Protein–Protein Interactions in Drug Discovery

Home , Epelsiban, Retosiban

Volume 56 Series Editors: R.Mann hold, H.Kubinyi, G.Folkers Methods and Principles in Medicinal Chemistry Edited by Alexander Dömling Protein–Protein Interactions in Drug Discovery

Protein–Protein Interactions Dömling (Ed.) 56 in Drug Discovery www.wiley-vch.de studied Chemistry and Biology at the Technical University Munich, Germany. The main part of the book describes examples of protein targets for Prediction of intra- and inter-species protein-proteinfacilitating interactions systems biology studies Modulators of protein-protein interactions: theof importance three-dimensionality Interactive technologies for leveraging theof known anchor chemistry residues SH3 domains as drug targets P53 MDM2 antagonists: towards nontreatments genotoxic anticancer Inhibition of LFA-1/ICAM interaction for treatmentdiseases of autoimmune The PIF-binding pocket of AGCPeptidic kinases inhibitors of protein-protein interactions forreceptors cell adhesion The REPLACE strategy for generatinginhibitors non-ATP competitive of cell-cycle protein kinases reating protein-protein interactions as a novel and highly promising T G G G G G G G G G class of drug targets, thisstep volume by introduces step, the from underlying the strategies methods biology for of their PPIs investigation. to biophysical and computational which small molecule modulators havediverse been fields developed, as covering cancer, such autoimmune disordersTailor-made for and the infectious practicing diseases. medicinal chemist,includes this a ready wide reference selection ofment case pipeline studies of taken major straight pharmaceutical fromand companies the potential to develop- of illustrate this the approach. power From the contents: and more After obtaining his PhD under theResearch supervision Institute of in Ivar La Ugi, Jolla he (USA) spentperformed in a his the postdoctoral habilitation group year at of at the Nobel the Technical Laureate Scripps Universityperforms Barry of research Sharpless. Munich. at In Since the 2004 2006 University he he of teachesappointments Pittsburgh and in in Chemistry the and Department Computational of Biology. Pharmacy Inhis with 2010 tenure secondary he in became Pittsburgh. full Since professor 2011 receiving of he Groningen/The is Netherlands. chairing Dr. the Dömling department is of founderMorphochem Drug of and Design several Carmolex. at His biotech the research companies, University centers including aroundprotein the interactions discovery and of other antagonists biologically of activeinfectious protein- compounds and in neglected the tropical therapeutic diseases areas using ofreaction “out-of-the-box” oncology, chemistries. software tools and multicomponent Alexander Dömling

Edited by Alexander D€omling

Protein–Protein Interactions in Drug Discovery Related Titles Methods and Principles in Medicinal Chemistry Edited by R. Mannhold, H. Kubinyi, G. Folkers Editorial Board H. Buschmann, H. Timmerman, H. van de Waterbeemd, T. Wieland

Previous Volumes of this Series

Kalgutkar, Amit S. / Dalvie, Deepak / De Clercq, Erik (Ed.) Obach, R. Scott / Smith, Dennis A. Antiviral Drug Strategies Reactive Drug Metabolites 2011 2012 ISBN: 978-3-527-32696-9, Vol. 50 ISBN: 978-3-527-33085-0, Vol. 55 Klebl, Bert / Muller,€ Gerhard / Hamacher, Brown, Nathan (Ed.) Michael (Eds.) Bioisosteres in Medicinal Protein Kinases as Drug Chemistry Targets 2012 2011 ISBN: 978-3-527-33015-7, Vol. 54 ISBN: 978-3-527-31790-5, Vol. 49

Gohlke, Holger (Ed.) Sotriffer, Christoph (Ed.) Protein-Ligand Interactions Virtual Screening 2012 Principles, Challenges, and Practical ISBN: 978-3-527-32966-3, Vol. 53 Guidelines 2011 Kappe, C. Oliver / Stadler, Alexander / ISBN: 978-3-527-32636-5, Vol. 48 Dallinger, Doris Microwaves in Organic and Rautio, Jarkko (Ed.) Medicinal Chemistry Prodrugs and Targeted Second, Completely Revised and Delivery Towards Better ADME Enlarged Edition Properties 2012 2011 ISBN: 978-3-527-33185-7, Vol. 52 ISBN: 978-3-527-32603-7, Vol. 47

Smith, Dennis A. / Allerton, Charlotte / Smit, Martine J. / Lira, Sergio A. / Leurs, Kalgutkar, Amit S. / van de Waterbeemd, Rob (Eds.) Han / Walker, Don K. Chemokine Receptors as Drug Pharmacokinetics and Targets Metabolism in Drug Design 2011 Third, Revised and Updated Edition ISBN: 978-3-527-32118-6, Vol. 46 2012 ISBN: 978-3-527-32954-0, Vol. 51 Edited by Alexander D€omling

Protein–Protein Interactions in Drug Discovery Series Editors All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and Prof. Dr. Raimund Mannhold publisher do not warrant the information contained Molecular Drug Research Group in these books, including this book, to be free of Heinrich-Heine-Universit€at errors. Readers are advised to keep in mind that Universit€atsstrasse 1 statements, data, illustrations, procedural details or 40225 Dusseldorf€ other items may inadvertently be inaccurate. Germany Library of Congress Card No.: applied for [email protected] British Library Cataloguing-in-Publication Data Prof. Dr. Hugo Kubinyi A catalogue record for this book is available from the Donnersbergstrasse 9 British Library. 67256 Weisenheim am Sand Bibliographic information published by the Deutsche Germany Nationalbibliothek [email protected] The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; Prof. Dr. Gerd Folkers detailed bibliographic data are available on the Collegium Helveticum Internet at < http:// dnb.d-nb.d e> . STW/ETH Zurich 8092 Zurich # 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Switzerland Boschstr. 12, 69469 Weinheim, Germany [email protected] All rights reserved (including those of translation into other languages). No part of this book may be Volume Editor reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or Prof. Dr. Alexander D€omling translated into a machine language without written University of Groningen permission from the publishers. Registered names, School of Pharmacy trademarks, etc. used in this book, even when not Drug Design specifically marked as such, are not to be considered Antonius Deusinglaan 1 unprotected by law. 9713 AV Groningen Netherlands Composition Thomson Digital, Noida, India

Printing and Binding Markono Print Media Pte Ltd, Cover Singapore Cut-away view of the co-crystal structure of an archetypical protein- protein interaction: the Cover Design Schulz Graﬁk-Design, Fugonheim€ tumor suppressor p53 and the oncogene Print ISBN: 978-3-527-33107-9 MDM2 (PDB ID 1YCR). The most deepest ePDF ISBN: 978-3-527-64823-8 buried p53 amino acid Trp23, anchoring the ePub ISBN: 978-3-527-64822-1 PPI, is shown in sticks presentation, the MDM2 mobi ISBN: 978-3-527-64821-4 receptor is shown as surface. Also shown the oBook ISBN: 978-3-527-64820-7 hydrogen bond between the Trp23 indole-NH and the receptor Leu54 carbonyl as green Printed in Singapore dotted line. The picture was rendered using Printed on acid-free paper PYMOL software. jV

Contents

List of Contributors XI Preface XV A Personal Foreword XVII

1 Protein–Protein Interactions: An Overview 1 Christian Ottman 1.1 Introduction 1 1.2 Role of PPIs in Human Physiology 2 1.3 Regulation of PPIs 3 1.4 Structural Features of PPI Interfaces 3 1.4.1 iNOS Homodimer 5 1.4.2 b-Catenin/Tcf4 Complex 5 1.4.3 LEDGF/HIV-IN Complex 6 1.4.4 HPV E1/E2 Complex 7 1.4.5 IFN-a/IFNAR Complex 8 1.4.6 TNF-a Trimer 9 1.5 Identiﬁcation of PPI Inhibitors 10 1.6 Conclusions and Outlook 13 References 14

2 Prediction of Intra- and Interspecies Protein–Protein Interactions Facilitating Systems Biology Studies 21 Sylvia Schleker, Seshan Ananthasubramanian, Judith Klein-Seetharaman, and Madhavi K. Ganapathiraju 2.1 Introduction: Relevance of Interactome Studies to Disease and Drug Discovery 21 2.2 Our Current Knowledge of Interactomes Identiﬁed from Experiments is Incomplete 23 2.3 Reliability of Interactions Identiﬁed Experimentally 24 2.4 Computational Methods for PPI Prediction 27 2.4.1 Conservation of Gene Neighborhood 27 2.4.2 Gene Fusion 28 2.4.3 Sequence-Based Coevolution 28 VIj Contents

2.4.4 Phylogenetic Proﬁling 28 2.4.5 Gene Expression 29 2.4.6 Structural Similarity 29 2.4.7 Integration Approaches 29 2.5 Sources of Biological Data in Use to Predict PPIs 30 2.6 Survey of Current Interactomes 32 2.6.1 Human Intraspecies Interactomes 32 2.6.2 Bacteria Intraspecies Interactomes 37 2.6.2.1 High-Throughput Experimental Approaches to Identify Intraspecies Bacterial Interactions 37 2.6.2.2 Modeling Intraspecies Bacterial Interactions 39 2.6.3 Bacteria–Human Interspecies Interactomes 40 2.6.3.1 Experimental Approaches to Identify Bacteria–Human PPIs 40 2.6.3.2 Modeling Bacteria–Human PPIs 40 2.6.4 Non-PPI Intraspecies Bacterial and Bacteria–Human Interspecies Interactome Models 41 2.6.5 Virus–Human Interspecies Interactomes 42 References 43

3 Modulators of Protein–Protein Interactions: Importance of Three-Dimensionality 55 David C. Fry and Sung-Sau So 3.1 Introduction 55 3.2 Study 56 3.3 Discussion 58 3.4 Summary 61 References 61

4 A Leap into the Chemical Space of Protein–Protein Interaction Inhibitors 63 Bruno O. Villoutreix, C. Labbe, David Lagorce, Guillaume Laconde, and Olivier Sperandio 4.1 Introduction 63 4.2 Types of Interaction 64 4.3 Properties of the Interface 65 4.4 Orthosteric versus Allosteric Modulation 66 4.5 Leap into the iPPI Chemical Space 66 4.5.1 Seminal Works 66 4.5.2 Road to a Rationalization of the iPPI Chemical Space 67 4.6 Case Study 68 4.6.1 Visualizing the iPPI Chemical Space 70 4.6.2 iPPI versus ADME/Tox Properties 71 4.6.3 iPPI versus Aromaticity 75 4.6.4 iPPI versus Chemical Complexity 77 Contents jVII

4.6.5 iPPI versus Molecular Shape 77 4.6.6 iPPI versus Potency 79 4.7 Conclusions 80 References 81

5 Interactive Technologies for Leveraging the Known Chemistry of Anchor Residues to Disrupt Protein Interactions 85 Carlos J. Camacho, David R. Koes, and Alexander S. D€omling 5.1 Introduction 85 5.2 Druggable Sites in PPIs 86 5.3 Structure-Based Library Design – A Powerful Alternative to High-Throughput Screening 87 5.4 New MCR Chemistry to Design PPI Antagonists 89 5.5 Virtual Screening 90 5.6 New Interactive Modeling Techniques for Medicinal Chemists 93 5.7 New Ideas: Hit Rate Validation of Anchor-Centered Screening of p53/MDM2/4 95 5.8 Summary 96 References 97

6 SH3 Domains as Drug Targets 101 James Luccarelli, Sam Thompson, and Andrew D. Hamilton 6.1 Introduction 101 6.2 Structure 101 6.3 Variability 102 6.4 SH3 Binding Motifs 104 6.4.1 Classical Binding Motifs 104 6.4.2 Tyrosine-Containing Motifs 107 6.4.3 RxxK Motif 108 6.4.4 Other Binding Motifs from Proteomic Screens 109 6.4.5 Tertiary Interactions 110 6.5 Selectivity 111 6.6 Drug Target Selection 114 6.7 Inhibition Strategies: Peptide and Peptoid Inhibitors 114 6.7.1 Peptide Ligands 114 6.7.2 Combinatorial Approaches 115 6.7.3 Peptide Dimers 116 6.7.4 Constrained Peptides 118 6.7.5 N-Substituted Peptoids 118 6.8 Small-Molecule Inhibitors 119 6.9 Conclusions 122 References 122 VIIIj Contents

7 p53/MDM2 Antagonists: Towards Nongenotoxic Anticancer Treatments 129 Kareem Khoury, Tad A. Holak, and Alexander D€omling 7.1 Introduction 129 7.2 p53/MDM2 PPI is Characterized by Many Cocrystal Structures 130 7.3 Nutlins: First-In-Class MDM2 Antagonists 131 7.4 Johnson & Johnson: Benzodiazepines 133 7.5 Amgen: Chromenotriazolopyrimidines & Piperidones 137 7.6 University of Michigan: Spirooxindole 148 7.7 University of Pittsburgh: Ugi Based Compounds 153 7.8 University of Newcastle: Some Scaffolds With No Structural Biology Information 155 7.9 Outlook 161 References 161

8 Inhibition of LFA-1/ICAM Interaction for the Treatment of Autoimmune Diseases 165 Kevin M. Guckian and Daniel M. Scott 8.1 Introduction 165 8.2 Integrin Structure and Activation 166 8.3 Direct Inhibition of the LFA-1/ICAM Interaction 168 8.4 Allosteric Inhibitors of the LFA-1/ICAM interaction – IDAS Site 171 8.4.1 Abbott/ICOS/Biogen Series 171 8.4.2 Boehringer Ingelheim/Tanabe Seiyaku/Bristol-Myers Squibb Series 178 8.5 Summary 183 References 183

9 The PIF Pocket of AGC Kinases: A Target Site for Allosteric Modulators and Protein–Protein Interaction Inhibitors 187 Matthias Engel 9.1 Introduction 187 9.2 Discovery and Physiological Functions of the PIF Pocket 190 9.3 Properties of the PIF Pocket Relevant to Drug Development 192 9.3.1 The PIF Pocket Offers the Potential to Develop Highly Selective Ligands 192 9.3.2 Molecular Interactions of the Natural HM Peptide Ligands 193 9.3.3 Allosteric Mechanism of the PIF Pocket 196 9.3.4 Structural Plasticity of the PIF Pocket 198 9.4 Small-Molecule PIF Pocket Ligands 199 9.4.1 Allosteric Activators and PPI Inhibitors of PDK1 199 9.4.2 Identiﬁcation of First Hit Compounds Using a Pharmacophore-Based Screening Approach 200 Contents jIX

9.4.3 Current State of Research on PIF Pocket-Directed PDK1 Modulators 203 9.4.4 Allosteric Inhibitors 207 9.5 Potential Supportive Effects Enhancing the Cellular Activity of PIF Pocket-Binding Modulators 209 9.5.1 Allosteric Activators of PDK1 209 9.5.2 PIF Pocket-Directed Inhibitors of AGC Kinases 210 9.6 Conclusions 212 9.6.1 Is the PIF Pocket a Druggable Site? 212 9.6.2 General Medicinal Chemistry Aspects and Outlook 213 References 215

10 Retosiban and Epelsiban: Potent and Selective Orally Available Oxytocin Antagonists 225 Alan D. Borthwick and John Liddle 10.1 Introduction 225 10.2 Aryl-2,5-DKP Template Discovery and Initial Structure–Activity Relationship Studies 227 10.3 Synthesis of the RRR and RRS 6-Indanyl-3-isobutyl-7-aryl-2,5-DKP Secondary Amides 231 10.4 Comparison of Crystal Structures of Oxytocin and 2,5-DKPs 231 10.5 Pharmacokinetics and Property-Based Design 232 10.6 In Vivo Potency of 20,40-Diflurophenyl Dimethylamide 22 235 10.7 Synthesis of Tertiary Amides 236 10.7.1 Synthesis of Five- and Six-Membered Heterocyclic 2,5-DKPs 237 10.8 Summary of Lead Oxytocin Antagonist 20,40-Diflurophenyl Dimethylamide 22 238 10.9 Further Modifications, Five- and Six-Membered Heterocyclic Derivatives 238 10.10 Five-Membered Heterocyclic Derivatives and Retosiban 239 10.10.1 Oxytocin Antagonist Activity and Selectivity versus Human Vasopressin Receptors 242 10.10.2 In Vivo Potency and Synthesis 243 10.11 Summary of Lead Oxytocin Antagonist Retosiban 56 244 10.12 Six-Membered Heterocyclic Derivatives and Epelsiban 244 10.12.1 Monosubstituted Pyridyl isoButyl Derivatives 246 10.12.2 Modification of isoButyl in 60-MePyridyl Derivatives 246 10.12.3 Dimethylpyridyl (S)-sec-Butyl Amides 246 10.12.4 Further Evaluation of 20,60-Dimethyl-30-pyridine Morpholine Amide 77 250 10.13 Summary of Lead Oxytocin Antagonist Epelsiban 77 252 10.14 Comparison of Lead Compounds 252 10.15 Conclusions 254 References 254 Xj Contents

11 Peptidic Inhibitors of Protein–Protein Interactions for Cell Adhesion Receptors: RGD Peptides and Beyond 257 Carlos Mas-Moruno and Horst Kessler 11.1 Introduction 257 11.2 From the Discovery of the RGD Motif in FN to the First Selective Cyclic RGD Peptide 258 11.2.1 RGD Sequence, Integrins, and Receptor Selectivity 258 11.2.2 Concept of Spatial Screening in Cyclic RGD Peptides 261 11.2.3 Conformational Aspects and Selectivity of c(RGDfV) 263 a b 265 11.2.4 Pharmacophoric Requirements of c(RGDfV) to Bind v 3 11.3 N-Methylation of c(RGDfV): Cilengitide and Beyond 267 11.3.1 Concept of N-Methylation 267 11.3.2 N-Methyl Scan of c(RGDV): Synthesis, Biological Activity, and Structural Considerations of Cilengitide 268 N a b 11.3.3 Beyond Cilengitide: di- -Methylated Analogs of c(RGDfV) and v 3 Selectivity 271 11.4 isoDGR Sequence as a New Integrin-Binding Motif 274 11.4.1 Formation of isoAsp Residues in Peptides and Proteins 274 11.4.2 NGR Deamidation to isoDGR Yields a New Integrin-Binding Motif 275 11.4.3 Design of Cyclic Peptides Containing the isoDGR Motif as New Integrin Antagonists 276 11.4.4 Receptor Selectivity of Cyclic isoDGR Peptides 279 11.5 Conclusions 281 References 282

12 REPLACE Strategy for Generating Non-ATP-Competitive Inhibitors of Cell Cycle Protein Kinases 291 Campbell McInnes 12.1 Introduction 291 12.2 Inhibition of CDKs Through the Cyclin Groove 291 12.3 Inhibitors of PLKs 298 12.3.1 PB Domain 298 12.4 Conclusions 301 References 302

Index 305 jXI

List of Contributors

Seshan Ananthasubramanian Alexander S. D€omling University of Pittsburgh University of Pittsburgh School of Medicine Department of Pharmacy and Department of Biomedical Informat- Computational and Systems Biology ics and Intelligent Systems Program 3501 Fifth Avenue 5607 Baum Blvd, Suite 401 Suite 3064 Pittsburgh Biomedical Science Tower 3 PA 15206 Pittsburgh, PA 15260 USA USA

Alan D. Borthwick and DrugMolDesign 15 Temple Grove University of Groningen London NW11 7UA Department of Drug Design UK A. Deusinglaan 1 9713 AV Groningen Carlos J. Camacho Netherlands University of Pittsburgh Department of Computational and Matthias Engel Systems Biology University of Saarland 3501 Fifth Avenue Research Group PhosphoSites Suite 3064 Department of Pharmaceutical and Biomedical Science Tower 3 Medicinal Chemistry Pittsburgh, PA 15260 Campus C2 2 USA PO Box 151150 66041 Saarbrucken€ Germany XIIj List of Contributors

David C. Fry Kareem Khoury Roche Research Center University of Pittsburgh Discovery Technologies School of Pharmacy 340 Kingsland Street 1104 Salk Hall Nutley, NJ 07110 3501 Terrace Street USA Pittsburgh, PA 15261 USA Madhavi K. Ganapathiraju University of Pittsburgh Judith Klein-Seetharaman School of Medicine Institute of Complex Systems Department of Biomedical Informatics ICS-5: Molecular Biophysics and Intelligent Systems Program 52425 Jülich 5607 Baum Blvd, Suite 401 Germany Pittsburgh, PA 15206 USA and

Kevin M. Guckian Department of Structural Biology BiogenIdec Inc. University of Pittsburgh Medicinal Chemistry School of Medicine Room 2051 14 Cambridge Center Biomedical Science Cambridge, MA 02142 Tower 3 USA 3501 Fifth Avenue Pittsburgh, PA 15261 Andrew D. Hamilton USA University of Oxford Chemistry Research Laboratory David R. Koes 12 Mansﬁeld Road University of Pittsburgh Oxford OX1 3TA Department of Computational and UK Systems Biology Tad A. Holak 3501 Fifth Avenue Max Planck Institute of Biochemistry Suite 3064 NMR Group Biomedical Science Tower 3 Am Klopferspitz 18 Pittsburgh, PA 15260 82152 Martinsried USA Germany C. Labbe Horst Kessler INSERM Technische Universit€at Munchen€ U973 Institute for Advanced Study and 35 rue H. Brion Center of Integrated Protein Science 75205 Paris Department Chemie France Lichtenbergstrasse 4 85747 Garching Germany List of Contributors jXIII and Carlos Mas-Moruno Technische Universit€at Munchen€ Universite Paris Diderot Institute for Advanced Study and Sorbonne Paris Cite Center of Integrated Protein Science UMRS 973 Department Chemie MTi Lichtenbergstrasse 4 35 rue H. Brion 85747 Garching 75205 Paris Germany France Campbell McInnes Guillaume Laconde University of South Carolina INSERM Pharmaceutical and Biomedical U973 Sciences 35 rue H. Brion South Carolina College of Pharmacy 75205 Paris Coker Life Science Building France 715 Sumter Street Columbia, SC 29208 and USA

Universite Paris Diderot Christian Ottmann Sorbonne Paris Cite Max Planck Society UMRS 973 Chemical Genomics Centre MTi Otto-Hahn-Strasse 15 35 rue H. Brion 44227 Dortmund 75205 Paris Germany France Sylvia Schleker John Liddle Institute of Complex Systems GlaxoSmithKline Research and ICS-5: Molekulare Biophysik Development 52425 Julich€ Department of Medicinal Chemistry Germany Medicines Research Centre Gunnels Wood Road and Stevenage SG1 2NY UK University of Pittsburgh School of Medicine James Luccarelli Department of Structural Biology University of Oxford Pittsburgh, PA 15260 Chemistry Research Laboratory USA 12 Mansﬁeld Road Oxford OX1 3TA UK XIVj List of Contributors

Sung-Sau So Sam Thompson Roche Research Center University of Oxford Discovery Chemistry Chemistry Research Laboratory 340 Kingsland Street 12 Mansﬁeld Road Nutley, NJ 07110 Oxford OX1 3TA USA UK

Daniel M. Scott Bruno O. Villoutreix BiogenIdec Inc. INSERM Medicinal Chemistry U973 14 Cambridge Center 35 rue H. Brion Cambridge, MA 02142 75205 Paris USA France

Olivier Sperandio and INSERM U973 Universite Paris Diderot 35 rue H. Brion Sorbonne Paris Cite 75205 Paris UMRS 973 France MTi 35 rue H. Brion and 75205 Paris France Universite Paris Diderot Sorbonne Paris Cite UMRS 973 MTi 35 rue H. Brion 75205 Paris France jXV

Preface

In the common definition of the proteome it is considered to be the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions. It is larger than the genome, due to special mechanisms in the biogenesis of proteins, which render them more numerous than the genes at the end. What, however is very special to the proteome is its enriched complexity which arises both from the protein’s 3D structure and the functional interaction of the 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, with modules connected through regulators, mediators or adaptors, the date hubs. Party hubs represent integral elements within distinct modules and, although important for the functions mediated by these modules (and therefore likely to be essential proteins), tend to function at a lower level of the organization of the proteome. We propose that date hubs participate in a wide range of integrated connections required for a global organization of biological modules in the whole proteome network (although some date hubs could simply be ‘shared’ between, and mediate local functions inside, overlapping modules). (. . .) Finally, it is possible that discriminating between date and party hubs might also help to define new therapeutic drug targets.1 Well, there we are in defining ambitious goals for drug development. Interfering with the party gossip and intruding the clandestine dates of proteins for sake of therapeutic 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 clearly undruggable for a long time. With the advent and success of monoclonal therapeutic antibodies (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. Evidence for dynamically organized modularity in the yeast protein–protein interaction network. Nature, 430, 2004, pp. 88-93 XVIj Preface

and immune-disease areas showed that interfering with protein-protein interactions (PPIs) is indeed a practicable approach. Unfortunately the proteins parties and dates are taken place inside the cell, with no admittance for mAbs. Hence, the challenge or the ”reaching for high-hanging fruit”2 is to interfere therapeutically by use of small molecules. In the present volume Alexander Domling€ and his co-authors depict a scenario that reveals more than a silver lining on the horizon. There is a variety of new (and old) targets and a plethora of new approaches and insights into molecular mechanisms which raise the hope that in the near future small molecule interference of protein-protein interactions will emerge as a new functional class of therapeutics. Twelve chapters illuminate opportunities, strategies, success stories and pitfalls in the 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 a hopefully greater commitment in drug discovery focusing PPIs. Not least because of this, the series editors are indebted to the authors and the editor who made this comprehensive issue possible. We are convinced that the book represents an important contribution to the body of knowledge in drug discovery and that it matches the interests of many researches who have adjourned to a promising but rocky ﬁeld. In addition, we are very much indebted to Frank Weinreich and Heike Nothe,€ both at Wiley-VCH. Their support and ongoing engagement, not only for this book but for the whole series “Methods and Principles in Medicinal Chemistry” adds to the success of this excellent collection of monographs on various topics, all related to drug research.

November 2012 Dusseldorf€ Raimund Mannhold Weisenheim am Sand Hugo Kubinyi Zurich€ Gerd Folkers

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

A Personal Foreword

Therapeutic targeting of protein–protein interactions (PPIs) has been in the past largely the domain of biotech industries. With their exquisite natural selection process, and the resulting high affinity and selectivity towards their targets, monoclonal antibodies (mAbs) and similar biologics have been the only way to target large protein interfaces to effectively compete with the endogenous interaction. 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 small molecules towards the biotech drugs has recently been observed based on the therapeutic and business success of the biologics; major takeovers of biotech companies by big pharma excite the industry and shareholders. Severe conditions can meanwhile be treated with mAbs, including some cancers, Crohn’s disease, rheumatoid arthritis, transplant rejection, or macular degeneration, just to name a few. However, large biomolecules like mAbs also have intrinsic disadvantages that are hard to overcome, such as lack of oral bioavailability, high cost of goods, and perhaps 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 with advantageous pharmacokinetics/pharmacodynamics properties, they can be produced orders of magnitude cheaper then biologics, and they can also be designed to penetrate cell membranes to reach intracellular targets. Therefore, small molecules should be ideal to disrupt PPIs. However, PPIs very often consist of large interfaces of greater than 1000 A2 and do not contain the deep pockets medicinal chemists are used to from other target areas, such as kinases, G-protein-coupled receptors, or proteases. Pocket dimension, form (concave versus convex), and hydrophobicity are, however, key features defining the druggability by small molecules. Analysis of the plethora of available crystal structures of PPIs reveals that only a fraction of PPIs fit the druggability criteria. Nevertheless, while PPIs as a class were considered as undruggable a decade ago, several small molecules advancing into clinical trials recently clearly support the notion that at least a fraction of the PPIs are not only attractive therapeutic targets, but can also be developed to drugs. It can be foreseen XVIIIj A Personal Foreword

that in the future many more small molecules targeting PPIs will emerge for the treatment 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 their fields I try to introduce the reader to the problems and opportunities associated with targeting PPIs by small molecules. Introductory overviews pave the way for specific medicinal chemistry showcases on developmental compounds. Additionally, several chapters introduce technologies relevant for PPI drug discovery, including the interactome and computational chemistry. Ottmann gives an excellent introductory overview on different PPI drug discovery approaches and compound classes, and the structural features of their interacting proteins. 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 the current experimental and computational identification approaches to obtain interactomes related to human are discussed. Cleary only careful comparison of the large-scale results of orthogonal experimental approaches can lead to meaningful results 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. From structural analysis based on the Protein Data Bank (PDB) database they deduce simple rules 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 why traditional chemical space is relatively unsuited to find PPI antagonists. The specifics of PPI-targeting compound classes are elaborated in a detailed analysis of many descriptors. Rather novel scaffold and privileged structures are needed to address 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 the disproportional high importance of anchoring residues in PPIs, and queries the very large 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 therapeutic areas. The structural biology is reviewed and, based on the understanding of specific 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 early clinical development. Khoury et al. review this area with a focus on small-molecule classes 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 A Personal Foreword jXIX treatment of autoimmune diseases. LFA-1/ICAM is a case where a recently approved 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 advantages of targeting non-ATP sites of kinases would be to obtain more selective compounds and to target kinases previously considered to be nondruggable. The recent discovery of different classes of AGC kinase pocket binders makes this an important area of research. The discovery and medicinal chemistry evolution of oxytocin receptor antagonists is reviewed by Borthwick and Liddle. Their efforts have led to Retosiban and Epelsiban – two orally bioavailable oxytocin receptor antagonists with superior potency 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 small molecules, much can be learned from the specific modes by which peptides interact with 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 recognition site. The REPLACE algorithm for the structure-based prediction of PPI binders has been applied to the Polo-box domain of PLK1 (Polo-like kinase 1), resulting in small peptides 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 the present book. Many interesting works could not be included. To finish, and in the tradition of the Editor’s choice, I want to point the reader to several more exciting recent 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 patients bear constitutively active Ras mutations that drive cancer growth. However, until recently no small-molecule drug has been known to directly bind to and effectively modify the downstream pathway. Lately, novel biophysical techniques have shown promise by identifying small molecules inhibiting the Ras effector complex Ras–Sos (Figure 1a) and cocrystal structures solved by two independent groups provide hope that the initial fragment-based hits can be further elaborated towards effective drugs [1,2]. Protein lysine methyltransferases are important regulators of epigenetic signaling. The oncogenic protein SMYD2 represses the tumor suppressors p53 and Rb by a PPI. The use of structural biology information cannot be overestimated, and the structure of apo-SMYD2, SMYD2 bound to a methylated p53 peptide, and in complex with a small molecule competitively inhibiting p53 binding was published recently (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. XXj A Personal Foreword

(a) (b) S HO

N H NH N N CI HN N H O O HO O CI

N N N OH S N

Figure 1 (a) Indole-3-thiocarbonylpiperidine molecule anchoring via the central fragment bound to Ras on the Sos interacting hydroxyproline ring to the VHL complex and interface (PDB ID: 4EPV). (b) Small molecule thus inhibiting the contact to HIF-1a (PDB ID: mimicking the p53 Lys-Me binding to SMYD2 3ZRC). (d) Cut-away view of the Gewald and extending with the dichlorophenyl moiety thiophenodiazepine JQ1 bound into the into a nearby hydrophobic pocket not occupied bromodomain of BRD4 into the Ac-Lys binding by the peptide (PDB ID: 3S7B). (c) Small site (PDB ID: 3MXF). A Personal Foreword jXXI

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 guided medicinal chemistry the first small molecule ligand for VHL was recently described. The authors used the hydroxyproline motif of HIF-1a as an “anchoring” starting point for their medicinal chemistry efforts (Figure 1c) [4]. Derivatives of the first- generation compounds might evolve into cell-penetrating chemical probes to test the 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 other proteins. Two small-molecule inhibitors of the LysAc-bromodomain PPI are currently undergoing clinical evaluation for the treatment of artherosclerotic cardio- vascular disease, and testis midline and other cancers (Figure 1d) [5,6]. As for many areas in PPIs, the availability of X-ray structures of Apo, Ac-Lys, and ligand-bound protein proved to be invaluable in the early discovery process. A major challenge of the area will be the design of selectivity into the small molecules targeting one of the 61 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 Domling€ Groningen

References

1 Maurer, T. et al. (2102) Proceedings of the 4 Buckley, D.L. et al. (2012) Journal of the National Academy of Sciences of the United American Chemical Society, 134, 4465. States of America, 109, 5299. 5 Filippakopoulos, P. et al. (2010) Nature, 2 Sun, Q. et al. (2102) Angewandte Chemie 468, 1067. International Edition, 51, 6140. 6 Nicodeme, E. et al. (2010) Nature, 468, 3 Ferguson, A.D. et al. (2011) Structure, 19, 1119. 1207.

1 Protein–Protein Interactions: An Overview Christian Ottmann

1.1 Introduction

Protein–protein interactions (PPIs) are implicated in almost all biological processes for any given protein engaged in complexes with other proteins for the majority of its lifetime. In this regard, proteins function not merely as single, isolated entities, but display their roles by interacting with other cellular components. The different interaction patterns are at least as important as the intrinsic biochemical activity status (e.g., of a protein kinase) of the protein itself. Therefore, to understand the biological role of a protein it is of the utmost importance to know the underlying PPI network. This holds especially true in the case of diseases where, for example, mutations in oncogene or tumor suppressor proteins are recognized as the cause for malignancies. An impressive recent example for the relevance of the PPI interplay is the finding that active-site inhibitors targeting the oncogenic kinase B-Raf can under 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 ultimately relies on regulating protein function by PPIs. In addition to taking into account this important concept for the drug development process, targeting PPIs significantly enlarges the “druggable genome” that was initially estimated to comprise around 1500 single protein targets [4]. While this number is still several times higher than the 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, the so-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] and 650 000 [7], successfully addressing PPIs will vastly expand our opportunities for pharmacological 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. 2j 1 Protein–Protein Interactions: An Overview

1.2 Role of PPIs in Human Physiology

Direct physical interactions of proteins are intricately implicated in the majority of processes in living organisms (Figure 1.1). For example, reception and propagation of growth signals can start with the binding of a proteinaceous signaling molecule like the epidermal growth factor (EGF) to its cell surface receptor (EGFR). This binding triggers the intracellular assembly and activation of signaling complexes comprised, for example, of adapter proteins like Grb2 and Sos and small G-proteins like 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 via transcription factors like Sp1 and Elk [8]. As each of these steps necessitates direct binding of the components of this signal transduction chain, small molecules inhibiting these interactions could disrupt this proproliferative signaling. Further- more, stabilization of the inhibitory binding of regulatory proteins like the Raf kinase 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 filaments are based on the interaction of actin with itself and with protein partners like ADF/cofilin and profilin [10]. Biological (surface) recognition, like in the immune system, is also mediated by PPIs as in the case of binding of lymphocyte function associated antigen (LFA)-1 presented on the surface of immune cells to intracellular adhesion molecule (ICAM)-1 found on the surface of endothelial cells [11]. This interaction enables immune cells to attach to the walls of blood vessels and to migrate into neighboring tissue to initiate inflammation.

Figure 1.1 Examples of the role of PPIs in human physiology. 1.4 Structural Features of PPI Interfaces j3

The control of subcellular localization is another important aspect of protein regulation 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 transcription factor family [13]. Also, direct regulation of biochemical activity by PPIs is performed many times by PPIs. The phosphatase calcineurin is activated upon complexation with þ Ca2 -activated calmodulin and repressed upon binding to cabin (calcineurin binding protein) or calcipressin [14]. Another important process involving PPIs is the functional constitution of transcriptional complexes. While transcription factors of the Tcf (Tcell factor) LEF (lymphoid enhancer factor) family can directly bind to DNA, transcription starts only when coactivators like b-catenin additionally interact with Tcf/LEF [15]. Many proteins of disease-causing organisms need host proteins as cofactors for their pathogenic activity. For example, exoenzyme S from Pseudomonas aeruginosa, an opportunistic, pneumonia-causing bacterium, has to interact with þ host 14-3-3 proteins to be able to transfer an ADP-ribose moiety from NAD to small G-proteins like Ras [16], thereby inhibiting its target proteins [17].

1.3 Regulation of PPIs

Given the importance and number of PPIs in the living cell it is no surprise that they have to be tightly orchestrated at any moment in time. The occurrence and perseverance of PPIs is governed by the two principal variables local concentration and intrinsic binding energy of the binary interaction [18]. The first is regulated by transcriptional and translational mechanisms, subcellular (co-)localization, degradation rates, and temporary storage. The second can be influenced by covalent modifications like phosphorylation, and by changes in pH, ionic strength, and temperature (Figure 1.2). Furthermore, additional PPIs can modulate binary interactions. They can be inhibitory when, for example, the interaction interface of one partner is masked by binding to the same interface or by simple sterical obstruction. They can also be stabilized, for example, when the third interacting protein binds simultaneously to both protein partners. Such a “bridging” or “assembly platform” function has been described for the A-kinase anchoring proteins (AKAPs) [19] and the kinase suppressor of Ras (KSR) [20]. It is now clear that the local architecture of such signaling complexes is one of the keys to understand regulation and specificity of signaling events.

1.4 Structural Features of PPI Interfaces

PPIs can be established between identical and nonidentical protomers leading to homo- or heterodimeric complexes, respectively. In the following, a number of examples are discussed in more detail. Small-molecule inhibitors have been identiﬁed for these PPIs (Table 1.1), strongly validating the general approach to pharmacologically interfere with the interaction of proteins. 4j 1 Protein–Protein Interactions: An Overview

Figure 1.2 Factors governing the occurrence partners’ local concentration and their mutual and perseverance of PPIs. Important control binding affinity. Additional cellular or mechanisms for the oligomerization state of pharmaceutical factors can compete for one interacting proteins. The association– partner or stabilize the dimeric complex. dissociation equilibrium between monomeric (Adapted from Nooren and Thornton [18].) and multimeric states is regulated by the

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 Ki ¼ 0.001 [21] Abbott ¼ HDM2 Nutlin-2 HTS IC50 0.14 [22] Roche (biochemical) a ¼ TNF- SP307 combinatorial IC50 22 [23] Sunesis fragment assembly

RSV BMS-433771 HTS (cellular) EC50 ¼ 0.012 [24] BMS F1 RSV JNJ 2408068 HTS (cellular) EC50 ¼ 0.00 [25] Johnson & F1 016 Johnson iNOS PPA250 HTS IC50 ¼ 0.082 [26] SSP iNOS compound 21b HTS Kd ¼ 0.00 029 [27] Berlex Biosciences

iNOS compound 6 HTS IC50 ¼ 0.012 [28] Adolor B7.1 compound 2 HTS IC50 ¼ 0.030 [29] Wyeth HPV compound 10 HTS IC50 ¼ 0.35 [30] Boehringer E1/E2 Ingelheim

ZipA pyridylpyrimidine HTS Ki ¼ 12 [31] Wyeth 1

IL-2 SP4206 tethering Kd ¼ 0.07 [32] Sunesis ¼ HDM2 TDP665759 HTS IC50 0.7 [33] Johnson & Johnson ¼ LFA-1 LFA878 HTS IC50 0.05 [34] Novartis LFA-1 compound 4 epitope transfer IC50 ¼ 0.0014 [35] Genentech/ Roche 1.4 Structural Features of PPI Interfaces j5

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

1.4.1 iNOS Homodimer

An example for a homodimeric protein complex is the inducible nitric oxide synthase (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- fl fl dopterin (H4B), avin adenine dinucleotide (FAD), avin mononucleotide (FMN), and iron protoporphyrin IX (heme). This enzyme is only active as a homodimer, and the crystal structures of the (dimeric) oxygenase domain [38] explained this fact by showing that the dimerization interface shapes the functional binding sites for the cofactors H4B and heme (Figure 1.3). It also displays a large intersubunit cavity of about 750 A3 that is separated from the surrounding bulk solvent when a zinc ion is coordinated by two cysteines from protomer A and two cysteines from protomer B. With 69% nonpolar and 31% polar amino acids, the interface of the iNOS dimer shows a distribution that is typical for the majority of known homodimers. The contact surface of roughly 2900 A2 is rather flat. Nonetheless, mainly due to the special situation characterized by interface-bound cofactors, inhibitors of dimer formation could be identified successfully.

1.4.2 b-Catenin/Tcf4 Complex

The Wnt pathway found to be constitutively activated in many colorectal cancers is dependent on the interaction of b-catenin with transcription factors of the Tcf/LEF family. Normally, the transcriptional coactivator b-catenin can be sequestered in the cytoplasm and the Tcf transcription factor is inhibited by complexation with negative regulators of the Groucho family [39]. Upon Wnt activation, b-catenin is translocated into the nucleus and binds to Tcf to constitute the active transcriptional complex [40]. 6j 1 Protein–Protein Interactions: An Overview

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

The crystal structure of the human b-catenin/Tcf4 complex [41,42] revealed the multisite binding nature of the interaction with three regions of Tcf4 to be important for 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-terminal a-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-catenin Lys435, a hydrophobic contact of Tcf4 Leu48 to Phe253 and Phe293 of b-catenin, and a 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 successfully abolish binding of Tcf4 to b-catenin.

1.4.3 LEDGF/HIV-IN Complex

For a productive infection HIV depends on the viral integrase (IN) that integrates the genetic material of the virus into the host cell’s DNA [43]. The human transcriptional coactivator LEDGF (lens epithelium-derived growth factor) is an essential host protein as cofactor for the function of IN that, among others, locates IN to the nucleus [44]. The interaction is mediated between the catalytic core domain (CCD) of IN and the IN-binding domain (IBD) of LEDGF [45]. The IN CCD/LEDGF IBD complex crystallized as an IN CCD dimer with two LEDGF IBD copies attached at opposing sites (Figure 1.5) [46]. An interhelical loop of IBD binds to a pocket at the IN dimer interface burying approximately 1300 A2 of protein surface. Binding is driven by the hydrophobic contact of LEDGF residue Ile365 to a pocket concomi- tantly established by IN residues Leu102, Ala128, Ala129, and Trp132 from one chain of the IN dimer (chain B), and Thr174 and Met178 from the other chain of the