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A DPhil Thesis completed at Christ Church College, University of Oxford, Department of Biochemistry.

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CompleteThesis_ChristianeRiedinger.pdf

  1. 1. 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 requirementsfor the degree of Doctor of Philosophy at the University of Oxford.
  2. 2. AbstractThe work towards this thesis comprises two independent projects that are linked through theprocess of carcinogenesis. In cancer cells, it is often the case that tumour suppressors,proteins that prevent uncontrolled cell division are down-regulated, while proto-oncogenespromoting cell growth are up-regulated. By applying structural biology in combination withbiophysical 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 oncogeneMDM2. Furthermore, this work has engaged in structural and functional characterisation ofthe potential tumour suppressor protein Doc1.Inhibiting the MDM2-p53 InteractionThe 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 cellproliferation, 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 mainantagonist, the ubiquitin ligase MDM2. Activation of wild type p53 in these cancer types hastherefore become a focus of cancer drug discovery. The MDM2-p53 interaction has beenlocalised to a relatively small hydrophobic pocket within the MDM2 N-terminal domain, towhich p53 makes contact through only three conserved amino acids. This renders theMDM2-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 Institutefor Cancer Research at the University of Newcastle. In this study, we have attempted toelucidate the molecular details of Isoindolinone binding to MDM2, in order to provide astructural basis for further rational inhibitor-design, with the ultimate aim of developingmore potent candidates for clinical trials.This goal was pursued applying two major methods: Crystallography and NMRSpectroscopy. Since MDM2 is a challenging target for protein crystallisation, we haveattempted to alter the surface-properties of MDM2 through protein-engineering in order toimprove its crystallisability. While standard NMR methods of structure determination cannotbe employed in this system, we have obtained a molecular model of Isoindolinone binding toMDM2 through detailed analysis of inhibitor-induced chemical shifts.While our efforts to obtain a high-resolution X-ray structure of MDM2 complexed to anIsoindolinone have been unsuccessful, the chemical shift analysis has provided insights intothe binding modes of Isoindolinone inhibitors. ii
  3. 3. Structure and Function of Doc1, a Potential Tumour SuppressorDeleted in Oral Cancer 1 (Doc1) is a potential tumour suppressor first identified in 1998. Itsname stems from the fact that the protein could not be detected in malignant human oralkeratinocytes, suggesting a potential involvement in cellular growth-control. Not muchinformation is available about the structure and function of Doc1 to date: The protein hasbeen shown to suppress DNA replication through association with DNA polymerase αprimase, and was later identified as a CDK2-binding protein. Furthermore, Doc1 is a 12kDaprotein consisting of an unstructured N-terminus and a helical C-terminal domain. The aimof this study was therefore to further characterise the interaction of Doc1 with CDK2 and todetermine its structure by NMR spectroscopy.Employing a variety of methods, we have not been able to demonstrate a direct interactionbetween Doc1 with CDK2. Furthermore, we have not been able to reproduce one of theinitial experiments establishing a Doc1/CDK2 interaction. We must therefore suspect that athird component not present in out assays mediates the interaction or that there is indeed nointeraction between Doc1 and CDK2 in vivo. Using a proteomics approach, we have notbeen able to identify a protein Doc1 ligand, but present preliminary evidence for a potentialDNA interaction. The structural characterisation of Doc1 has progressed to the initial stagesof NMR structure calculations. Combining all structural evidence available at this point, wepropose a structural model for Doc1. iii
  4. 4. To Prof Justus Henatsch (). iv
  5. 5. AcknowledgementsAbstractI would like to thank my supervisors Prof Jane Endicott, Dr Jim McDonnell and ProfMartin Noble for supervision and guidance throughout the four years of my DPhil.IntroductionMany people deserve to be mentioned here, and I will try my best to point out thespecial contribution that each individual person has made.MethodsFor precious NMR advice on many occasions, I would like to thank Dr ChristinaRedfield, Dr Jonathan Boyd, Mr Nick Soffe, Dr to-be Charlie Taylor and Dr IoannisVakonakis. Each of them has always been very generous with their help and support.Another important person to be mentioned here is Dr Emma Boswell. Emmasupervised me during my Diploma Thesis in 2003. Very patiently, she turned a crudechemist into a potential structural biologist. She taught me the daily business ofstructural biology (yes, it’s cloning and protein expression!) and I would have beenmuch less successful without such great supervision at the beginning of my scientificcareer.I would also like to thank the members of the LMB, in particular Dr Aude Echalierand 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 DrAnette Medhurst, both working in Dr Nick Lakin’s lab at the Oxford BiochemistryDepartment.ResultsI 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 trioof Harefield’s housemates to pick me up: Dr Tee, Dr Why and Dr Kay. Doctors, Iowe you a great deal… you mean a lot to me! Doctors Loco, Pea and π are also haveto be included in this round of thanks.Non-scientists (Claudilaudi, Lisa, my family, Katrin et al.) must have had a hard timecoping with endless discussions about mutants, clones, magnets and dead proteinsover the years. I thank you for not rejecting the geek-ness and for even showing realinterest in my work.Finally: Charlie (most special), Duc, Fiona, Franziska and Loukas deserve the mostgratitude 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 vonganzem Herzen – Ihr seid echt super! v
  6. 6. AbbreviationsAUC Analytical UltracentrifugationBSA Bovine serum albuminCDK Cyclin dependent kinaseC-terminal Carboxy terminalCyclinA Human CyclinADMSO DimethylsulfoxideDNA Deoxyribonucleic acidDoc1 Deleted in Oral CancerDSS 2,2-Dimethyl-2-silapentane-5-sulfonate sodium saltDTT Dithio-threitolE.coli Escherichia ColiEDTA Ethylene diamine tetra-acetic acidESI-MS Electrospray Ionization MSFPLC Fast protein liquid chromatographyGST Glutathione S-transferaseHBS HEPES-buffered salineHEPES N-[2-Hydroxyethyl]piperazine-N-[2- ethanesulphonic acid]HPLC High performance liquid chromatographyHSQC Heteronuclear Single Quantum CorrelationIPTG Iso-propyl–D-thiogalactosideITC Isothermal titration calorimetrykDa Kilo DaltonLB Luria Bertani BrothMALDI-TOF Matrix Assisted Laser ionisationMAPK Mitogen activated protein kinaseMDM2 Mouse Double Minute 2MTG Mono-thioglycerol vi
  7. 7. MS Mass SpectrometryNMR Nuclear Magnetic ResonanceNOE Nuclear Overhauser EffectN-terminal Amino terminalMS Mass spectrometryOD Optical densityPBS Phosphate buffered salinePCR Polymerase chain reactionPDB Protein data bankPEG Polyethylene glycolppm Parts per millionProzac Fluoxetine hydrochlorideRmsd Root mean square deviationRNA Ribonucleic acidrpm Revolutions per minuteSAR Structure-Activity-RelationshipsSDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresisSPR Surface Plasmon ResonanceSw Sweep widtht1dw t1dwell timeTFA Triflouroacetic acidTm Melting temperatureTricine N-tris[hydroxymethyl]methylglycineTris Tris[hydroxymethyl]aminomethaneUV Ultraviolet radiationWT Wild typeX. laevis Xenopus laevis vii
  8. 8. Foreword – Tumour Suppressors: Structure, Function and Drug DesignForeword Tumour Suppressors and Oncogenes: Structure, Function and Drug Design viii
  9. 9. Foreword – Tumour Suppressors: Structure, Function and Drug DesignCancer 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 greatneed for improvement in cancer therapies. This will require the development of newmethods, 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 promotecell growth. Therefore, it is essential for the understanding of cancer development toelucidate underlying mechanisms of cell growth and division. Tight regulation of theactivities of proteins that regulate cell proliferation, and the presence of pathways that detectdamage are essential to prevent cells from dividing uncontrollably, and to minimise the riskof genetic defects being passed on to the daughter cells. The loss of these growth controlmechanisms leads to uncontrolled cell proliferation, which is the main characteristic ofcancer.In cancer cells, it is often observed that negative regulators of the cell cycle (tumoursuppressors) are inhibited in their function, either through mutations or deregulation of theassociated 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-typeprotein or mutations that result in increased activity {Felsher, 2004 #85}. The aim of thisthesis is to further our understanding of the structural mechanisms underlying tumour-suppressor/oncogene interactions, with the view of correcting their imbalance throughrational structure-based drug development.One focus of my D.Phil. is the inhibition of the interaction between the tumour suppressorp53 and its antagonist MDM2 using small molecules. P53 is the cell’s major tumoursuppressor 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 beachieved through elevated levels of MDM2. In these cancer types, wild-type p53 fails to ix
  10. 10. Foreword – Tumour Suppressors: Structure, Function and Drug Designcarry out its function as a tumour suppressor, which is the essential step towards thedevelopment of malignant cells. Thus, inhibiting the MDM2/p53 interaction is a promisingtarget for cancer drug development in these cases, as releasing wild-type p53 would enablethe 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 theMDM2-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 noveltumour suppressor protein Deleted-in-Oral-Cancer 1 (Doc1), a potential CDK2 inhibitor.Cyclin-dependent kinases (CDKs) are the main coordinators of cell-cycle progression andare therefore often affected during cancer development. The name Deleted-in-Oral-Cancerresults from the failure to detect the Doc1 in human oral cancer cells. The fact that theprotein is absent in the examined tumour tissues indicates that it may play an important rolein the healthy cell and makes it a very interesting target for basic characterisation. The mainaims of this project were therefore to characterise the structural properties of Doc1, as wellas to study its interaction with CDK2. This project, if successful, will lead to a more detailedunderstanding of the causes of oral cancer in particular, and can hopefully serve as a startingpoint for the development of new treatments against this disease.As these two projects are independent of each other, this thesis has been divided into twomajor parts: The first one describes my studies of the MDM2-p53 interaction, the secondpart describes the structural characterisation of Doc1. x
  11. 11. Part I Inhibiting the MDM2-p53 Interaction
  12. 12. Table of Contents – Part I Table of Contents.............................................................................................................. 1 Table of Figures ................................................................................................................ 4 Table of Tables ................................................................................................................. 6 Table of Equations............................................................................................................ 7Section 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................................. 35Section 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 1
  13. 13. 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 2.2.4.3 Materials and Methods............................................................................. 52 2.2.4.4 Results....................................................................................................... 52 2.2.5 P53-Binding of MDM2 Surface Mutants........................................................ 55 2.2.5.1 Materials and Methods............................................................................. 55 2.2.5.2 Results....................................................................................................... 56 2.3 Generation of a Truncated MDM2 Point Mutant................................................ 59 2.3.1 MDM225-108 L33E ............................................................................................. 59 2.3.1.1 Materials and Methods............................................................................. 60 2.3.1.2 Results....................................................................................................... 63 2.4 Methylation of MDM2 ......................................................................................... 64 2.4.1 Reductive Methylation of Lysine Residues .................................................... 64 2.4.1.1 Materials and Methods............................................................................. 65 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.......................................................... 69Section 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 3.3.2.2 Materials and Methods............................................................................. 78 3.3.2.3 Results....................................................................................................... 79 3.3.3 Sample Preparation and Data Acquisition ...................................................... 80 3.3.4 Results ............................................................................................................... 80 2
  14. 14. 3.3.4.1 Backbone Assignments............................................................................ 80 3.3.4.2 Interpretation of p53-induced Chemical Shift Changes ........................ 843.4 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 ......................................................................... 1113.5 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 ............................................................... 1483.6 Summary of Results............................................................................................ 151 3
  15. 15. 3.7 Conclusions ......................................................................................................... 154 3.8 Future Work......................................................................................................... 158Section 4............................................................................................................... 161 Final Discussion............................................................................................................ 161Section 5............................................................................................................... 163 Bibliography.................................................................................................................. 163Table of FiguresFigure 1-1. Cellular responses induced by p53 as a result of cytotoxic stress.. ...................... 12Figure 1-2. The MDM2-p53 auto-regulatory feedback loop.................................................... 17Figure 1-3. Schematic representation of the domain organisation of p53. .............................. 18Figure 1-4. Schematic representation of the domain organisation of MDM2......................... 19Figure 1-5. Pseudo-symmetrical arrangement of MDM2’s N-terminal domain, forming a hydrophobic pocket at their interface................................................................................ 21Figure 1-6. The NMR structure of MDM2’s N-terminal domain (1Z1M). ............................. 22Figure 1-7. Crystal structure of a p53 peptide bound to MDM2 at 2.6Å resolution (1YCR). 23Figure 1-8. Triad of p53 amino acids forming the main contacts to MDM2. ......................... 24Figure 1-9. Inhibitory peptides of the MDM2-p53 interaction ................................................ 26Figure 1-10. Crystal structure of peptide 8 (an 8-mer p53 peptide analogue) bound to MDM2 ............................................................................................................................................. 28Figure 1-11. Natural inhibitors of the MDM2-p53 interaction. ............................................... 29Figure 1-12. Small-molecule inhibitors of the MDM2-p53 interaction. ................................. 31Figure 1-13. Inhibitors of the Nutlin-series.. ............................................................................. 32Figure 1-14. Binding modes of Nutlin-inhibitors atomic resolution........................................ 33Figure 1-15. Benzodiazepine-inhibitors of the MDM2-p53 interaction. ................................. 34Figure 1-16. Benzodiadepinedione inhibitor in complex with MDM2. .................................. 35Figure 1-17. Initial Isoindolinone inhibitors of the MDM2-p53 interaction. .......................... 36Figure 1-18. SAR of the Isoindolinone Series........................................................................... 37Figure 2-1. Crystal contacts from MDM2 crystal structure 1YCR, 1T4E and 1RV1. ........... 40Figure 2-2. Clusters of Glutamate-Lysine pairs within MDM217-125 amenable to surface engineering by site-directed mutagenesis. ........................................................................ 42Figure 2-3. PCR 1 and 2 of Gene Synthesis. ............................................................................. 43Figure 2-4. Assembly PCR products for gene assembly PCRs 1 and 2................................... 47 4
  16. 16. Figure 2-5. Example of an expression trial of MDM2 K94E95A surface mutant produced by gene synthesis.. ................................................................................................................... 48Figure 2-6: Overlap Extension PCR........................................................................................... 50Figure 2-7. Example of overlap extension PCRs to generate MDM2 surface mutants. ......... 53Figure 2-8. Preparative scale S200 gel filtration profiles of MDM2 surface mutants. ........... 54Figure 2-9. 1 H/15N-HSQC spectra of MDM2 surface mutants and WT. ................................. 56Figure 2-10. 1 H/15N-HSQC spectra of MDM2 surface mutants and WT bound to the p53- peptide................................................................................................................................. 57Figure 2-11. Crystal structure of the MDM2 L33E mutant (1RV1). ....................................... 59Figure 2-12. Introducing the L33E mutation into pGEX6P1. .................................................. 60Figure 2-13. Expression of MDM225-108-L33E. ......................................................................... 63Figure 2-14. Gel filtration profile of 1mg WT and methylated MDM2 on an S75 K16 column................................................................................................................................. 66Figure 2-15. Ensemble of 20 NMR structures of apo-NMR (1Z1M)...................................... 69Figure 3-1. Expression and purification of MDM217-125. .......................................................... 76Figure 3-2. Previous backbone assignments of MDM2 and application to the MDM217-125 construct.............................................................................................................................. 77Figure 3-3. Backbone assignment of apo- and holo-MDM2.................................................... 82Figure 3-4. Assigned 1 H/15N HSQC of apo-MDM2. ................................................................ 83Figure 3-5. Assigned 1 H/15N HSQC of holo-MDM2 bound to a p53-peptide. ....................... 84Figure 3-6. Chemical shift changes induced by p53. ................................................................ 86Figure 3-7. Chemical shift changes induced by p53 mapped onto the structure of MDM2 (1YCR)................................................................................................................................ 87Figure 3-8. Overlay of the apo and holo-structure of MDM2 (1Z1M and 1YCR)................. 89Figure 3-9. The 1H/15N HSQC pulse sequence.......................................................................... 92Figure 3-10. Exchange phenomena in NMR spectroscopy. ..................................................... 94Figure 3-11. Residues excluded from analysis, highlighted in blue. ....................................... 99Figure 3-12. Isoindolinone-induced chemical shift changes in MDM217-125......................... 101Figure 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....................... 103Figure 3-14. Average per-residue chemical shift change for all Isoindolinones tested plotted onto structure of MDM217-125 (1YCR). ........................................................................... 104Figure 3-15. Degree of line broadening / intermediate exchange per residue caused by Isoindolinone binding. ..................................................................................................... 106Figure 3-16. Movement of K93 and H96 side-chain upon ligand binding to MDM2. ......... 107 5
  17. 17. Figure 3-17. Difference in Isoindolinone and p53 induced chemical shift changes. ............ 110Figure 3-18. Original titration spectra and chemical shift changes mapped onto MDM2’s primary structure for Nutlin-3.. ....................................................................................... 111Figure 3-19. Chemical shift changes induced by Nutlin-3. .................................................... 113Figure 3-20. Difference in chemical shift changes induced by Nutlin-3 and Isoindolinones ........................................................................................................................................... 115Figure 3-21. Structural relationships between the Isoindolinone inhibitors tested in this thesis.................................................................................................................................. 123Figure 3-22. Chemical shift perturbations induced by Isoindolinone inhibitors mapped onto the MDM2 structure (1YCR). ......................................................................................... 125Figure 3-23. Magnitude of chemical shift change for inhibitors 8247 and 8248. ................. 125Figure 3-24. “Per-residue” plots for amino acids V75, L82, K93 and L103......................... 129Figure 3-25. Comparison of chemical shift perturbations by all inhibitors on each residue of MDM2’s hydrophobic pocket. ........................................................................................ 131Figure 3-26. Comparison of chemical shift change within group C of isodindolinone inhibitors. .......................................................................................................................... 133Figure 3-27. Per-residue plot for residue Y100 of MDM2..................................................... 134Figure 3-28. Possible orientations of Isoindolinones in MDM2’s hydrophobic pocket....... 135Figure 3-29. Selection of a structure for docking of Isoindolinones...................................... 138Figure 3-30. Nomenclature of Isoindolinone enantiomers using inhibitor 8231 as the example. ............................................................................................................................ 141Figure 3-31. Possible binding modes derived from the NMR data and manual docking..... 143Figure 3-32. Binding models of Isoindolinone inhibitors derived from NMR data and molecular docking with the program GOLD.................................................................. 150Figure 3-33. Structural relationship of Isoindolinone inhibitors............................................ 152Figure 3-34. Summary of Isoindolinone binding models derived from chemical shift data and molecular docking. ........................................................................................................... 154Figure 3-35. Different inhibitors bound to MDM2. ................................................................ 155Figure 3-36. Binding model for inhibitors of group B obtained from manual docking.. ..... 156Figure 3-37. SAR of Isoindolinones similar to group B. ........................................................ 158Table of TablesTable 2-1. Primers for gene synthesis of the MDM2 gene.. ..................................................... 45Table 2-2. Primer combination for gene synthesis.................................................................... 45 6
  18. 18. Table 2-3. Primers for side-directed mutagenesis. .................................................................... 49Table 2-4. Primers for overlap-extension PCR. . ...................................................................... 52Table 2-5. Primers for the MDM225-108-L33E mutant............................................................... 61Table 2-6. ESI-MS results for methylated versus WT MDM2. ............................................... 65Table 2-7. Summary of all MDM2 constructs and crystallisation conditions......................... 67Table 3-1. Dialysis test to improve MDM2 stability. ............................................................... 79Table 3-2. Inhibitors used for NMR titrations........................................................................... 97Table 3-3. Average chemical shift changes observed in inhibitor titrations. ........................ 102Table 3-4. Criteria for the analysis of magnitude of chemical shift....................................... 123Table 3-5. Analysis of per-residue plots. ................................................................................. 127Table 3-6. Structural restrains obtained from the NMR data.. ............................................... 136Table 3-7. Selection of MDM2 Structures available in the protein data bank. ..................... 136Table 3-8. Docking results from manual docking. .................................................................. 142Table 3-9. Comparison of GOLD docking results to manual docking results. ..................... 148Table of EquationsEquation 2-1. Reductive methylation of amides. ...................................................................... 64Equation 3-1. Calculation of chemical shift differences........................................................... 99Equation 3-2. Calculation of combined chemical shifts ........................................................... 99Equation 3-3. Definition of chemical shift. ............................................................................. 116 7
  19. 19. Part I: Section 1 - Introduction to the MDM2-p53 InteractionSection 1 Introduction to the MDM2-p53 Interaction 8
  20. 20. Part I: Section 1 - Introduction to the MDM2-p53 Interaction1.1 The MDM2-p53 Interaction is an Important Target in Drug DesignThe transcription factor p53 is the cell’s major tumor suppressor (Levine 1997) and found tobe mutated in more than 50% of human cancers (Hainaut and Hollstein 2000). Targetingtumour cells with altered p53 is therefore an important strategy in drug discovery, both inacademia and industry (Blagosklonny 2002). Since overcoming p53 function is one of themajor steps in carcinogenesis, cancer cells with wild-type p53 have found other means ofinhibiting its activity. In many cancer cells, disruption of p53 function is achieved throughover-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 majoradvantage for therapeutic intervention: once p53 inhibition is alleviated, its tumoursuppressor activity is unleashed, triggering apoptosis in such cells. In fact, three very recentpublications have stated that restoration of p53 function alone is sufficient to cause tumourregression (Martins, Brown-Swigart et al. 2006; Ventura, Kirsch et al. 2007; Xue, Zender etal. 2007). Disruption of the MDM2-p53 interaction in MDM2-amplified cells with WT-p53has therefore become an important target in drug discovery. The molecular details ofMDM2-p53 binding present one of the few cases in which protein-protein interactions areamenable to inhibition by small molecules (Kussie, Gorina et al. 1996).During the course of this thesis, I have studied Isoindolinone-inhibitors of the MDM2-p53interaction, gathering structural information about Isoindolinone binding to MDM2 bycrystallography and NMR. These structural insights were required to provide the basis forfurther development of compounds with increased specificity and affinity, with the ultimateaim of generating more suitable candidates for clinical trials. 9
  21. 21. Part I: Section 1 - Introduction to the MDM2-p53 Interaction1.2 The p53 Tumour SuppressorActivation of the p53 protein protects the organism against the propagation of cells that carrydamaged DNA with potentially oncogenic mutations. Because of its essential function ingrowth control, the p53 tumour suppressor is sometimes termed the “Guardian of theGenome” (Lane 1992). Currently, over 19,000 publications discussing p53 are available onPubmed; a result of nearly 30 years of dedicated research into the subject. Unfortunately, itwill be impossible to cover such a large body of work in this context. Instead, this sectionwill give a brief general overview about p53 function, and then focus on the aspects of theMDM2-p53 interaction relevant to this work.1.2.1 The History of p53 DiscoveryEven though p53 was first discovered in 1979, it took another 10 years for its true function tobe revealed. P53 was first identified in complex with the SV40 tumour antigen, a proteinencoded by the SV40 DNA virus that causes malignant transformation in mice (Lane andCrawford 1979), (Linzer and Levine 1979). Subsequently, the p53 gene product was reportedto cause immortalisation upon over-expression and transformation in conjunction with theRas 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 cDNAused 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 aresult 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 ofp53 function might offer an advantage in tumour development. In 1989, WT-p53 was shownto prevent transformation (Finlay, Hinds et al. 1989), leading the way for a reinterpretationof earlier results and subsequent classification of p53 as a tumour suppressor. 10
  22. 22. Part I: Section 1 - Introduction to the MDM2-p53 InteractionSoon after, p53 was found to activate transcription (Fields and Jang 1990), (Raycroft, Wu etal. 1990), and was identified as a sequence-specific DNA-binding protein (Kern, Kinzler etal. 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 frequentlyinactivated gene in human cancers (Vousden and Lu 2002).1.2.2 The p53 NetworkP53 has so far been best understood in its function as a tumour suppressor, responding tosignals of DNA damage and aberrant growth, initiating DNA repair, cell cycle arrest and, ifnecessary, apoptosis. These functions are mainly provided through p53’s activity as atranscription 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 itsinvolvement in cancer prevention (Vousden and Lane 2007), suggesting an importantcontribution of p53 to other aspects of cellular life. The various pathways of p53 control andfunction 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 ofp53 activation and the resulting down-stream events. 11
  23. 23. Part I: Section 1 - Introduction to the MDM2-p53 InteractionFigure 1-1. Cellular responses induced by p53 as a result of cytotoxic stress. This figure has beenextended 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 throughdifferent pathways. P53 activation occurs according to three major principles: Activation of p53through post-translational modifications, sub-cellular localisation of the protein and stabilisation ofp53 due to reduced proteasomal degradation. Depending on the type of stress, the activation pathwayand the cell type, p53 then initiates the cellular response (yellow). Initiation of cell cycle arrest, DNArepair and apoptosis represent its most classical functions providing tumour suppression. Otherfunctions, such as maintenance of genomic stability, inhibition of angiogenesis, involvement insenescence and cell survival are also part of the repertoire of p53-induced down-stream events. 12
  24. 24. Part I: Section 1 - Introduction to the MDM2-p53 Interaction1.2.2.1 P53 ActivationDue to the potentially suicidal response that can be triggered by p53, a low cellularconcentration is maintained during normal cell development. Tight regulation of p53 levelsis achieved in three different ways addressing the protein’s activity, stability and sub-cellularlocalisation. P53’s activity can be controlled through various post-translational modificationssuch as phosphorylation, acetylation or glycosylation. The stability of p53 levels is mainlydependent on the rate of ubiquitin-mediated degradation, while localisation of p53 indifferent sub-sections of the cell can be regulated through shuttling the transcription factor inand 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). IfDNA damage is caused by ionising radiation, then the central signalling protein in DNAdamage control, the kinase ATM (Ataxia telangiectasia mutated) activates p53 either throughdirect 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 andRad3 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 ofMDM2. The function of p14/p19ARF is described further in Section 1.3.1.2.2.2 P53 FunctionKeeping in mind additional functions mentioned in Figure 1-1, the major effects of p53activation are the initiation of cell cycle arrest, DNA repair and apoptosis, accomplishedthrough regulation of DNA transcription. One important transcriptional target of p53 is the 13
  25. 25. Part I: Section 1 - Introduction to the MDM2-p53 Interactioncyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1, which serves to prevent entry from G1to S phase, as well as G2 to mitosis transitions. In terms of triggering apoptosis, the proteinBax and the tumour necrosis factor (TNF) are amongst the most important proteins activatedthrough the transcription factor p53. For a more complete discussion of transcriptionalregulation 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 themselvesmodulate the activity of p53. One of these genes is MDM2, p53’s main antagonist, which isable to return p53 levels back to normality at the end of a successful stress response.1.3 The Oncogene MDM2The Murine Double Minute Clone 2 (MDM2) is the main antagonist of p53, and was firstidentified in 1987 as one of three genes amplified in a spontaneously transformed mouse cellline (3T3DM) (Cahilly-Snyder, Yang-Feng et al. 1987). Amplification of the MDM2 gene inrodent cells was shown to result in high tumourigenic potential, suggesting MDM2 is anoncogene (Fakharzadeh, Trusko et al. 1991). A 90kDa protein found to co-immunoprecipitate with the p53 tumour suppressor was later identified as the MDM2 geneproduct 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 theMDM2 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 moietyonto 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 foundin several viral proteins (Roth, Dobbelstein et al. 1998). By shuttling in an out of the nucleus, 14
  26. 26. Part I: Section 1 - Introduction to the MDM2-p53 InteractionMDM2 transports p53 to the cytoplasm where ubiquitin-mediated degradation can occur andwhere 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 untransformedcells is the regulation of p53. MDM2-/- mice are not viable and die within the first days ofgestation (Jones, Roe et al. 1995). Deletion of the p53 gene in addition to MDM2 provides arescue from cell death (Montes de Oca Luna, Wagner et al. 1995), indicating that anincreased level of p53 in the absence of MDM2 drives the cells into apoptosis. p53-- micedevelop 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 ofother proteins, which either regulate MDM2 activity in upstream events, or are affected byMDM2 in downstream events. These interactions are beyond the scope of this thesis; so onlyone other MDM2-binding protein in addition to p53 will be mentioned here. This protein isthe p14/p19ARF tumour suppressor, which was one of the first proteins shown to interact withMDM2 (Quelle, Zindy et al. 1995; Zhang, Xiong et al. 1998). P14/p19ARF is expressed fromthe INK4a locus in an alternate reading frame (Quelle, Zindy et al. 1995) and indirectlyactivates p53 in response to oncogene activation by inhibiting MDM2’s ubiquitin ligaseactivity (Silva, Silva et al. 2003). Furthermore, p14/p19ARF blocks MDM2-dependent nuclearexport 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 MDM2MDM2 and p53 are mutually regulated through an auto-regulatory feedback-loop (Picksleyand Lane 1993; Wu, Bayle et al. 1993), first described in 1993. In normal cells, the 15
  27. 27. Part I: Section 1 - Introduction to the MDM2-p53 Interactionconcentration 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 andThreonine residues (Oren, Maltzman et al. 1981; Reich, Oren et al. 1983; Ashcroft, Taya etal. 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/p19ARFpathway, releasing MDM2’s tight control of p53 function. Elevated levels of p53 promotetranscription of MDM2 (Barak, Juven et al. 1993) and the MDM2 protein inhibits p53activity 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
  28. 28. Part I: Section 1 - Introduction to the MDM2-p53 InteractionFigure 1-2. The MDM2-p53 auto-regulatory feedback loop. In response to cytotoxic stress, p53 isactivated and transported to the nucleus, where it acts as a transcription factor of genes implicated incell cycle control. MDM2 is one of the genes controlled by p53 and is expressed upon activation ofp53. MDM2 inhibits p53 function by (1.) blocking p53’s transcriptional activity through directbinding 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 inhibitthe 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 awindow for p53-activity, but also offer the possibility of aborting the p53-response aftersuccessful recovery from DNA damage.In tumour cells where increased activity of MDM2 creates an imbalance in the complexequilibrium between MDM2 and p53, a compound designed to bind MDM2 in order to 17
  29. 29. Part I: Section 1 - Introduction to the MDM2-p53 Interactiondissociate p53 could rescue p53 function and induce cell cycle arrest or apoptosis in theaffected cells.1.5 Structural Details of MDM2 and p53The sites of interaction between MDM2 and p53 were first identified in 1993 by yeast-twohybrid 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. Thefollowing section is giving a brief overview of the overall domain organisation of MDM2and p53, in order to illustrate the context in which the two interacting N-termini exist in vivo.1.5.1 Overall Structure of p53P53 is a tetrameric 393 amino acid protein (Stenger, Mayr et al. 1992), composed of fivemajor 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 circulardiochroism studies of a fragment comprising the transactivation domain and the adjacentProline rich region showed that this region is natively unfolded, lacking tertiary andsecondary structure elements (Dawson, Muller et al. 2003). However, upon binding toMDM2, the transactivation domain adopts a helical conformation (Kussie, Gorina et al. 18
  30. 30. Part I: Section 1 - Introduction to the MDM2-p53 Interaction1996). The DNA binding domain comprises residues 102-292 (Cho, Gorina et al. 1994) andis 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 atetramer in vivo. The NMR structure of this domain has been deposited under the PDB code2J0Z. The C-terminal domain (residues 367-393) is capable of binding to single-strandedDNA and RNA (Figure 1-3) (Lee, Elenbaas et al. 1995).1.5.2 Overall Structure of MDM2MDM2 is a 491 amino acid protein composed of several functional domains interlinked byless structured regions, not all of which are well characterised in terms of their structure andfunction.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 bynuclear 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 andX-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 theremainder of this introduction. The central region of the protein, often referred to as theacidic domain (residues 222-272), is rich in negatively charged residues. This region is 19
  31. 31. Part I: Section 1 - Introduction to the MDM2-p53 Interactionfollowed by a Zinc finger domain (residues 290-335), for which the NMR structure has beendetermined (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. TheNMR structure of this domain was revealed by NMR spectroscopy (2HDP) (Kostic, Matt etal. 2006).1.5.3 The MDM2 N-terminal DomainThe first structure of MDM2’s p53-binding domain was solved by X-ray crystallography in1996, in complex with a 15-residue p53 peptide (Kussie, Gorina et al. 1996). In addition tothe details of MDM2-p53 binding (described in Section 1.5.4), this structure revealed thatMDM2’s N-terminal section is composed of two major repeats of secondary structure. Theserepeats are arranged pseudo-symmetrically along an approximate dyad axis, as shown inFigure 1-5a. The interface of the repeats on either side of the symmetry axis is lined withhydrophobic residues that form a deep cleft at the surface of MDM2 (Figure 1-5b), to whichthe 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 andwill be applied throughout the remainder of this thesis.The hydrophobic pocket arising at the interface of the two repeats is composed of thefollowing structural elements: The bottom of the cleft is composed of two helices, α1 andα1’. Helices α2 and α2’ form the sides of the pocket, and two β-sheets seal the ends at eitherside of the pocket. Even though the molecule is built from symmetrically arranged elementsof secondary structure, the cleft itself is asymmetrical, as a result of different lengths of thehelices forming the walls of the pocket. 20
  32. 32. Part I: Section 1 - Introduction to the MDM2-p53 InteractionFigure 1-5. Pseudo-symmetrical arrangement of MDM2’s N-terminal domain, forming ahydrophobic pocket at their interface. Elements of secondary structure have been labelledaccording 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) Schematicrepresentation of the secondary structure highlighting the axis of symmetry. (b) Three-dimensionalstructure highlighting the hydrophobic pocket formed at the interface of the two repeats (grey). 21
  33. 33. Part I: Section 1 - Introduction to the MDM2-p53 InteractionIn 2005, Uhrinova et al. solved the NMR structure of the MDM2 N-terminal in the absenceof a ligand (Uhrinova, Uhrin et al. 2005). This structure revealed that the p53-bindingdomain is less structured in the holo-state, consisting of shorter elements of secondarystructure than observed for the p53-bound form. The decrease in structural definition wasalso reflected in the restraint-density, as the structure is based on only 8 inter-residue NOEsper residue. Furthermore, the heteronuclear NOE ratio (for more information, see Part II ofthis thesis) for the well-structured residues was relatively low (0.7±0.1), indicating that eventhe structured parts of the protein show increased flexibility. Overall, the decreased structuraldefinition of the apo-domain, as well as problems due to protein instability, signal overlapand 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 theelements of secondary structure is identical to Figure 1-5. N and C-termini of MDM2 were found tobe unstructured.When comparing p53-bound MDM2 to the holo-structure (Figures 1-5b and 1-6), it becomesevident 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 ofMDM2 forms a “flexible lid” folding over the hydrophobic pocket in the absence of a ligand 22
  34. 34. Part I: Section 1 - Introduction to the MDM2-p53 Interaction(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-terminaltail of MDM2 and the hydrophobic cleft (Uhrinova, Uhrin et al. 2005).1.6 The MDM2-p53 InteractionThe molecular details of p53-binding to MDM2 were revealed in the crystal structure byKussie et al. (Kussie, Gorina et al. 1996), which formed the basis for structure-based designof MDM2-antagonists. Upon binding to MDM2, the p53-peptide forms an amphipathic helixhighly complementary to the hydrophobic pocket at the surface of MDM2. This helixextends 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 highlyconserved p53 residues, namely Phe19, Trp23 and Leu26, which insert deeply into the 23
  35. 35. Part I: Section 1 - Introduction to the MDM2-p53 Interactionhydrophobic pocket of MDM2 (Figure 1-8). These three residues occupy three sub-pocketsat 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 Section3.5.4. 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 formedbetween the NH-group of the Trp23-sidechain of p53 and the carbonyl group of Leu54 ofMDM2. The second hydrogen bond is formed between the backbone amide of p53’s Phe19and 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 thethree hydrophobic acids Phe19, Trp23 and Leu26 to the hydrophobic pocket of MDM2. (b) Thetryptophan-side chain of p53 is inserted into the deepest sub-pocket of MDM2. Stars indicate thelocation of the two hydrogen bonds contributing to the interaction.The importance of the three side-chains of residues Phe19, Trp23 and Leu26 as maincontributors 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 affinitycompared to the WT-peptide (Sakurai, Chung et al. 2004). This supports the hypothesis thatbinding is merely driven by the side-chain contacts, and the backbone and helix of the p53peptide 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 mostcontacts 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
  36. 36. Part I: Section 1 - Introduction to the MDM2-p53 Interactionthe main contacts to MDM2 have a molecular weight of approximately 300 Daltons, whichrenders this protein-protein interaction one of the few targets amenable to inhibition bysmall-molecules.1.7 Inhibitors of the MDM2-p53 InteractionSince the publication of the crystal structure of the MDM2-p53 complex (Kussie, Gorina etal. 1996), many resources have been invested in the development of a pharmacophore modelfor this protein-protein interaction. A pharmacophore is a three-dimensional substructure of amolecule that carries the essential features responsible for a drug’s biological activity. It doesnot represent a real molecule or a real association of functional groups, but a purely abstractconcept that accounts for the common molecular interaction capacities of a group ofcompounds towards their target structure. Detailed exploration of the 1YCR crystal structurewas an obvious starting point for the development of a pharmacophore:Comparing the binding epitopes of MDM2 and p53, MDM2 presents a more well-definedbinding site, to which the p53 peptide, normally unstructured in solution, binds in acomplementary shape. Inhibitors should aim to mimic the less structured binding partner, inthis case the p53-peptide. Since the interaction is mainly hydrophobic, MDM2-antagonistshave to be lipophilic. This is favourable, on the one hand, since burial of lipophilic groups inthe binding pocket is accompanied by partial desolvation of the inhibitor, which makes thebinding-event entropically favourable. On the other hand, hydrophobicity is accompanied bya decrease in bioavailability of the inhibitor through decreased solubility. Finally, it isdesirable 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 theUniversity of Dundee engaged in the development of a more detailed pharmacophore model, 25
  37. 37. Part I: Section 1 - Introduction to the MDM2-p53 Interactiondetermining amino acid preferences of MDM2’s hydrophobic pocket. This work resulted inthe 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), muchmore 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 InhibitorsIn the year 2000, the Novartis group published the most potent MDM2 inhibitor to date, an8-residue p53-peptide analogue, named peptide 8 (Figure 1-9) (Garcia-Echeverria, Chene etal. 2000). This peptide was developed from a phage display peptide library (Bottger, Bottgeret al. 1996), based on synthetic peptides derived from p53’s MDM2-binding region(Picksley, Vojtesek et al. 1994), and subsequent structure-based design. Phage displayresulted in the identification of peptide 2, displaying 28-fold increased potency compared tothe 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 residuesforming the main contacts with MDM2 are indicated in bold. Residues represented in blue arederivatives of natural amino acids: α-aminoisobutyric acid (Aib), phosphonomethylphenylalanine(Pmp), 6-chlorotryptophane (6-Cl-Trp) and 1-amino-cyclopropanecarboxylic acid (Ac3C). IC50 valuesare stated as nanomolar concentrations.Assuming that it is entropically favourable for the peptide to adopt a helical conformationprior to MDM2-binding, non-natural amino acids which are known to stabilise helices, suchas α-aminoisobutyric acid and 1-amino-cyclopropanecarboxylic acid (Figure 1-9), were 26
  38. 38. Part I: Section 1 - Introduction to the MDM2-p53 Interactionintroduced, resulting in a further increase of affinity. In the next step, attempts to improvebinding of the Tyr22 and Trp23 side-chains were made. Tyr22 was replaced byphosphonomethylphenylalanine (Figure 1-9), in order to allow salt-bridge formation betweenthis 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 achloro-moiety at position 6 of the Trp23 side-chain. The chloro-substituent is able to exploitthe Trp23-pocket of MDM2 more fully, resulting in a 60-fold increase in affinity. In total, a1700-fold increase in binding capacity compared to the initial WT-peptide was achieved bythis 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. Afteraddition of full-length p53, the amount of full-length p53 bound to MDM2 in the presence ofthe inhibitory peptide was determined by Western Blotting (Garcia-Echeverria, Chene et al.2000). By using different concentrations of inhibitors, the concentration of inhibitor requiredto replace 50% of the reporter ligand, in this case full-length p53, can be determined. Thisvalue is called the median inhibitory concentration, or IC50. It is a useful parameter tocompare 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 madeby the three conserved residues, Phe19, Trp23 and Leu26 (Figure 1-8), but that peptide 8 showsincreased steric complementarity compared to the WT peptide. The chloro-substituent of theTrp23-sidechain is able to explore a void in the deep pocket of MDM2, making additionalcontacts 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 insteadprojecting into the solvent. 27
  39. 39. Part I: Section 1 - Introduction to the MDM2-p53 InteractionFigure 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 Leu26side-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 shownin grey. Lower panel: On the left, the Trp23 side-chain of the WT peptide is shown in the sameorientation 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
  40. 40. Part I: Section 1 - Introduction to the MDM2-p53 Interaction1.7.2 Natural InhibitorsIn 2001, the first natural inhibitors of the MDM2-p53 interaction were published (Stoll,Renner et al. 2001). These inhibitors were chalcone derivatives, compounds derived from1,3-diphenyl-2-propen-1-one, and had previously been known as substances with tumoursuppressing activity (Dore and Viel 1974). Now, these compounds could be identified asMDM2 antagonists, binding in the same location as p53, as established by chemical shiftmapping (Stoll, Renner et al. 2001). It is assumed that the dicholorophenyl-moiety resides inthe Trp23-pocket of MDM2. The IC50’s of these naturally occurring compounds lay in themicromolar range, with the potent compound (compound B) having an IC50 of approximately50-70µM (Figure 1-11).Figure 1-11. Natural inhibitors of the MDM2-p53 interaction. 29
  41. 41. Part I: Section 1 - Introduction to the MDM2-p53 InteractionIn the same year, Chlorofusin, a natural substance identified from over 53000 microbialextracts, was identified as an MDM2-antagonist (Duncan, Gruschow et al. 2001).Chlorofusin is a fungal metabolite, inhibiting the MDM2-p53 interaction with an IC50 of4.6µM (Figure 1-11). To date, there is no information about the binding-mode ofChlorofusin.In 2006, a third natural inhibitor was reported (Tsukamoto, Yoshida et al. 2006). Thecompound 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 of50µM.1.7.3 Small Molecule InhibitorsThe first non-natural MDM2-antagonist, named Syc (non-peptidic small-moleculesynthesised compound), was reported in 2002 (Zhao, Wang et al. 2002). Followingcomputer-aided design and subsequent synthesis of 23 compounds, five compounds werefound to exert an inhibitory effect on the MDM2-p53 interaction in cellular assays. The mainscaffold of these compounds is bicyclic with two varying aromatic substituents, hoped tomimic 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 asa 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 compoundswith an oxindole ring, in order to mimic the Trp23 side-chain of p53. The compoundspiro(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
  42. 42. Part I: Section 1 - Introduction to the MDM2-p53 Interactiongroups 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 sameassay (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
  43. 43. Part I: Section 1 - Introduction to the MDM2-p53 Interaction1.7.4 Nutlin-InhibitorsThe 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 ofsynthetic chemicals (Vassilev, Vu et al. 2004). The compounds were synthesised as racemicmixtures 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 were260nM, 140nM and 90nM for Nutlin-1, Nutlin-2 and Nutlin-3, respectively. These values weredetermined by SPR, in a competition assay measuring the binding of MDM2 in the presence ofinhibitors to an immobilised p53 peptide (Vassilev, Vu et al. 2004).In order to investigate the molecular details of Nutlin-binding to MDM2, the crystal structureof 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 humanisedXenopus MDM2 was also published (1TTV) (Fry, Emerson et al. 2004). Both structuresshow 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
  44. 44. Part I: Section 1 - Introduction to the MDM2-p53 Interactionin to the Trp23-pocket of MDM2, while the other one projects into the sub-pocketaccommodating the Leu26-sidechain of p53. Both halogeno-phenyl groups are held inposition 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 thePhe19 side-chain of p53. The N-piperazinyl moiety points into the solvent. Its position couldnot 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 XenopusMDM2 (1TTV). Note that the N-piperazinyl moiety of the inhibitor is not displayed due to a lack ofNOEs 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 potentcompounds 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 InhibitorsIn 2005, a second high-resolution inhibitor-MDM2 structure was reported (Grasberger, Lu etal. 2005). A Benzodiazepinedione antagonist of MDM2 was identified through screening ofover 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
  45. 45. Part I: Section 1 - Introduction to the MDM2-p53 Interactionpotential inhibitor of the MDM2-p53 interaction. In 2001, a benzodiazepine-2-one had beenidentified by Majeux et al., as part of the establishment of a computational method toevaluate 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 afluorescent peptide displacement assay. This assay was able to show thatBenzodiazeponediones 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 crystalstructure (PDB code 1TAE). The two para-chlorophenyl moieties point into the Trp23 andLeu26 sub-pockets, held in place by the benzodiazepine heterocycle (Figure 1-16). A similarorientation 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
  46. 46. Part I: Section 1 - Introduction to the MDM2-p53 Interactionhydrophobic 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 theinhibitor. (b) The N-terminus of MDM2 (residues 17-25, shown in blue) folds over the binding-siteand extends the Leu26 sub-pocket accommodating the para-chlorobenzoyl-moiety.Due to the close superposition of elements of the Benzodiazepinediole-substituents with thethree crucial side-chains of p53, it was suggested that the Benzodiazepinediole-scaffoldcould 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 InteractionThis thesis focuses on the elucidation of the binding modes of Isoindolinone inhibitors of theMDM2-p53 interaction, developed by our collaborators at the Northern Institute for CancerResearch at the University of Newcastle upon Tyne. Hardcastle et al. first reported inhibitorswith 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 ofapproximately 200µM (Figure 1-17). These compounds were further developed in a programof focused library synthesis guided by virtual screening. Initial development was based on 35
  47. 47. Part I: Section 1 - Introduction to the MDM2-p53 Interactionbinding modes predicted by easyDock (Mancera, Kallblad et al. 2004) followed by virtualscreening, 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 bindingmodes obtained by prediction with easyDock and GOLD (Verdonk, Cole et al. 2003) wereused as “seeds” for further virtual screening (Hardcastle, Ahmed et al. 2006). In thisapproach, the position of the Isoindolinone-scaffold was preserved during simulatedannealing with the program Skelgen (Stahl, Todorov et al. 2002). From the compoundsgenerated in this way, 57 were selected as “virtual hits” and 43 of these were synthesised andtested for their activity by an ELISA competition assay. Amongst this generation ofcompounds, five inhibitors displayed a significant loss in activity, and only two were moreactive than the most potent seed compound.In the light of these results, combinatorial chemistry was chosen as an approach for furtheroptimisation of compounds. The most favourable substituents of the Isoindolinone scaffoldwere selected based on initial SAR, and an array of 36 new compounds covering everypossible combination of substituents was designed. 24 of these compounds were thensynthesised and assayed. SAR results for this inhibitor generation were contradictory: Forexample, the introduction of a 4-chloro-substituent at the phenyl-moiety of two compoundsresulted in both an increase and decrease in potency (Figure 1-18). This result suggested thatdifferent binding modes for this class of inhibitors are possible and are induced by subtledifferences in the chemical structure. 36
  48. 48. Part I: Section 1 - Introduction to the MDM2-p53 InteractionFigure 1-18. SAR of the Isoindolinone Series. The introduction of a 4-chloro-substituent at thephenyl-moiety of the Isoindolinone causes contradictory SAR results. NU8231 was the most potentIsoindolinone at the time.The most potent compound at this time (NU8231, Figure 1-18) had an IC50 of 5µM anddisplayed promising biological activity. In SJSA cells with amplified MDM2, transcriptionof p21WAF1/CIP1, a CDK/cyclin inhibitor under transcriptional control of p53, as well asMDM2 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 beincreased (Hardcastle, Ahmed et al. 2006).At this point in time, three different strategies to generate more potent inhibitor generationshad not resulted in a significant increase in affinity. The availability of a high-resolutionstructure of Isoindolinone-binding to MDM2 was therefore thought to provide a majoradvance to assist the design of more potent inhibitor generations. My work seeks to providethese insights, with the hope of enabling the design of more potent candidates suitable forclinical trials. 37
  49. 49. Part I: Section 2 - Towards a Crystallisable Form of MDM2Section 2 Towards a Crystallisable Form of MDM2 38
  50. 50. Part I: Section 2 - Towards a Crystallisable Form of MDM22.1 Crystallising MDM2Protein crystallisation still remains the rate-limiting step in the determination of X-raystructures. For proteins that are “stubborn crystallisers”, the formation of crystals is oftenless likely due to intrinsic flexibility or unstructured sections within the protein of interest, ordue to surface patches of flexible, solvent-exposed amino acids of lower conformationalentropy. 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 domainis 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. Themobility of MDM2 in the absence of a ligand might explain why no crystallisationconditions have so far been established that allow formation of apo-crystals amenable tosoaking of a small molecule ligand into the crystal.Furthermore, all X-ray structures of MDM2 published to date show the N-terminal domainin 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 newset of crystallisation conditions. Furthermore, it has been shown that high-affinity ligandscan 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 tocrystallise MDM2, one therefore requires ligands able to stabilise MDM2 as well as tocontribute to the formation of crystal contacts. 39
  51. 51. Part I: Section 2 - Towards a Crystallisable Form of MDM2This part of my thesis attempts to enable MDM2-crystallisation in the presence and absenceof small-molecule ligands through protein engineering. The establishment of suitable proteinconstructs and crystallisation conditions that consistently yield diffraction quality crystalswill hopefully allow rapid determination of Isoindolinone binding-modes enabling furthercompound design.Figure 2-1. Crystal contacts from MDM2 crystal structure 1YCR, 1T4E and 1RV1. In the 1YCRstructure, one way by which the p53-peptide contributes to the crystal contacts is through packing oftwo Lys24 side-chains. In the 1T4E structure, an MDM2-bound Benzodiazepinedione forms ahydrogen bond with an adjacent MDM2 molecule. In the 1RV1 structure, Nutlin-2 contributes to thecrystal contacts through unspecific binding to the surface of MDM2, bringing together three MDM2molecules. 40
  52. 52. Part I: Section 2 - Towards a Crystallisable Form of MDM22.2 MDM2 Surface Engineering2.2.1 Decreasing the Entropic Penalty of CrystallisationBased on a current estimate, only 30% of proteins expressed solubly from E.coli willeventually crystallise (Dale, Oefner et al. 2003). Approximately 80% of these crystallisableproteins will form crystals from screening of only 50 different conditions (Jancarik and Kim1991). Testing a huge number of conditions when initial screens were unsuccessful istherefore not likely to increase success. A much more efficient way to obtain crystals isrational 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 ofthe protein (Derewenda 2004). Since the incorporation of large extended side-chains into thecrystal lattice puts an entropic penalty on the process of crystallisation, it was suggested toreplace these with small residues, such as Alanines. Surface-alanines are thought to provide aconformationally more homogeneous surface and could potentially form crystal contacts. Itwas suggested that clusters of Lysine and Glutamic acids are the most suitable amino acidsfor mutagenesis, since they occur with high incidence at protein surfaces (Baud and Karlin1999), possess high conformational entropy (Avbelj and Fele 1998) and occur rarely at theinterface of protein-protein interactions (Conte, Chothia et al. 1999).The MDM217-125 sequence contains three clusters of Lysines and Glutamic acids amenable toprotein engineering, namely K51 and E52, E69 and K70, K94 and E95. All three clusters arepotential targets, as they do not directly interfere with p53 binding (Figure 2-2). 41

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