virtual screening. Initial development was based on

35


binding modes predicted by easyDock (Mancera, Kallblad et al. 2004) followed by virtual

screening, and did not prove to be more successful than random selection of substituents.

Figure 1-17. Initial Isoindolinone inhibitors of the MDM2-p53 interaction.

In a revised strategy for the development of more potent compounds, multiple binding

modes obtained by prediction with easyDock and GOLD (Verdonk, Cole et al. 2003) were

used as “seeds” for further virtual screening (Hardcastle, Ahmed et al. 2006). In this

approach, the position of the Isoindolinone-scaffold was preserved during simulated

annealing with the program Skelgen (Stahl, Todorov et al. 2002). From the compounds

generated in this way, 57 were selected as “virtual hits” and 43 of these were synthesised and

tested for their activity by an ELISA competition assay. Amongst this generation of

compounds, five inhibitors displayed a significant loss in activity, and only two were more

active than the most potent seed compound.

In the light of these results, combinatorial chemistry was chosen as an approach for further

optimisation of compounds. The most favourable substituents of the Isoindolinone scaffold

were selected based on initial SAR, and an array of 36 new compounds covering every

possible combination of substituents was designed. 24 of these compounds were then

synthesised and assayed. SAR results for this inhibitor generation were contradictory: For

example, the introduction of a 4-chloro-substituent at the phenyl-moiety of two compounds

resulted in both an increase and decrease in potency (Figure 1-18). This result suggested that

different binding modes for this class of inhibitors are possible and are induced by subtle

differences in the chemical structure.

36


Figure 1-18. SAR of the Isoindolinone Series. The introduction of a 4-chloro-substituent at the
phenyl-moiety of the Isoindolinone causes contradictory SAR results. NU8231 was the most potent
Isoindolinone at the time.

The most potent compound at this time (NU8231, Figure 1-18) had an IC50 of 5µM and

displayed promising biological activity. In SJSA cells with amplified MDM2, transcription

of p21WAF1/CIP1, a CDK/cyclin inhibitor under transcriptional control of p53, as well as

MDM2 levels were increased in a dose-dependent manner after incubation with the inhibitor.

Furthermore, in a luciferase-based reporter gene assay, p53 activity was found to be

increased (Hardcastle, Ahmed et al. 2006).

At this point in time, three different strategies to generate more potent inhibitor generations

had not resulted in a significant increase in affinity. The availability of a high-resolution

structure of Isoindolinone-binding to MDM2 was therefore thought to provide a major

advance to assist the design of more potent inhibitor generations. My work seeks to provide

these insights, with the hope of enabling the design of more potent candidates suitable for

clinical trials.

37

Part I: Section 2 - Towards a Crystallisable Form of MDM2

Section 2

Towards a Crystallisable Form of MDM2

38


2.1 Crystallising MDM2

Protein crystallisation still remains the rate-limiting step in the determination of X-ray

structures. For proteins that are “stubborn crystallisers”, the formation of crystals is often

less likely due to intrinsic flexibility or unstructured sections within the protein of interest, or

due to surface patches of flexible, solvent-exposed amino acids of lower conformational

entropy. Unfortunately, the N-terminal domain of MDM2, whether in the apo or holo state,

can be described as “stubborn” when attempting to crystallise the domain.

In 2005, a series of NMR relaxation experiments showed that MDM2’s p53-binding domain

is flexible and rather poorly structured in the absence of a ligand (Uhrinova, Uhrin et al.

2005). The C- and N-terminal portions of the protein were shown to undergo fast motions,

while the residues in the p53-binding site undergo motions on a slower timescale. The

mobility of MDM2 in the absence of a ligand might explain why no crystallisation

conditions have so far been established that allow formation of apo-crystals amenable to

soaking of a small molecule ligand into the crystal.

Furthermore, all X-ray structures of MDM2 published to date show the N-terminal domain

in complex with a ligand that contributes significantly to the observed crystal contacts

(Figure 2-1) (Kussie, Gorina et al. 1996; Vassilev, Vu et al. 2004; Grasberger, Lu et al. 2005;

Sakurai, Schubert et al. 2006). For each ligand, it was therefore necessary to determine a new

set of crystallisation conditions. Furthermore, it has been shown that high-affinity ligands

can have a stabilising effect on the N-terminal domain of MDM2 (Uhrinova, Uhrin et al.

2005), (Grasberger, Lu et al. 2005), increasing the likelihood of crystallisation. In order to

crystallise MDM2, one therefore requires ligands able to stabilise MDM2 as well as to

contribute to the formation of crystal contacts.

39


This part of my thesis attempts to enable MDM2-crystallisation in the presence and absence

of small-molecule ligands through protein engineering. The establishment of suitable protein

constructs and crystallisation conditions that consistently yield diffraction quality crystals

will hopefully allow rapid determination of Isoindolinone binding-modes enabling further

compound design.

Figure 2-1. Crystal contacts from MDM2 crystal structure 1YCR, 1T4E and 1RV1. In the 1YCR
structure, one way by which the p53-peptide contributes to the crystal contacts is through packing of
two Lys24 side-chains. In the 1T4E structure, an MDM2-bound Benzodiazepinedione forms a
hydrogen bond with an adjacent MDM2 molecule. In the 1RV1 structure, Nutlin-2 contributes to the
crystal contacts through unspecific binding to the surface of MDM2, bringing together three MDM2
molecules.

40


2.2 MDM2 Surface Engineering

2.2.1 Decreasing the Entropic Penalty of Crystallisation

Based on a current estimate, only 30% of proteins expressed solubly from E.coli will

eventually crystallise (Dale, Oefner et al. 2003). Approximately 80% of these crystallisable

proteins will form crystals from screening of only 50 different conditions (Jancarik and Kim

1991). Testing a huge number of conditions when initial screens were unsuccessful is

therefore not likely to increase success. A much more efficient way to obtain crystals is

rational modification of the target protein in order to favour crystallisation. Derewenda et al.

have proposed one such method, using site-directed mutagenesis to engineer the surface of

the protein (Derewenda 2004). Since the incorporation of large extended side-chains into the

crystal lattice puts an entropic penalty on the process of crystallisation, it was suggested to

replace these with small residues, such as Alanines. Surface-alanines are thought to provide a

conformationally more homogeneous surface and could potentially form crystal contacts. It

was suggested that clusters of Lysine and Glutamic acids are the most suitable amino acids

for mutagenesis, since they occur with high incidence at protein surfaces (Baud and Karlin

1999), possess high conformational entropy (Avbelj and Fele 1998) and occur rarely at the

interface of protein-protein interactions (Conte, Chothia et al. 1999).

The MDM217-125 sequence contains three clusters of Lysines and Glutamic acids amenable to

protein engineering, namely K51 and E52, E69 and K70, K94 and E95. All three clusters are

potential targets, as they do not directly interfere with p53 binding (Figure 2-2).

41


Figure 2-2. Clusters of Glutamate-Lysine pairs within MDM217-125 amenable to surface
engineering by site-directed mutagenesis. (a) MDM2 primary sequence spanning residues 17 to
125, highlighting three Glutamate/Lysine clusters. (b) Structure of p53-bound MDM2 (1YCR)
highlighting the location of the three Glutamate/Lysine clusters. Glutamates are shown in dark blue,
Lysines in light blue.

42


2.2.2 Generating MDM2 Surface Mutants by Gene Synthesis

2.2.2.1 Cloning by Assembly PCR or Gene Synthesis

WT-MDM21-125, WT-MDM217-125 and a series of mutants (K51E52A, E69K70A, K94E95A,

K51E52K94E95A, E69K70K94E95A, K51E52E69K70K94E95A) were initially generated

by gene synthesis from short overlapping primers. One way to generate a desired DNA

template is by synthesising a gene through assembly Polymerase Chain Reaction (PCR) or

gene synthesis. Instead of amplifying the desired DNA from a cDNA clone, the template is

formed from short overlapping oligonucleotides in an initial PCR. Since the initial PCR

results in products of varying sizes, the full-length gene is obtained in a second PCR by

amplification with flanking primers. This method is suitable for the quick generation of

many constructs of different lengths and desired mutations by choosing the appropriate

flanking and mutant primers.

Figure 2-3. PCR 1 and 2 of Gene Synthesis.

43

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