Döm

ling(Ed.)

Edited by Alexander Dömling

56 Methods and Principles in Medicinal Chemistry

Protein–ProteinInteractions inDrug Discovery

Protein–ProteinInteractions

inD

rugD

iscovery

Volume 56

Series Editors:R. Mannhold,H. Kubinyi,G. Folkers

www.wiley-vch.de

Treating protein-protein interactions as a novel and highly promisingclass of drug targets, this volume introduces the underlying strategiesstep by step, from the biology of PPIs to biophysical and computationalmethods for their investigation.

The main part of the book describes examples of protein targets forwhich small molecule modulators have been developed, covering suchdiverse fields as cancer, autoimmune disorders and infectious diseases.Tailor-made for the practicing medicinal chemist, this ready referenceincludes a wide selection of case studies taken straight from the develop-ment pipeline of major pharmaceutical companies to illustrate the powerand potential of this approach.

Prediction of intra- and inter-species protein-protein interactionsfacilitating systems biology studiesModulators of protein-protein interactions: the importanceof three-dimensionalityInteractive technologies for leveraging the known chemistryof anchor residuesSH3 domains as drug targetsP53 MDM2 antagonists: towards non genotoxic anticancertreatmentsInhibition of LFA-1/ICAM interaction for treatment of autoimmunediseasesThe PIF-binding pocket of AGC kinasesPeptidic inhibitors of protein-protein interactions for cell adhesionreceptorsThe REPLACE strategy for generating non-ATP competitiveinhibitors of cell-cycle protein kinases

and more

From the contents:�

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Alexander Dömling studied Chemistry and Biology at the Technical University Munich, Germany.After obtaining his PhD under the supervision of Ivar Ugi, he spent a postdoctoral year at the ScrippsResearch Institute in La Jolla (USA) in the group of Nobel Laureate Barry Sharpless. In 2004 heperformed his habilitation at the Technical University of Munich. Since 2006 he teaches andperforms research at the University of Pittsburgh in the Department of Pharmacy with secondaryappointments in Chemistry and Computational Biology. In 2010 he became full professor receivinghis tenure in Pittsburgh. Since 2011 he is chairing the department of Drug Design at the Universityof Groningen/The Netherlands. Dr. Dömling is founder of several biotech companies, includingMorphochem and Carmolex. His research centers around the discovery of antagonists of protein-protein interactions and other biologically active compounds in the therapeutic areas of oncology,infectious and neglected tropical diseases using “out-of-the-box” software tools and multicomponentreaction chemistries.

Edited by

Alexander D€omling

Protein–Protein Interactionsin Drug Discovery

Related Titles

Methods and Principles in Medicinal ChemistryEdited by R. Mannhold, H. Kubinyi, G. FolkersEditorial BoardH. Buschmann, H. Timmerman, H. van de Waterbeemd, T. Wieland

Previous Volumes of this Series

Kalgutkar, Amit S. / Dalvie, Deepak /Obach, R. Scott / Smith, Dennis A.

Reactive Drug Metabolites2012

ISBN: 978-3-527-33085-0, Vol. 55

Brown, Nathan (Ed.)

Bioisosteres in MedicinalChemistry2012

ISBN: 978-3-527-33015-7, Vol. 54

Gohlke, Holger (Ed.)

Protein-Ligand Interactions2012

ISBN: 978-3-527-32966-3, Vol. 53

Kappe, C. Oliver / Stadler, Alexander /Dallinger, Doris

Microwaves in Organic andMedicinal ChemistrySecond, Completely Revised andEnlarged Edition

2012

ISBN: 978-3-527-33185-7, Vol. 52

Smith, Dennis A. / Allerton, Charlotte /Kalgutkar, Amit S. / van de Waterbeemd,Han / Walker, Don K.

Pharmacokinetics andMetabolism in Drug DesignThird, Revised and Updated Edition

2012

ISBN: 978-3-527-32954-0, Vol. 51

De Clercq, Erik (Ed.)

Antiviral Drug Strategies2011

ISBN: 978-3-527-32696-9, Vol. 50

Klebl, Bert / M€uller, Gerhard / Hamacher,Michael (Eds.)

Protein Kinases as DrugTargets2011

ISBN: 978-3-527-31790-5, Vol. 49

Sotriffer, Christoph (Ed.)

Virtual ScreeningPrinciples, Challenges, and PracticalGuidelines

2011

ISBN: 978-3-527-32636-5, Vol. 48

Rautio, Jarkko (Ed.)

Prodrugs and TargetedDelivery Towards Better ADMEProperties

2011

ISBN: 978-3-527-32603-7, Vol. 47

Smit, Martine J. / Lira, Sergio A. / Leurs,Rob (Eds.)

Chemokine Receptors as DrugTargets2011

ISBN: 978-3-527-32118-6, Vol. 46

Edited by Alexander D€omling

Protein–Protein Interactionsin Drug Discovery

Series Editors

Prof. Dr. Raimund MannholdMolecular Drug Research GroupHeinrich-Heine-Universit€atUniversit€atsstrasse 140225 D€usseldorfGermanymannhold@uni-duesseldorf.de

Prof. Dr. Hugo KubinyiDonnersbergstrasse 967256 Weisenheim am SandGermanykubinyi@t-online.de

Prof. Dr. Gerd FolkersCollegium HelveticumSTW/ETH Zurich8092 ZurichSwitzerlandfolkers@collegium.ethz.ch

Volume Editor

Prof. Dr. Alexander D€omlingUniversity of GroningenSchool of PharmacyDrug DesignAntonius Deusinglaan 19713 AV GroningenNetherlands

CoverCut-away view of the co-crystal structure of anarchetypical protein- protein interaction: thetumor suppressor p53 and the oncogeneMDM2 (PDB ID 1YCR). The most deepestburied p53 amino acid Trp23, anchoring thePPI, is shown in sticks presentation, the MDM2receptor is shown as surface. Also shown thehydrogen bond between the Trp23 indole-NHand the receptor Leu54 carbonyl as greendotted line. The picture was rendered usingPYMOL software.

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Print ISBN: 978-3-527-33107-9ePDF ISBN: 978-3-527-64823-8ePub ISBN: 978-3-527-64822-1mobi ISBN: 978-3-527-64821-4oBook ISBN: 978-3-527-64820-7

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Contents

List of Contributors XIPreface XVA Personal Foreword XVII

1 Protein–Protein Interactions: An Overview 1Christian Ottman

1.1 Introduction 11.2 Role of PPIs in Human Physiology 21.3 Regulation of PPIs 31.4 Structural Features of PPI Interfaces 31.4.1 iNOS Homodimer 51.4.2 b-Catenin/Tcf4 Complex 51.4.3 LEDGF/HIV-IN Complex 61.4.4 HPV E1/E2 Complex 71.4.5 IFN-a/IFNAR Complex 81.4.6 TNF-a Trimer 91.5 Identification of PPI Inhibitors 101.6 Conclusions and Outlook 13

References 14

2 Prediction of Intra- and Interspecies Protein–Protein InteractionsFacilitating Systems Biology Studies 21Sylvia Schleker, Seshan Ananthasubramanian, Judith Klein-Seetharaman,and Madhavi K. Ganapathiraju

2.1 Introduction: Relevance of Interactome Studies to Disease and DrugDiscovery 21

2.2 Our Current Knowledge of Interactomes Identified fromExperiments is Incomplete 23

2.3 Reliability of Interactions Identified Experimentally 242.4 Computational Methods for PPI Prediction 272.4.1 Conservation of Gene Neighborhood 272.4.2 Gene Fusion 282.4.3 Sequence-Based Coevolution 28

jV

2.4.4 Phylogenetic Profiling 282.4.5 Gene Expression 292.4.6 Structural Similarity 292.4.7 Integration Approaches 292.5 Sources of Biological Data in Use to Predict PPIs 302.6 Survey of Current Interactomes 322.6.1 Human Intraspecies Interactomes 322.6.2 Bacteria Intraspecies Interactomes 372.6.2.1 High-Throughput Experimental Approaches to Identify Intraspecies

Bacterial Interactions 372.6.2.2 Modeling Intraspecies Bacterial Interactions 392.6.3 Bacteria–Human Interspecies Interactomes 402.6.3.1 Experimental Approaches to Identify Bacteria–Human PPIs 402.6.3.2 Modeling Bacteria–Human PPIs 402.6.4 Non-PPI Intraspecies Bacterial and Bacteria–Human Interspecies

Interactome Models 412.6.5 Virus–Human Interspecies Interactomes 42

References 43

3 Modulators of Protein–Protein Interactions: Importanceof Three-Dimensionality 55David C. Fry and Sung-Sau So

3.1 Introduction 553.2 Study 563.3 Discussion 583.4 Summary 61

References 61

4 A Leap into the Chemical Space of Protein–Protein InteractionInhibitors 63Bruno O. Villoutreix, C. Labb�e, David Lagorce, Guillaume Laconde,and Olivier Sperandio

4.1 Introduction 634.2 Types of Interaction 644.3 Properties of the Interface 654.4 Orthosteric versus Allosteric Modulation 664.5 Leap into the iPPI Chemical Space 664.5.1 Seminal Works 664.5.2 Road to a Rationalization of the iPPI Chemical Space 674.6 Case Study 684.6.1 Visualizing the iPPI Chemical Space 704.6.2 iPPI versus ADME/Tox Properties 714.6.3 iPPI versus Aromaticity 754.6.4 iPPI versus Chemical Complexity 77

VIj Contents

4.6.5 iPPI versus Molecular Shape 774.6.6 iPPI versus Potency 794.7 Conclusions 80

References 81

5 Interactive Technologies for Leveraging the Known Chemistryof Anchor Residues to Disrupt Protein Interactions 85Carlos J. Camacho, David R. Koes, and Alexander S. D€omling

5.1 Introduction 855.2 Druggable Sites in PPIs 865.3 Structure-Based Library Design – A Powerful Alternative

to High-Throughput Screening 875.4 New MCR Chemistry to Design PPI Antagonists 895.5 Virtual Screening 905.6 New Interactive Modeling Techniques for Medicinal

Chemists 935.7 New Ideas: Hit Rate Validation of Anchor-Centered Screening

of p53/MDM2/4 955.8 Summary 96

References 97

6 SH3 Domains as Drug Targets 101James Luccarelli, Sam Thompson, and Andrew D. Hamilton

6.1 Introduction 1016.2 Structure 1016.3 Variability 1026.4 SH3 Binding Motifs 1046.4.1 Classical Binding Motifs 1046.4.2 Tyrosine-Containing Motifs 1076.4.3 RxxK Motif 1086.4.4 Other Binding Motifs from Proteomic Screens 1096.4.5 Tertiary Interactions 1106.5 Selectivity 1116.6 Drug Target Selection 1146.7 Inhibition Strategies: Peptide and Peptoid

Inhibitors 1146.7.1 Peptide Ligands 1146.7.2 Combinatorial Approaches 1156.7.3 Peptide Dimers 1166.7.4 Constrained Peptides 1186.7.5 N-Substituted Peptoids 1186.8 Small-Molecule Inhibitors 1196.9 Conclusions 122

References 122

Contents jVII

7 p53/MDM2 Antagonists: Towards NongenotoxicAnticancer Treatments 129Kareem Khoury, Tad A. Holak, and Alexander D€omling

7.1 Introduction 1297.2 p53/MDM2 PPI is Characterized by Many Cocrystal

Structures 1307.3 Nutlins: First-In-Class MDM2 Antagonists 1317.4 Johnson & Johnson: Benzodiazepines 1337.5 Amgen: Chromenotriazolopyrimidines & Piperidones 1377.6 University of Michigan: Spirooxindole 1487.7 University of Pittsburgh: Ugi Based Compounds 1537.8 University of Newcastle: Some Scaffolds With No Structural Biology

Information 1557.9 Outlook 161

References 161

8 Inhibition of LFA-1/ICAM Interaction for the Treatmentof Autoimmune Diseases 165Kevin M. Guckian and Daniel M. Scott

8.1 Introduction 1658.2 Integrin Structure and Activation 1668.3 Direct Inhibition of the LFA-1/ICAM Interaction 1688.4 Allosteric Inhibitors of the LFA-1/ICAM interaction – IDAS Site 1718.4.1 Abbott/ICOS/Biogen Series 1718.4.2 Boehringer Ingelheim/Tanabe Seiyaku/Bristol-Myers Squibb

Series 1788.5 Summary 183

References 183

9 The PIF Pocket of AGC Kinases: A Target Site for Allosteric Modulatorsand Protein–Protein Interaction Inhibitors 187Matthias Engel

9.1 Introduction 1879.2 Discovery and Physiological Functions of the PIF Pocket 1909.3 Properties of the PIF Pocket Relevant to Drug

Development 1929.3.1 The PIF Pocket Offers the Potential to Develop Highly

Selective Ligands 1929.3.2 Molecular Interactions of the Natural HM Peptide Ligands 1939.3.3 Allosteric Mechanism of the PIF Pocket 1969.3.4 Structural Plasticity of the PIF Pocket 1989.4 Small-Molecule PIF Pocket Ligands 1999.4.1 Allosteric Activators and PPI Inhibitors of PDK1 1999.4.2 Identification of First Hit Compounds Using a Pharmacophore-Based

Screening Approach 200

VIIIj Contents

9.4.3 Current State of Research on PIF Pocket-Directed PDK1Modulators 203

9.4.4 Allosteric Inhibitors 2079.5 Potential Supportive Effects Enhancing the Cellular Activity of PIF

Pocket-Binding Modulators 2099.5.1 Allosteric Activators of PDK1 2099.5.2 PIF Pocket-Directed Inhibitors of AGC Kinases 2109.6 Conclusions 2129.6.1 Is the PIF Pocket a Druggable Site? 2129.6.2 General Medicinal Chemistry Aspects and

Outlook 213References 215

10 Retosiban and Epelsiban: Potent and Selective Orally AvailableOxytocin Antagonists 225Alan D. Borthwick and John Liddle

10.1 Introduction 22510.2 Aryl-2,5-DKP Template Discovery and Initial Structure–Activity

Relationship Studies 22710.3 Synthesis of the RRR and RRS 6-Indanyl-3-isobutyl-7-aryl-2,5-DKP

Secondary Amides 23110.4 Comparison of Crystal Structures of Oxytocin and 2,5-DKPs 23110.5 Pharmacokinetics and Property-Based Design 23210.6 In Vivo Potency of 20,40-Diflurophenyl Dimethylamide 22 23510.7 Synthesis of Tertiary Amides 23610.7.1 Synthesis of Five- and Six-Membered Heterocyclic 2,5-DKPs 23710.8 Summary of Lead Oxytocin Antagonist 20,40-Diflurophenyl

Dimethylamide 22 23810.9 Further Modifications, Five- and Six-Membered Heterocyclic

Derivatives 23810.10 Five-Membered Heterocyclic Derivatives and Retosiban 23910.10.1 Oxytocin Antagonist Activity and Selectivity versus Human

Vasopressin Receptors 24210.10.2 In Vivo Potency and Synthesis 24310.11 Summary of Lead Oxytocin Antagonist Retosiban 56 24410.12 Six-Membered Heterocyclic Derivatives and Epelsiban 24410.12.1 Monosubstituted Pyridyl isoButyl Derivatives 24610.12.2 Modification of isoButyl in 60-MePyridyl Derivatives 24610.12.3 Dimethylpyridyl (S)-sec-Butyl Amides 24610.12.4 Further Evaluation of 20,60-Dimethyl-30-pyridine Morpholine

Amide 77 25010.13 Summary of Lead Oxytocin Antagonist Epelsiban 77 25210.14 Comparison of Lead Compounds 25210.15 Conclusions 254

References 254

Contents jIX

11 Peptidic Inhibitors of Protein–Protein Interactions for Cell AdhesionReceptors: RGD Peptides and Beyond 257Carlos Mas-Moruno and Horst Kessler

11.1 Introduction 25711.2 From the Discovery of the RGD Motif in FN to the First Selective

Cyclic RGD Peptide 25811.2.1 RGD Sequence, Integrins, and Receptor Selectivity 25811.2.2 Concept of Spatial Screening in Cyclic RGD Peptides 26111.2.3 Conformational Aspects and Selectivity of c(RGDfV) 26311.2.4 Pharmacophoric Requirements of c(RGDfV) to Bind avb3 26511.3 N-Methylation of c(RGDfV): Cilengitide and Beyond 26711.3.1 Concept of N-Methylation 26711.3.2 N-Methyl Scan of c(RGDV): Synthesis, Biological Activity, and

Structural Considerations of Cilengitide 26811.3.3 Beyond Cilengitide: di-N-Methylated Analogs of c(RGDfV) and avb3

Selectivity 27111.4 isoDGR Sequence as a New Integrin-Binding Motif 27411.4.1 Formation of isoAsp Residues in Peptides and Proteins 27411.4.2 NGR Deamidation to isoDGR Yields a New Integrin-Binding

Motif 27511.4.3 Design of Cyclic Peptides Containing the isoDGR Motif as

New Integrin Antagonists 27611.4.4 Receptor Selectivity of Cyclic isoDGR Peptides 27911.5 Conclusions 281

References 282

12 REPLACE Strategy for Generating Non-ATP-Competitive Inhibitorsof Cell Cycle Protein Kinases 291Campbell McInnes

12.1 Introduction 29112.2 Inhibition of CDKs Through the Cyclin Groove 29112.3 Inhibitors of PLKs 29812.3.1 PB Domain 29812.4 Conclusions 301

References 302

Index 305

Xj Contents

List of Contributors

Seshan AnanthasubramanianUniversity of PittsburghSchool of MedicineDepartment of Biomedical Informat-ics and Intelligent Systems Program5607 Baum Blvd, Suite 401PittsburghPA 15206USA

Alan D. BorthwickDrugMolDesign15 Temple GroveLondon NW11 7UAUK

Carlos J. CamachoUniversity of PittsburghDepartment of Computational andSystems Biology3501 Fifth AvenueSuite 3064Biomedical Science Tower 3Pittsburgh, PA 15260USA

Alexander S. D€omlingUniversity of PittsburghDepartment of Pharmacy andComputational and Systems Biology3501 Fifth AvenueSuite 3064Biomedical Science Tower 3Pittsburgh, PA 15260USA

and

University of GroningenDepartment of Drug DesignA. Deusinglaan 19713 AV GroningenNetherlands

Matthias EngelUniversity of SaarlandResearch Group PhosphoSitesDepartment of Pharmaceutical andMedicinal ChemistryCampus C2 2PO Box 15115066041 Saarbr€uckenGermany

jXI

David C. FryRoche Research CenterDiscovery Technologies340 Kingsland StreetNutley, NJ 07110USA

Madhavi K. GanapathirajuUniversity of PittsburghSchool of MedicineDepartment of Biomedical Informaticsand Intelligent Systems Program5607 Baum Blvd, Suite 401Pittsburgh, PA 15206USA

Kevin M. GuckianBiogenIdec Inc.Medicinal Chemistry14 Cambridge CenterCambridge, MA 02142USA

Andrew D. HamiltonUniversity of OxfordChemistry Research Laboratory12 Mansfield RoadOxford OX1 3TAUK

Tad A. HolakMax Planck Institute of BiochemistryNMR GroupAm Klopferspitz 1882152 MartinsriedGermany

Horst KesslerTechnische Universit€at M€unchenInstitute for Advanced Study andCenter of Integrated Protein ScienceDepartment ChemieLichtenbergstrasse 485747 GarchingGermany

Kareem KhouryUniversity of PittsburghSchool of Pharmacy1104 Salk Hall3501 Terrace StreetPittsburgh, PA 15261USA

Judith Klein-SeetharamanInstitute of Complex SystemsICS-5: Molecular Biophysics52425 JülichGermany

and

Department of Structural BiologyUniversity of PittsburghSchool of Medicine Room 2051Biomedical ScienceTower 33501 Fifth AvenuePittsburgh, PA 15261USA

David R. KoesUniversity of PittsburghDepartment of Computational andSystems Biology3501 Fifth AvenueSuite 3064Biomedical Science Tower 3Pittsburgh, PA 15260USA

C. Labb�eINSERMU97335 rue H. Brion75205 ParisFrance

XIIj List of Contributors

and

Universit�e Paris DiderotSorbonne Paris Cit�eUMRS 973MTi35 rue H. Brion75205 ParisFrance

Guillaume LacondeINSERMU97335 rue H. Brion75205 ParisFrance

and

Universit�e Paris DiderotSorbonne Paris Cit�eUMRS 973MTi35 rue H. Brion75205 ParisFrance

John LiddleGlaxoSmithKline Research andDevelopmentDepartment of Medicinal ChemistryMedicines Research CentreGunnels Wood RoadStevenage SG1 2NYUK

James LuccarelliUniversity of OxfordChemistry Research Laboratory12 Mansfield RoadOxford OX1 3TAUK

Carlos Mas-MorunoTechnische Universit€at M€unchenInstitute for Advanced Study andCenter of Integrated Protein ScienceDepartment ChemieLichtenbergstrasse 485747 GarchingGermany

Campbell McInnesUniversity of South CarolinaPharmaceutical and BiomedicalSciencesSouth Carolina College of PharmacyCoker Life Science Building715 Sumter StreetColumbia, SC 29208USA

Christian OttmannMax Planck SocietyChemical Genomics CentreOtto-Hahn-Strasse 1544227 DortmundGermany

Sylvia SchlekerInstitute of Complex SystemsICS-5: Molekulare Biophysik52425 J€ulichGermany

and

University of PittsburghSchool of MedicineDepartment of Structural BiologyPittsburgh, PA 15260USA

List of Contributors jXIII

Sung-Sau SoRoche Research CenterDiscovery Chemistry340 Kingsland StreetNutley, NJ 07110USA

Daniel M. ScottBiogenIdec Inc.Medicinal Chemistry14 Cambridge CenterCambridge, MA 02142USA

Olivier SperandioINSERMU97335 rue H. Brion75205 ParisFrance

and

Universit�e Paris DiderotSorbonne Paris Cit�eUMRS 973MTi35 rue H. Brion75205 ParisFrance

Sam ThompsonUniversity of OxfordChemistry Research Laboratory12 Mansfield RoadOxford OX1 3TAUK

Bruno O. VilloutreixINSERMU97335 rue H. Brion75205 ParisFrance

and

Universit�e Paris DiderotSorbonne Paris Cit�eUMRS 973MTi35 rue H. Brion75205 ParisFrance

XIVj List of Contributors

Preface

In the common definition of the proteome it is considered to be the set of expressedproteins in a given type of cells or an organism at a given time under definedconditions. It is larger than the genome, due to special mechanisms in thebiogenesis of proteins, which render them more numerous than the genes atthe end. What, however is very special to the proteome is its enriched complexitywhich arises both from the protein’s 3D structure and the functional interaction ofthe proteins.Proteins talk to each other. They do it very frequently and in different ways and

obviously in large communication networks of sophisticated structure. For yeast,recent research has suggested kind of social life in protein communication:

This distinction suggests a model of organized modularity for the yeast proteome, withmodules connected through regulators, mediators or adaptors, the date hubs. Party hubsrepresent integral elements within distinct modules and, although important for thefunctions mediated by these modules (and therefore likely to be essential proteins), tendto function at a lower level of the organization of the proteome. We propose that datehubs participate in a wide range of integrated connections required for a globalorganization of biological modules in the whole proteome network (although somedate hubs could simply be ‘shared’ between, and mediate local functions inside,overlapping modules). (. . .) Finally, it is possible that discriminating between dateand party hubs might also help to define new therapeutic drug targets.1

Well, there we are in defining ambitious goals for drug development. Interferingwith the party gossip and intruding the clandestine dates of proteins for sake oftherapeutic benefits is still the freestyle of drug design. The complexity is huge.Large interfaces that resemble flat landscapes, lacking cosy caves or deep pockets,where small molecules might dock made protein-protein interactions clearlyundruggable for a long time.With the advent and success of monoclonal therapeuticantibodies (mAbs) however, this view changed. The benefits of mAbs in the cancer

1 Jing-Dong J. Han, Nicolas Bertin, Tong Hao, Debra S. Goldberg, Gabriel F. Berriz, Lan V. Zhang,Denis Dupuy, Albertha J. M. Walhout�, Michael E. Cusick, Frederick P. Roth & Marc Vidal. Evidencefor dynamically organized modularity in the yeast protein–protein interaction network. Nature, 430,2004, pp. 88-93

jXV

and immune-disease areas showed that interfering with protein-protein interactions(PPIs) is indeed a practicable approach. Unfortunately the proteins parties and datesare taken place inside the cell, with no admittance for mAbs. Hence, the challengeor the ”reaching for high-hanging fruit”2 is to interfere therapeutically by use ofsmall molecules.In the present volume Alexander D€omling and his co-authors depict a scenario

that reveals more than a silver lining on the horizon. There is a variety of new (andold) targets and a plethora of new approaches and insights into molecular mecha-nisms which raise the hope that in the near future small molecule interference ofprotein-protein interactions will emerge as a new functional class of therapeutics.Twelve chapters illuminate opportunities, strategies, success stories and pitfalls inthe discovery of small molecules targeting protein-protein-interactions.While this volume grants an encompassing view on the state-of-the-art in the PPI

field, it is at the same time structuring the future, since it paves the way for ahopefully greater commitment in drug discovery focusing PPIs.Not least because of this, the series editors are indebted to the authors and the

editor whomade this comprehensive issue possible. We are convinced that the bookrepresents an important contribution to the body of knowledge in drug discoveryand that it matches the interests of many researches who have adjourned to apromising but rocky field.In addition, we are verymuch indebted to FrankWeinreich andHeike N€othe, both

at Wiley-VCH. Their support and ongoing engagement, not only for this book butfor the whole series “Methods and Principles in Medicinal Chemistry” adds to thesuccess of this excellent collection of monographs on various topics, all related todrug research.

November 2012D€usseldorf Raimund MannholdWeisenheim am Sand Hugo KubinyiZ€urich Gerd Folkers

2 James A. Wells and Christopher L. McClendon. Reaching for high-hanging fruit in drug discovery atprotein–protein interfaces. Nature 450, 2007, 1001-1009

XVIj Preface

A Personal Foreword

Therapeutic targeting of protein–protein interactions (PPIs) has been in the pastlargely the domain of biotech industries. With their exquisite natural selectionprocess, and the resulting high affinity and selectivity towards their targets,monoclonal antibodies (mAbs) and similar biologics have been the only way totarget large protein interfaces to effectively compete with the endogenous interac-tion. Meanwhile, mAbs have overcome their early issues, and the generation of safe,specific, high-affinity, and nonimmunogenic antibodies has become a reality. In fact,a switch of the pharmaceutical industry from focusing traditionally on smallmolecules towards the biotech drugs has recently been observed based on thetherapeutic and business success of the biologics; major takeovers of biotechcompanies by big pharma excite the industry and shareholders. Severe conditionscan meanwhile be treated with mAbs, including some cancers, Crohn’s disease,rheumatoid arthritis, transplant rejection, or macular degeneration, just to name afew. However, large biomolecules like mAbs also have intrinsic disadvantages thatare hard to overcome, such as lack of oral bioavailability, high cost of goods, andperhaps most importantly mAbs can only target extracellular accessible structures.The majority of PPI drug targets, are, however, localized intracellularly.The strength of small molecules, in principle, is to overcome exactly the issues of

mAbs and similar biologics: they can be made orally bioavailable and decorated withadvantageous pharmacokinetics/pharmacodynamics properties, they can be pro-duced orders of magnitude cheaper then biologics, and they can also be designed topenetrate cell membranes to reach intracellular targets. Therefore, small moleculesshould be ideal to disrupt PPIs. However, PPIs very often consist of large interfacesof greater than 1000A

� 2 and do not contain the deep pockets medicinal chemists areused to from other target areas, such as kinases, G-protein-coupled receptors, orproteases. Pocket dimension, form (concave versus convex), and hydrophobicity are,however, key features defining the druggability by small molecules. Analysis of theplethora of available crystal structures of PPIs reveals that only a fraction of PPIs fitthe druggability criteria. Nevertheless, while PPIs as a class were considered asundruggable a decade ago, several small molecules advancing into clinical trialsrecently clearly support the notion that at least a fraction of the PPIs are not onlyattractive therapeutic targets, but can also be developed to drugs. It can be foreseen

jXVII

that in the future many more small molecules targeting PPIs will emerge for thetreatment of unmet medical needs.This book aims to address an audience including medicinal chemists, organic

chemists, and other interested readers. In 12 chapters written by experts in theirfields I try to introduce the reader to the problems and opportunities associated withtargeting PPIs by small molecules. Introductory overviews pave the way for specificmedicinal chemistry showcases on developmental compounds. Additionally, severalchapters introduce technologies relevant for PPI drug discovery, including theinteractome and computational chemistry.Ottmann gives an excellent introductory overview on different PPI drug discovery

approaches and compound classes, and the structural features of their interactingproteins.Next, Schleker et al. introduce the reader to different techniques used in systems

biology to define the interactome of different species. Scope and limitations of thecurrent experimental and computational identification approaches to obtain inter-actomes related to human are discussed. Cleary only careful comparison of thelarge-scale results of orthogonal experimental approaches can lead to meaningfulresults and a deeper understanding of the interactome’s wide regulation of life.Fry and So stress the importance of three-dimensionality in the modulation of

PPIs – a shape feature often under-represented in company screening libraries. Fromstructural analysis based on theProteinData Bank (PDB) database they deduce simplerules of thumb for when a particular PPI is druggable by small molecules.Villoutreix et al. introduce the reader to computational chemistry approaches

towards PPI drug discovery. Detailed discussions on the relationship and comple-mentarity of the shape of the receptor binding site and the scaffold shape clarify whytraditional chemical space is relatively unsuited to find PPI antagonists. Thespecifics of PPI-targeting compound classes are elaborated in a detailed analysisof many descriptors. Rather novel scaffold and privileged structures are needed toaddress the PPI interfaces.Web-based techniques to computationally predict small-molecule PPI inhibitors

are introduced by Camacho et al. The AnchorQueryTM approach makes use of thedisproportional high importance of anchoring residues in PPIs, and queries the verylarge chemical space and one-pot accessibility of multicomponent reactions (MCR).Again, the importance of suitably designed libraries for PPIs is stressed.Luccarelli et al. review the Src homology 3 (SH3) domain as a drug target. SH3

domain proteins comprise a very large class with implications in multiple thera-peutic areas. The structural biology is reviewed and, based on the understanding ofspecific features of the binding pocket, current inhibitor design is discussed.The PPI of the transcription factor p53 and the oncogene MDM2 is amongst the

most intensively studied PPIs with several compounds in late preclinical and earlyclinical development. Khoury et al. review this area with a focus on small-moleculeclasses supported by structural biology information, their structure–activity relation-ships (SARs), and the methods of initial hit finding.Guckian reviews the area of inhibitors of the lymphocyte function-associated

antigen-1/intracellular adhesion molecule (LFA-1/ICAM) interaction for the

XVIIIj A Personal Foreword

treatment of autoimmune diseases. LFA-1/ICAM is a case where a recentlyapproved mAb had to be withdrawn from the market due to fatal side-effects.Structural biology as well as different inhibitor classes and their SAR are discussed.Several small molecules are currently being evaluated in clinical trials.Engel reviews the PIF-binding pocket of the subclass of AGC kinases, which

serves as a target site for allosteric modulators and PPI inhibitors. Major advantagesof targeting non-ATP sites of kinases would be to obtain more selective compoundsand to target kinases previously considered to be nondruggable. The recentdiscovery of different classes of AGC kinase pocket binders makes this an importantarea of research.The discovery and medicinal chemistry evolution of oxytocin receptor antagonists

is reviewed by Borthwick and Liddle. Their efforts have led to Retosiban andEpelsiban – two orally bioavailable oxytocin receptor antagonists with superiorpotency and selectivity for the treatment of preterm birth and related disorders.Mas-Moruno and Kessler describe peptidic inhibitors targeting cell adhesion

receptors (e.g., RGD peptides). Although peptides are not considered as smallmolecules, much can be learned from the specific modes by which peptides interactwith their targets for the development of small-molecule PPI inhibitors.Last, but not least, McInnes reviews another method to allosterically inhibit the

cyclin-dependent kinase (CDK) kinase class by targeting the substrate recognitionsite. The REPLACE algorithm for the structure-based prediction of PPI binders hasbeen applied to the Polo-box domain of PLK1 (Polo-like kinase 1), resulting in smallpeptides that are N- and C-capped by organic residues.The area of small molecules successfully targeting PPIs has recently exploded and

therefore only a glimpse of the exciting research in the area can be reflected by thepresent book. Many interesting works could not be included. To finish, and in thetradition of the Editor’s choice, I want to point the reader to several more excitingrecent targets.The small G-protein and oncogene Ras has been recognized for several decades as

a major oncology target. A large portion of lung, colon, and pancreas cancer patientsbear constitutively active Ras mutations that drive cancer growth. However, untilrecently no small-molecule drug has been known to directly bind to and effectivelymodify the downstream pathway. Lately, novel biophysical techniques have shownpromise by identifying small molecules inhibiting the Ras effector complex Ras–Sos(Figure 1a) and cocrystal structures solved by two independent groups provide hopethat the initial fragment-based hits can be further elaborated towards effectivedrugs [1,2].Protein lysine methyltransferases are important regulators of epigenetic signal-

ing. The oncogenic protein SMYD2 represses the tumor suppressors p53 and Rb bya PPI. The use of structural biology information cannot be overestimated, and thestructure of apo-SMYD2, SMYD2 bound to a methylated p53 peptide, and incomplex with a small molecule competitively inhibiting p53 binding was publishedrecently (Figure 1b) [3].The von Hippel–Lindau (VHL) syndrome is a rare autosomal dominant genetic

orphan disease characterized by abnormal angiogenesis in certain parts of the body.

A Personal Foreword jXIX

N NH

S

HO

HN

OO

NH

N

HO

HN CI

CIO

N

O

NH

O

N

NOO

OH

O O

N

N

N

NS

CI

(a) (b)

(c) (d)

Figure 1 (a) Indole-3-thiocarbonylpiperidinefragment bound to Ras on the Sos interactinginterface (PDB ID: 4EPV). (b) Small moleculemimicking the p53 Lys-Me binding to SMYD2and extending with the dichlorophenyl moietyinto a nearby hydrophobic pocket not occupiedby the peptide (PDB ID: 3S7B). (c) Small

molecule anchoring via the centralhydroxyproline ring to the VHL complex andthus inhibiting the contact to HIF-1a (PDB ID:3ZRC). (d) Cut-away view of the Gewaldthiophenodiazepine JQ1 bound into thebromodomain of BRD4 into the Ac-Lys bindingsite (PDB ID: 3MXF).

XXj A Personal Foreword

The E3 ubiquitin ligase VHL complex primarily targets the hypoxia-inducible factor(HIF)-1a – a transcription factor involved in the regulation of numerous genes (i.e.,involved in angiogenesis and cancer). Using in silico methods and structure guidedmedicinal chemistry the first small molecule ligand for VHLwas recently described.The authors used the hydroxyproline motif of HIF-1a as an “anchoring” startingpoint for their medicinal chemistry efforts (Figure 1c) [4]. Derivatives of the first-generation compounds might evolve into cell-penetrating chemical probes to testthe involvement of the VHL complex in disease conditions such as chronic anemia,acute ischemia, and stroke.Bromodomains are reader elements in the framework of epigenetic control and

recognize sequence-specific acetylated lysine side-chains of histones and otherproteins. Two small-molecule inhibitors of the LysAc-bromodomain PPI are cur-rently undergoing clinical evaluation for the treatment of artherosclerotic cardio-vascular disease, and testis midline and other cancers (Figure 1d) [5,6]. As for manyareas in PPIs, the availability of X-ray structures of Apo, Ac-Lys, and ligand-boundprotein proved to be invaluable in the early discovery process. A major challenge ofthe area will be the design of selectivity into the small molecules targeting one of the61 very similar bromodomains found in the human genome.I have enjoyed editing this book, and I hope that readers will benefit from the

expert reviews on cutting-edge small-molecule PPI research and development.

October 2012 Alexander D€omlingGroningen

References

1 Maurer, T. et al. (2102) Proceedings of theNational Academy of Sciences of the UnitedStates of America, 109, 5299.

2 Sun, Q. et al. (2102) Angewandte ChemieInternational Edition, 51, 6140.

3 Ferguson, A.D. et al. (2011) Structure, 19,1207.

4 Buckley, D.L. et al. (2012) Journal of theAmerican Chemical Society, 134, 4465.

5 Filippakopoulos, P. et al. (2010) Nature,468, 1067.

6 Nicodeme, E. et al. (2010) Nature, 468,1119.

A Personal Foreword jXXI

1Protein–Protein Interactions: An OverviewChristian Ottmann

1.1Introduction

Protein–protein interactions (PPIs) are implicated in almost all biological processesfor any given protein engaged in complexes with other proteins for themajority of itslifetime. In this regard, proteins function not merely as single, isolated entities, butdisplay their roles by interacting with other cellular components. The differentinteraction patterns are at least as important as the intrinsic biochemical activitystatus (e.g., of a protein kinase) of the protein itself. Therefore, to understand thebiological role of a protein it is of the utmost importance to know the underlying PPInetwork. This holds especially true in the case of diseases where, for example,mutations in oncogene or tumor suppressor proteins are recognized as the cause formalignancies. An impressive recent example for the relevance of the PPI interplayis the finding that active-site inhibitors targeting the oncogenic kinase B-Raf canunder certain circumstances activate the underlying signal transduction pathway(mitogen-activated protein kinase (MAPK) pathway) instead of inhibiting it [1–3].This finding is a strong reminder that nature in the majority of cases ultimatelyrelies on regulating protein function by PPIs. In addition to taking into account thisimportant concept for the drug development process, targeting PPIs significantlyenlarges the “druggable genome” that was initially estimated to comprise around1500 single protein targets [4]. While this number is still several times higher thanthe 266 human protein targets actually addressed by currently approved drugs [5],there are diseases that lack a good “conventional” target like an enzyme, receptor,or ion channel. By adding the number of PPIs occurring in the human body, theso-called protein–protein “interactome,” this situation will definitely be improved.As the size of the interactome has been estimated to lie between 130 000 [6] and650 000 [7], successfully addressing PPIs will vastly expand our opportunities forpharmacological intervention.

Protein–Protein Interactions in Drug Discovery, First Edition. Edited by Alexander Dömling.# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

j1

1.2Role of PPIs in Human Physiology

Direct physical interactions of proteins are intricately implicated in the majority ofprocesses in living organisms (Figure 1.1). For example, reception and propagationof growth signals can start with the binding of a proteinaceous signaling moleculelike the epidermal growth factor (EGF) to its cell surface receptor (EGFR). Thisbinding triggers the intracellular assembly and activation of signaling complexescomprised, for example, of adapter proteins like Grb2 and Sos and small G-proteinslike Ras that – again by physically interacting – activate protein kinases like Raf.Activated Raf then stimulates a phosphorylation cascade via the kinases MEK(mitogen-activated protein kinase/extracellular signal-related kinase) and ERK(extracellular signal-related kinase) that ultimately leads to gene activation viatranscription factors like Sp1 and Elk [8]. As each of these steps necessitates directbinding of the components of this signal transduction chain, small moleculesinhibiting these interactions could disrupt this proproliferative signaling. Further-more, stabilization of the inhibitory binding of regulatory proteins like the Rafkinase inhibitory protein (RKIP) [9] and 14-3-3 to components of the pathway (e.g.,Raf) might also produce a therapeutic benefit.Many cellular functions like motility are related to functional changes in the

cytoskeleton. For example, dynamic assembly and disassembly of actin filamentsare based on the interaction of actin with itself and with protein partners likeADF/cofilin and profilin [10]. Biological (surface) recognition, like in the immunesystem, is also mediated by PPIs as in the case of binding of lymphocyte functionassociated antigen (LFA)-1 presented on the surface of immune cells to intracellularadhesion molecule (ICAM)-1 found on the surface of endothelial cells [11]. Thisinteraction enables immune cells to attach to the walls of blood vessels and tomigrate into neighboring tissue to initiate inflammation.

Figure 1.1 Examples of the role of PPIs in human physiology.

2j 1 Protein–Protein Interactions: An Overview

The control of subcellular localization is another important aspect of proteinregulation performed by PPIs. For example, the transcription factor NFkB is pre-vented from nuclear import upon complexation with its negative regulator IkB [12].The 14-3-3 adapter proteins play a similar role in the case of the FoxO transcriptionfactor family [13]. Also, direct regulation of biochemical activity by PPIs is performedmany times byPPIs. Thephosphatase calcineurin is activatedupon complexationwithCa2þ-activated calmodulin and repressed upon binding to cabin (calcineurin bindingprotein) or calcipressin [14]. Another important process involving PPIs is thefunctional constitution of transcriptional complexes. While transcription factors ofthe Tcf (Tcell factor) LEF (lymphoid enhancer factor) family can directly bind to DNA,transcription starts only when coactivators like b-catenin additionally interact withTcf/LEF [15]. Many proteins of disease-causing organisms need host proteins ascofactors for their pathogenic activity. For example, exoenzyme S from Pseudomonasaeruginosa, an opportunistic, pneumonia-causing bacterium, has to interact withhost 14-3-3 proteins to be able to transfer an ADP-ribose moiety from NADþ tosmall G-proteins like Ras [16], thereby inhibiting its target proteins [17].

1.3Regulation of PPIs

Given the importance and number of PPIs in the living cell it is no surprise that theyhave to be tightly orchestrated at any moment in time. The occurrence and persever-ance of PPIs is governed by the two principal variables local concentration and intrinsicbinding energy of the binary interaction [18]. The first is regulated by transcriptionaland translational mechanisms, subcellular (co-)localization, degradation rates, andtemporary storage. The second can be influenced by covalent modifications likephosphorylation, and by changes in pH, ionic strength, and temperature (Figure1.2). Furthermore, additional PPIs can modulate binary interactions. They canbe inhibitory when, for example, the interaction interface of one partner is maskedby binding to the same interface or by simple sterical obstruction. They can also bestabilized, for example, when the third interacting protein binds simultaneously toboth protein partners. Such a “bridging” or “assembly platform” function has beendescribed for the A-kinase anchoring proteins (AKAPs) [19] and the kinase suppressorof Ras (KSR) [20]. It is now clear that the local architecture of such signaling complexesis one of the keys to understand regulation and specificity of signaling events.

1.4Structural Features of PPI Interfaces

PPIs can be established between identical and nonidentical protomers leading tohomo- or heterodimeric complexes, respectively. In the following, a number ofexamples are discussed in more detail. Small-molecule inhibitors have beenidentified for these PPIs (Table 1.1), strongly validating the general approach topharmacologically interfere with the interaction of proteins.

1.4 Structural Features of PPI Interfaces j3

Figure 1.2 Factors governing the occurrenceand perseverance of PPIs. Important controlmechanisms for the oligomerization state ofinteracting proteins. The association–dissociation equilibrium between monomericand multimeric states is regulated by the

partners’ local concentration and their mutualbinding affinity. Additional cellular orpharmaceutical factors can compete for onepartner or stabilize the dimeric complex.(Adapted from Nooren and Thornton [18].)

Table 1.1 Small-molecule PPI inhibitors from the pharmaceutical industry.

Target Compound Identification Affinity (mM) Reference Company

Bcl-2 ABT-737 SAR by NMR Ki¼ 0.001 [21] AbbottHDM2 Nutlin-2 HTS

(biochemical)IC50¼ 0.14 [22] Roche

TNF-a SP307 combinatorialfragmentassembly

IC50¼ 22 [23] Sunesis

RSVF1

BMS-433771 HTS (cellular) EC50¼ 0.012 [24] BMS

RSVF1

JNJ 2408068 HTS (cellular) EC50¼ 0.00016

[25] Johnson &Johnson

iNOS PPA250 HTS IC50¼ 0.082 [26] SSPiNOS compound 21b HTS Kd¼ 0.00 029 [27] Berlex

BiosciencesiNOS compound 6 HTS IC50¼ 0.012 [28] AdolorB7.1 compound 2 HTS IC50¼ 0.030 [29] WyethHPVE1/E2

compound 10 HTS IC50¼ 0.35 [30] BoehringerIngelheim

ZipA pyridylpyrimidine1

HTS Ki¼ 12 [31] Wyeth

IL-2 SP4206 tethering Kd¼ 0.07 [32] SunesisHDM2 TDP665759 HTS IC50¼ 0.7 [33] Johnson &

JohnsonLFA-1 LFA878 HTS IC50¼ 0.05 [34] NovartisLFA-1 compound 4 epitope transfer IC50¼ 0.0014 [35] Genentech/

Roche

4j 1 Protein–Protein Interactions: An Overview

1.4.1iNOS Homodimer

An example for a homodimeric protein complex is the inducible nitric oxidesynthase (iNOS) that produces the signaling molecule NO from L-arginine [36–38].To perform its catalytic activity NOS depends on the tightly bound cofactors tetrahy-dopterin (H4B),flavin adeninedinucleotide (FAD),flavinmononucleotide (FMN), andiron protoporphyrin IX (heme). This enzyme is only active as a homodimer, and thecrystal structures of the (dimeric) oxygenase domain [38] explained this fact byshowing that the dimerization interface shapes the functional binding sites for thecofactors H4B and heme (Figure 1.3). It also displays a large intersubunit cavity ofabout 750A

� 3 that is separated from the surrounding bulk solvent when a zinc ion iscoordinated by two cysteines from protomer A and two cysteines from protomer B.With69%nonpolar and31%polar aminoacids, the interface of the iNOSdimer showsa distribution that is typical for the majority of known homodimers. The contactsurface of roughly 2900A

� 2 is rather flat. Nonetheless, mainly due to the specialsituation characterized by interface-bound cofactors, inhibitors of dimer formationcould be identified successfully.

1.4.2b-Catenin/Tcf4 Complex

The Wnt pathway found to be constitutively activated in many colorectal cancers isdependent on the interaction of b-catenin with transcription factors of the Tcf/LEFfamily. Normally, the transcriptional coactivator b-catenin can be sequestered in thecytoplasm and the Tcf transcription factor is inhibited by complexation with negativeregulators of the Groucho family [39]. UponWnt activation, b-catenin is translocatedinto the nucleus and binds to Tcf to constitute the active transcriptional complex [40].

Figure 1.3 Structure of the iNOS homodimer. General topology of the iNOS dimer, andexpanded, detailed view of the dimer interface with the cysteine-coordinated zinc ion and thecofactors H4B and heme.

1.4 Structural Features of PPI Interfaces j5

The crystal structure of the human b-catenin/Tcf4 complex [41,42] revealed themultisite binding nature of the interaction with three regions of Tcf4 to be importantfor binding to b-catenin (Figure 1.4): (i) an extended N-terminal sequence,(ii) a kinked a-helix, and (iii) a second extended segment followed by the C-terminala-helix. The binding module of Tcf4 wraps around the 12-membered armadillo-repeat region of b-catenin. Three essential interaction “hotspots” have been identi-fied in the b-catenin/Tcf4 interface; a salt bridge between Tcf4 Asp16 and b-cateninLys435, a hydrophobic contact of Tcf4 Leu48 to Phe253 and Phe293 of b-catenin, anda second salt bridge between Glu29 of Tcf4 and b-catenin Lys312. Disruption of one(or several) of these contacts by a small-molecule PPI inhibitor may successfullyabolish binding of Tcf4 to b-catenin.

1.4.3LEDGF/HIV-IN Complex

For a productive infectionHIVdepends on the viral integrase (IN) that integrates thegeneticmaterial of the virus into the host cell’s DNA [43]. The human transcriptionalcoactivator LEDGF (lens epithelium-derived growth factor) is an essential hostprotein as cofactor for the function of IN that, among others, locates IN to thenucleus [44]. The interaction ismediated between the catalytic core domain (CCD) ofIN and the IN-binding domain (IBD) of LEDGF [45]. The IN CCD/LEDGF IBDcomplex crystallized as an IN CCD dimer with two LEDGF IBD copies attached atopposing sites (Figure 1.5) [46]. An interhelical loop of IBD binds to a pocket at theIN dimer interface burying approximately 1300A

� 2 of protein surface. Binding isdriven by the hydrophobic contact of LEDGF residue Ile365 to a pocket concomi-tantly established by IN residues Leu102, Ala128, Ala129, and Trp132 from onechain of the IN dimer (chain B), and Thr174 and Met178 from the other chain of the

Figure 1.4 Complex of Tcf4 (black ribbon) bound to b-catenin (gray ribbon). Three hotspots ofthe interaction are presented in structural detail with key residues of Tcf4 (black sticks) andb-catenin (light gray sticks) labeled. Polar contacts are shown as black dotted lines.

6j 1 Protein–Protein Interactions: An Overview