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De

Clercq

(Ed.)

Edited by Holger Gohlke

50 Methods and Principles in Medicinal Chemistry

Protein-LigandInteractions

AntiviralD

rugStrategies

Volume 53

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

www.wiley-vch.de

New viruses can arise very quickly and, if unchecked, result in majorpandemics. Obvious examples being the AIDS and SARS virus. In orderto deal with such imminent threats, drug development times need to becut short. This is only possible by relying on proven strategies andadapting them to the specific features of any new virus or virus variant.

By focusing on general molecular mechanisms of antiviral drugsrather than therapies for individual viruses, this ready reference providesthe critical knowledge needed to develop entirely novel therapeutics andto target new viruses. It is edited by Erik de Clercq, a world authority onantiviral drug discovery.

The volume covers a general discussion of antiviral strategies,followed by a broad survey of known viral targets, such as reversetranscriptases, proteases, neuraminidases, RNA polymerases, helicases,and primases, as well as their known inhibitors. The book also containsseveral case studies of recent successful antiviral drug development.

As a result, medicinal and pharmaceutical chemists, as well asvirologists will be able to pinpoint strategies for combating future viralpandemics.

Erik De Clercq, M.D., PhD, is currently President of the RegaFoundation, a member of the Belgian (Flemish) Royal Academyof Medicine and of the Academia Europaea, and a Fellow of theAmerican Association for the Advancement of Science. He is anactive Emeritus Professor of the Katholieke Universiteit Leuven(K.U.Leuven), Belgium. He is honorary doctor of the Universitiesof Ghent, Belgium, Athens, Greece, Ferrara, Italy, Jinan(Shandong), China, Charles (Prague), Czech Republic, andJihoceska (Ceské Budejovice), Czech Republic, and Tours, France.

For his pioneering efforts in antiviral research, Professor DeClercq received in 1996 the Aventis award from the AmericanSociety for Microbiology, and in 2000 the Maisin Prize forBiomedical Sciences from the Belgian National ScienceFoundation. In 2008 he was elected Inventor of the Year by theEuropean Union. Jointly with Dr. Anthony Fauci, Prof. De Clercqreceived the Dr. Paul Janssen Award for Biomedical Research in2010.

He is the (co)inventor of a number of antiviral drugs, used forthe treatment of HSV (valaciclovir, Valtrex , Zelitrex ), VZV(brivudin, Zostex , Brivirac , Zerpex ), CMV (cidofovir, Vistide ),HBV (adefovir dipivoxil, Hepsera ), and HIV infections (AIDS)(tenofovir disoproxil fumarate, Viread ).

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57268File AttachmentCover.jpg

Edited by

Holger Gohlke

Protein-Ligand Interactions

Methods and Principles in Medicinal ChemistryEdited by R. Mannhold, H. Kubinyi, G. Folkers

Editorial Board

H. Buschmann, H. Timmerman, H. van

de Waterbeemd, T. Wieland

Previous Volumes of this Series:

Kappe, C. Oliver / Stadler, Alexander /

Dallinger, Doris

Microwaves in Organic andMedicinal ChemistrySecond, Completely Revised

and Enlarged 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 and Metabolism inDrug DesignThird, Revised and Updated Edition

2012

ISBN: 978-3-527-32954-0

Vol. 51

De Clercq, Erik (Ed.)

Antiviral Drug Strategies

2011

ISBN: 978-3-527-32696-9

Vol. 50

Klebl, Bert / Müller, Gerhard / Hamacher, Michael

(Eds.)

Protein Kinases as Drug Targets

2011

ISBN: 978-3-527-31790-5

Vol. 49

Sotriffer, Christoph (Ed.)

Virtual ScreeningPrinciples, Challenges, and Practical

Guidelines

2011

ISBN: 978-3-527-32636-5

Vol. 48

Rautio, Jarkko (Ed.)

Prodrugs and Targeted Delivery

Towards Better ADME Properties

2011

ISBN: 978-3-527-32603-7

Vol. 47

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

Chemokine Receptors as Drug Targets

2011

ISBN: 978-3-527-32118-6

Vol. 46

Ghosh, Arun K. (Ed.)

Aspartic Acid Proteases as TherapeuticTargets

2010

ISBN: 978-3-527-31811-7

Vol. 45

Ecker, Gerhard F. / Chiba, Peter (Eds.)

Transporters as Drug Carriers

Structure, Function, Substrates

2009

ISBN: 978-3-527-31661-8

Vol. 44

Faller, Bernhard / Urban, Laszlo (Eds.)

Hit and Lead Profiling

Identification and Optimization of Drug-like

Molecules

2009

ISBN: 978-3-527-32331-9

Vol. 43

Edited by Holger Gohlke

Protein-Ligand Interactions

Series Editors

Prof. Dr. Raimund MannholdMolecular Drug Research GroupHeinrich-Heine-UniversitätUniversitätsstrasse 140225 Dü[email protected]

Prof. Dr. Hugo KubinyiDonnersbergstrasse 967256 Weisenheim am [email protected]

Prof. Dr. Gerd FolkersCollegium HelveticumSTW/ETH Zurich8092 [email protected]

Volume Editor

Prof. Dr. Holger GohlkeHeinrich-Heine-UniversitätInst.für Pharmaz. + Med.ChemieUniversitätsstr. 140225 DüsseldorfGermany

Cover DescriptionAldose reductase bound to NADP (PDB code: 1ads;dark blue) as well as bound to NADP and theinhibitor tolrestat (PDB code: 2fzd; light blue). Theprotein shows pronounced movements of Phe122and Leu300 to accomodate hydrophobic parts of theinhibitor. Such movements can be detected byperturbations of NMR chemical shifts, asschematically shown around the protein.

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Contents

List of Contributors XIIIPreface XVIIA Personal Foreword XIX

Part I Binding Thermodynamics 1

1 Statistical Thermodynamics of Binding and MolecularRecognition Models 3Kim A. Sharp

1.1 Introductory Remarks 31.2 The Binding Constant and Free Energy 31.3 A Statistical Mechanical Treatment of Binding 41.3.1 Binding in a Square Well Potential 61.3.2 Binding in a Harmonic Potential 71.4 Strategies for Calculating Binding Free Energies 91.4.1 Direct Association Simulations 91.4.2 The Quasi-Harmonic Approximation 101.4.3 Estimation of Entropy Contributions to Binding 111.4.4 The Molecule Mechanics Poisson–Boltzmann Surface Area Method 131.4.5 Thermodynamic Work Methods 141.4.6 Ligand Decoupling 151.4.7 Linear Interaction Methods 151.4.8 Salt Effects on Binding 161.4.9 Statistical Potentials 171.4.10 Empirical Potentials 18

References 19

2 Some Practical Rules for the Thermodynamic Optimizationof Drug Candidates 23Ernesto Freire

2.1 Engineering Binding Contributions 252.2 Eliminating Unfavorable Enthalpy 252.3 Improving Binding Enthalpy 26

V

2.4 Improving Binding Affinity 272.5 Improving Selectivity 282.6 Thermodynamic Optimization Plot 28

Acknowledgments 30References 31

3 Enthalpy–Entropy Compensation as Deduced from Measurementsof Temperature Dependence 33Athel Cornish-Bowden

3.1 Introduction 333.2 The Current Status of Enthalpy–Entropy Compensation 343.3 Measurement of the Entropy and Enthalpy of Activation 343.4 An Example 353.5 The Compensation Temperature 383.6 Effect of High Correlation on Estimates of Entropy and Enthalpy 393.7 Evolutionary Considerations 403.8 Textbooks 40

References 42

Part II Learning from Biophysical Experiments 45

4 Interaction Kinetic Data Generated by Surface Plasmon ResonanceBiosensors and the Use of Kinetic Rate Constants in LeadGeneration and Optimization 47U. Helena Danielson

4.1 Background 474.2 SPR Biosensor Technology 484.2.1 Principles 484.2.2 Sensitivity 494.2.3 Kinetic Resolution 504.2.4 Performance for Drug Discovery 514.3 From Interaction Models to Kinetic Rate Constants and Affinity 534.3.1 Determination of Interaction Kinetic Rate Constants 534.3.2 Determination of Affinities 544.3.3 Steady-State Analysis versus Analysis of Complete Sensorgrams 544.4 Affinity versus Kinetic Rate Constants for Evaluation of Interactions 554.5 From Models to Mechanisms 564.5.1 Irreversible Interactions 574.5.2 Induced Fit 574.5.3 Conformational Selection 584.5.4 Unified Model for Dynamic Targets 584.5.5 Heterogeneous Systems/Parallel Reactions 594.5.6 Mechanism-Based Inhibitors 604.5.7 Multiple Binding Sites and Influence of Cofactors 614.6 Structural Information 61

VI Contents

4.7 The Use of Kinetic Rate Constants in Lead Generation andOptimization 62

4.7.1 Structure–Kinetic Relationships 624.7.2 Selectivity/Specificity and Resistance 634.7.3 Chemodynamics 634.7.4 Thermodynamics 644.8 Designing Compounds with Optimal Properties 654.8.1 Correlation between Kinetic and Thermodynamic Parameters

and Pharmacological Efficacy 654.8.2 Structural Modeling 664.9 Conclusions 67


5 NMR Methods for the Determination of Protein–Ligand Interactions 71Bernd W. Koenig, Sven Schünke, Matthias Stoldt, and Dieter Willbold

5.1 Experimental Parameters from NMR 725.2 Aspects of Protein–Ligand Interactions That Can Be Addressed

by NMR 775.2.1 Detection and Verification of Ligand Binding 775.2.2 Interaction Site Mapping 785.2.3 Interaction Models and Binding Affinity 805.2.4 Molecular Recognition 815.2.5 Structure of Protein–Ligand Complexes 825.3 Ligand-Induced Conformational Changes of a Cyclic Nucleotide

Binding Domain 845.4 Ligand Binding to GABARAP Binding Site and Affinity Mapping 865.5 Transient Binding of Peptide Ligands to Membrane Proteins 88

References 90

Part III Modeling Protein–Ligand Interactions 99

6 Polarizable Force Fields for Scoring Protein–Ligand Interactions 101Jiajing Zhang, Yue Shi, and Pengyu Ren

6.1 Introduction and Overview 1016.2 AMOEBA Polarizable Potential Energy Model 1026.2.1 Bond, Angle, and Cross-Energy Terms 1026.2.2 Torsional Energy Term 1036.2.3 Van der Waals Interactions 1036.2.4 Permanent Electrostatic Interactions 1036.2.5 Electronic Polarization 1046.2.6 Polarization Energy 1056.3 AMOEBA Explicit Water Simulation Applications 1066.3.1 Small-Molecule Hydration Free Energy Calculations 1066.3.2 Ion Solvation Thermodynamics 108

Contents VII

6.3.3 Binding Free Energy of Trypsin and Benzamidine Analogs 1106.4 Implicit Solvent Calculation Using AMOEBA Polarizable

Force Field 1136.5 Conclusions and Future Directions 115

References 116

7 Quantum Mechanics in Structure-Based Ligand Design 121Pär Söderhjelm, Samuel Genheden, and Ulf Ryde

7.1 Introduction 1217.2 Three MM-Based Methods 1227.3 QM-Based Force Fields 1237.4 QM Calculations of Ligand Binding Sites 1257.5 QM/MM Calculations 1267.6 QM Calculations of Entire Proteins 1277.6.1 Linear Scaling Methods 1287.6.2 Fragmentation Methods 1297.7 Concluding Remarks 133


8 Hydrophobic Association and Volume-ConfinedWater Molecules 145Riccardo Baron, Piotr Setny, and J. Andrew McCammon

8.1 Introduction 1458.2 Water as a Whole in Hydrophobic Association 1468.2.1 Background 1468.2.2 Computational Modeling of Hydrophobic Association 1508.2.2.1 Explicit versus Implicit Solvent: Is the Computational

Cost Motivated? 1528.3 Confined Water Molecules in Protein–Ligand Binding 1538.3.1 Protein Hydration Sites 1538.3.2 Thermodynamics of Volume-Confined Water Localization 1548.3.3 Computational Modeling of Volume-Confined Water Molecules 1568.3.4 Identifying Hydration Sites 1588.3.5 Water in Protein–Ligand Docking 160


9 Implicit Solvent Models and Electrostatics in Molecular Recognition 171Tyler Luchko and David A. Case

9.1 Introduction 1719.2 Poisson–Boltzmann Methods 1739.3 The Generalized Born Model 1759.4 Reference Interaction Site Model of Molecular Solvation 1769.5 Applications 179

VIII Contents

9.5.1 The ‘‘MM-PBSA’’ Model 1809.5.2 Rescoring Docking Poses 1829.5.3 MM/3D-RISM 182


10 Ligand and Receptor Conformational Energies 191Themis Lazaridis

10.1 The Treatment of Ligand and Receptor Conformational Energyin Various Theoretical Formulations of Binding 191

10.1.1 Double Decoupling Free Energy Calculations 19210.1.2 MM-PB(GB)SA 19210.1.3 Mining Minima 19310.1.4 Free Energy Functional Approach 19410.1.5 Linear Interaction Energy Methods 19510.1.6 Scoring Functions 19610.2 Computational Results on Ligand Conformational Energy 19610.3 Computational Results on Receptor Conformational Energy 19810.4 Concluding Remarks 199


11 Free Energy Calculations in Drug Lead Optimization 207Thomas Steinbrecher

11.1 Modern Drug Design 20711.1.1 In Silico Drug Design 21011.2 Free Energy Calculations 21211.2.1 Considerations for Accurate and Precise Results 21511.3 Example Protocols and Applications 21711.3.1 Example 1: Disappearing an Ion 21911.3.2 Example 2: Relative Ligand Binding Strengths 22111.3.3 Applications 22311.4 Discussion 226

References 227

12 Scoring Functions for Protein–Ligand Interactions 237Christoph Sotriffer

12.1 Introduction 23712.2 Scoring Protein–Ligand Interactions: What for and How to? 23712.2.1 Knowledge-Based Scoring Functions 23812.2.2 Force Field-Based Methods 24012.2.3 Empirical Scoring Functions 24212.2.4 Further Approaches 24412.3 Application of Scoring Functions: What Is Possible and

What Is Not? 246

Contents IX

12.4 Thermodynamic Contributions and Intermolecular Interactions:Which Are Accounted for and Which Are Not? 248

12.5 Conclusions or What Remains to be Done and WhatCan be Expected? 254Acknowledgments 255References 255

Part IV Challenges in Molecular Recognition 265

13 Druggability Prediction 267Daniel Alvarez-Garcia, Jesus Seco, Peter Schmidtke, and Xavier Barril

13.1 Introduction 26713.2 Druggability: Ligand Properties 26713.3 Druggability: Ligand Binding 26813.4 Druggability Prediction by Protein Class 27013.5 Druggability Predictions: Experimental Methods 27013.5.1 High-Throughput Screening 27013.5.2 Fragment Screening 27113.5.3 Multiple Solvent Crystallographic Screening 27213.6 Druggability Predictions: Computational Methods 27213.6.1 Cavity Detection Algorithms 27213.6.2 Empirical Models 27313.6.2.1 Training Sets 27313.6.2.2 Applicability and Prediction Performance 27413.6.3 Physical Chemistry Predictions 27513.7 A Test Case: PTP1B 27613.8 Outlook and Concluding Remarks 278

References 278

14 Embracing Protein Plasticity in Ligand Docking 283Manuel Rueda and Ruben Abagyan

14.1 Introduction 28314.2 Docking by Sampling Internal Coordinates 28414.3 Fast Docking to Multiple Receptor Conformations 28514.4 Single Receptor Conformation 28514.5 Multiple Receptor Conformations 28614.5.1 Exploiting Existing Experimental Conformational Diversity 28614.5.2 Selecting ‘‘Important’’ Conformations 28814.5.3 Generating In Silico Models 28814.6 Improving Poor Homology Models of the Binding Pocket 28914.7 State of the Art: GPCR Dock 2010 Modeling and Docking

Assessment 29014.8 Conclusions and Outlook 290


X Contents

15 Prospects of Modulating Protein–Protein Interactions 295Shijun Zhong, Taiji Oashi, Wenbo Yu, Paul Shapiro, andAlexander D. MacKerell Jr.

15.1 Introduction 29515.2 Thermodynamics of Protein–Protein Interactions 29715.3 CADD Methods for the Identification and Optimization

of Small-Molecule Inhibitors of PPIs 29815.3.1 Identifying Inhibitors of PPIs Using SBDD 29915.3.1.1 Protein Structure Preparation 29915.3.1.2 Binding Site Identification 30015.3.1.3 Virtual Chemical Database 30215.3.1.4 Virtual Screening of Compound Database 30215.3.1.5 Rescoring 30415.3.1.6 Final Selection of Ligands for Experimental Assay 30615.3.2 Lead Optimization 30715.3.2.1 Ligand-Based Optimization 30715.3.2.2 Computation of Binding Free Energy 30815.4 Examples of CADD Applied to PPIs 30815.4.1 ERK 30915.4.2 BCL6 31115.4.3 S100B 31315.4.4 p56Lck Kinase SH2 Domain 31315.5 Summary 315


Index 331

Contents XI

List of Contributors

XIII

Ruben AbagyanUniversity of California, San DiegoSkaggs School of Pharmacy andPharmaceutical Sciences9500 Gilman DriveLa Jolla, CA 92093USA

and

University of California, San DiegoSan Diego Supercomputer CenterLa Jolla, CA 92093USA

and

Molsoft LLC11199 Sorrento Valley Road, S209San Diego, CA 92121USA

Daniel Alvarez-GarciaUniversitat de BarcelonaFacultat de FarmàciaDepartament de FisicoquímicaAv. Joan XXIII s/n08028 BarcelonaSpain

and

Institut de Biomedicina de la Universitatde Barcelona (IBUB)BarcelonaSpain

Riccardo BaronThe University of UtahDepartment of Medicinal ChemistryCollege of Pharmacy

and

TheHenry Eyring Center for TheoreticalChemistrySalt Lake CityUT 84112-5820USAEmail: [email protected]

Xavier BarrilUniversitat de BarcelonaFacultat de FarmàciaDepartament de FisicoquímicaAv. Joan XXIII s/n08028 BarcelonaSpain

and


and

Catalan Institution for Research andAdvanced Studies (ICREA)Passeig Lluís Companys 2308010 BarcelonaSpain

David A. CaseRutgers UniversityDepartment of Chemistry and ChemicalBiology and BioMaPS Institute610 Taylor RoadPiscataway, NJ 08854-8087USA

Athel Cornish-BowdenCNRSBioénergétique et Ingénierie desProtéinesB.P. 71, 31 chemin Joseph-Aiguier13402 Marseille Cedex 20France

U. Helena DanielsonUppsala UniversityDepartment of Biochemistry andOrganic ChemistryBMC, Box 57675123 UppsalaSweden

Ernesto FreireJohns Hopkins UniversityDepartment of Biology114A Mudd Hall, 3400 North CharlesStreetBaltimoreMD 21218-2685USA

Samuel GenhedenLund UniversityDepartment of Theoretical ChemistryChemical CentreP.O. Box 124221 00 LundSweden

Bernd W. KoenigForschungszentrum JülichIBI-252425 JülichGermany

Themis LazaridisCity University of New YorkCity College of New YorkDepartment of Chemistry138th Street & Convent AvenueNew York, NY 10031USA

Tyler LuchkoRutgers UniversityDepartment of Chemistry and ChemicalBiology and BioMaPS Institute610 Taylor RoadPiscataway, NJ 08854-8087USA

Alexander D. MacKerell Jr.University of Maryland BaltimoreSchool of PharmacyDepartment of Pharmaceutical Sciences20 Penn StreetBaltimore, MD 21201USA

J. Andrew McCammonUniversity of CaliforniaDepartment of Chemistry andBiochemistryCenter for Theoretical BiologicalPhysicsLa JollaCA 92093-0365USA

XIV List of Contributors

Taiji OashiUniversity of Maryland BaltimoreSchool of PharmacyDepartment of Pharmaceutical Sciences20 Penn StreetBaltimore, MD 21201USA

Pengyu RenThe University of Texas at AustinDepartment of Biomedical EngineeringBME 5.202M, 1 University Station,C0800AustinTX 78712-1062USA

Manuel RuedaUniversity of California, San DiegoSkaggs School of Pharmacy andPharmaceutical Sciences9500 Gilman DriveLa Jolla, CA 92093USA

Ulf RydeLund UniversityDepartment of Theoretical ChemistryChemical CentreP.O. Box 124221 00 LundSweden

Peter SchmidtkeUniversitat de BarcelonaFacultat de FarmàciaDepartament de FisicoquímicaAv. Joan XXIII s/n08028 BarcelonaSpain

and


Sven SchünkeForschungszentrum JülichIBI-252425 JülichGermany

Jesus SecoUniversitat de BarcelonaFacultat de FarmàciaDepartament de FisicoquímicaAv. Joan XXIII s/n08028 BarcelonaSpain

and


Piotr SetnyTechnical University MunichDepartment of PhysicsMunichGermany

Paul ShapiroUniversity of Maryland BaltimoreSchool of PharmacyDepartment of Pharmaceutical Sciences20 Penn StreetBaltimore, MD 21201USA

List of Contributors XV

Kim A. SharpUniversity of PennsylvaniaDepartment of Biochemistry andBiophysics37th and Hamilton WalkPhiladelphiaPA 19104-6059USA

Yue ShiThe University of Texas at AustinDepartment of Biomedical EngineeringBME 5.202M, 1 University Station,C0800AustinTX 78712-1062USA

Pär SöderhjelmETH ZürichDepartment of Chemistry and AppliedBiosciencesComputational ScienceVia Giuseppe Buffi 136900 LuganoSwitzerland

Christoph SotrifferUniversity of WürzburgInstitute of Pharmacy and FoodChemistryDepartment of PharmaceuticalChemistryAm Hubland97074 WürzburgGermany

Thomas SteinbrecherKarlsruher Institut für TechnologieInstitut für Physikalische ChemieGebäude 30.45, Kaiserstr. 1276131 KarlsruheGermany

Matthias StoldtForschungszentrum JülichIBI-252425 JülichGermany

Dieter WillboldForschungszentrum JülichIBI-252425 JülichGermany

Wenbo YuUniversity of Maryland BaltimoreSchool of PharmacyDepartment of Pharmaceutical Sciences20 Penn StreetBaltimore, MD 21201USA

Jiajing ZhangThe University of Texas at AustinDepartment of Biomedical EngineeringBME 5.202M, 1 University Station,C0800AustinTX 78712-1062USAShijun ZhongSchool of Life Science and TechnologyDalian University of Technology2 Linggong Road, DalianLiaoning Province, 116024, ChinaPhone: (086)0411-84707913Email: [email protected],[email protected]

XVI List of Contributors

Preface

‘‘Um ein Bild zu gebrauchen, will ich sagen, dass Enzym und Glucosid wie Schloss undSchlüssel zueinander passen müssen, um eine chemische Wirkung aufeinander ausübenzu können’’ [To use a picture, I would like to say that enzyme and glucoside have to fitlike a lock and a key, in order to exert a chemical action on each other] wrote EmilFischer in 1894, to illustrate his concept on protein-ligand interactions. Well, ourpicture of the protein-ligand interaction has developed further. Instead of a rigid fit,the concepts of induced fit and, later, flexible fit were formulated. Indeed, we have tounderstand the interaction like a handshake, where the one partner adapts to theother, in a mutual fit. Of course, this accommodation should not waste too muchconformational energy, otherwise the affinity of the ligand would be significantlyreduced. Correspondingly experience shows that compounds where the bioactiveconformation is fixed or at least stabilized are most often high-affinity ligands. Onthe other hand, ligands that cannot achieve such a conformation will have no affinityat all. However, these geometric requirements are only part of the story. In addition,there must be complementary properties - similia similibus. Lipophilic groupsshould find their counterpart, charges or partial charges should have opposite signs,and hydrogen bond donor and acceptor groups should find together. The better thiscomplementarity, the higher will be the affinity, provided that there are no stericclashes. Now, whereas this is a correct description of the requirements of a protein-ligand interaction, it is a very simple one.

Holger Gohlke assembled a team of leading experts in this field to describenot only the thermodynamics of binding but also the underlying biophysicalapproaches. The major part of the book is devoted to the discussion of factors thatare responsible for the intermolecular interactions. Finally some challenges inmolecular recognition are discussed. In a logical and didactic way, this volume isorganized in four sections. The three introductory chapters review statistical ther-modynamics of binding and molecular recognition models, practical rules for thethermodynamic optimization of drug candidates, and the puzzling concept ofenthalpy-entropy compensation, as deduced from measurements of temperaturedependence. A section on the impact of biophysical experiments focuses, in parti-cular, on interaction kinetic data generated by surface plasmon resonance biosen-sors as well as NMR methods for the determination of protein-ligand interactions.The central and most comprehensive section is dedicated to aspects of modeling

XVII

protein-ligand interactions including polarizable force fields, quantum mechanicsin structure-based ligand design, the role of water in hydrophobic association,implicit solvation models and electrostatics in molecular recognition, conforma-tional aspects, free energy calculations in drug lead optimization, as well as scoringfunctions for protein-ligand interactions. The final section on challenges for protein-ligand interaction modelling considers druggability prediction, protein plasticity,and protein-protein interactions.The series editors are grateful to Holger Gohlke for his enthusiasm to organize

this volume and to work with such a selection of excellent authors. We believe thatthis book adds a fascinating new facet to our book series on ‘‘Methods and Principlesin Medicinal Chemistry’’. Last, but not least we thank the publisher Wiley-VCH, inparticular Frank Weinreich and Heike Nöthe, for their valuable contributions to thisproject and the entire series.

February 2012Düsseldorf Raimund MannholdWeisenheim am Sand Hugo KubinyiZürich Gerd Folkers

XVIII Preface

A Personal Foreword

Ever since I started my scientific work I have been fascinated by the questions whatmakes two molecules bind to each other and how can one make use of thisknowledge to modulate biological processes: After all, corpora non agunt nisifixata, as Paul Ehrlich put it almost 100 years ago. Ehrlichs statement that bodiesdo not act if they are not bound is strikingly exemplified by about 130,000 binaryinteractions in the human protein-protein interactome [1]. Along the same linesand more oriented towards the topic of this book, the famous wall chartBiochemical Pathways introduced by Gerhard Michal [2] is a vivid picture inthe eyes of a life science scientist, with its comprehensive view on metabolicpathways and cellular and molecular processes, particularly involving interactionsbetween proteins and endogenous small-molecules.

For a xenobiotic ligand to exert an influence on an organism, it must bind to abiological target, too.While this statement seems unspectacular nowadays, it still hasfar-reaching consequences because it provides a valuable handle to explain andpredict biological activity, both beneficial and detrimental, in terms of affinity, a well-defined thermodynamic property, of a ligand towards a (or multiple) targets. In fact,the fields of medicinal chemistry and drug design have made use of differentvariations of this theme over time depending on which type of information aboutbinding was available in each case.

From an inductive point of view, already since the very early days of modernchemistry [3] structures of ligands and, hence, their binding properties, have beencorrelated to activities. Further milestones on this route were the establishment ofquantitative structure-activity relationships and the comparison of ligands based onconcepts of similarity or dissimilarity of shape and chemical properties. From adeductive point of view, the above theme summons medicinal chemists to look atbiological activity from the perspective of processes and contributions that lead tobinding,with structural information of the binding partners being of invaluable help.As such, the event of (reversible) binding is a consequence of association anddissociation processes and involves enthalpic and entropic components. If and howthese separate processes and components can be modulated by modification of aligands structure for the sake of overall optimized binding properties is a hot topicat present in drug research. Furthermore, the deductive point of view allows one to

XIX

apply a divide-and-conquer strategy when it comes to understanding and predictingbinding from a theoretical perspective. As a first approximation, binding can beattributed to direct interactions between the binding partners mediated by anaqueous environment, to which contributions due to changes in the conformationand configuration of the binding partners add. While this approximation is valuablein that it allows optimizing the description of each of these terms separately, it alsoprovokes the question to what extent does it hold or, phrased differently, when docooperativity or compensation effects prevail over additivity?

Themajority of topics for this book were selected following this deductive point ofview, with an emphasis on rigorous approaches because I believe that these will bemore successful in the long term than ad hoc ones. The selection also focused ontopics that, at that time, had most quickened interests, had seen considerableprogress, or had still been major stumbling blocks in the description and predictionof binding. Unsurprisingly, while much has been achieved in all of the covered areasas undoubtedly laid out in each of the chapters, not in all cases have methods orapproaches lived up to ones expectations so far. I am grateful to the authors forpointing this out clearly – such insights will drive further developments that aim atimproving our understanding of protein-ligand interactions. Finally, I also tried tobalance topics related to biophysical experiments against theoretical and computa-tional approaches, because I have learnt frommy own work how well both sides cancomplement and enhance each other, and what joy this gives.

Last but not least, I expressmy gratitude to all contributors for providing insightfulaccounts on the topic of protein-ligand interactions, to the series editors RaimundMannhold,HugoKubinyi, andGerd Folkers for givingme the opportunity to addressthis topic, to my current working group for providing fruitful comments about thechapters, and to Frank Weinreich and Heike N€othe from the publisher Wiley-VCHfor their continuous support, great help, and even greater patience.

D€usseldorf, Germany Holger Gohlke

References

1 Venkatesan, K., Rual, J.F., Vazquez, A.,Stelzl, U., Lemmens, I.,Hirozane-Kishikawa, T., Hao, T.,Zenkner, M., Xin, X.F., Goh, K.I.,Yildirim, M.A., Simonis, N.,Heinzmann, K., Gebreab, F., Sahalie, J.M.,Cevik, S., Simon, C., de Smet, A.S.,Dann, E., Smolyar, A., Vinayagam, A.,Yu, H.Y., Szeto, D., Borick, H., Dricot, A.,Klitgord, N., Murray, R.R., Lin, C.,Lalowski, M., Timm, J., Rau, K., Boone, C.,

Braun, P., Cusick, M.E., Roth, F.P.,Hill, D.E., Tavernier, J., Wanker, E.E.,Barabasi, A.L. and Vidal, M., An empiricalframework for binary interactomemapping. Nature Methods 2009. 6:83–90.

2 Michal,G.,Biochemical Pathways: Biochemie-Atlas. Spektrum Akademischer Verlag,Heidelberg: 1999.

3 Borman, S., New QSAR techniques eyedfor environmental assessments.Chem EngNews 1990. 68:20–23.

XX A Personal Foreword

Part IBinding Thermodynamics

Protein-Ligand Interactions, First Edition. Edited by Holger Gohlke.� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

j1

1Statistical Thermodynamics of Binding and MolecularRecognition ModelsKim A. Sharp

1.1Introductory Remarks

Equilibrium binding or association of twomolecules to form a bimolecular complex,AþB,AB, is a thermodynamic event. This chapter will cover some of thefundamental thermodynamics and statistical mechanics aspects of this event. Theaim is to introduce general principles and broad theoretical approaches to thecalculation of binding constants, while later chapters will provide examples. Onlythe noncovalent, bimolecular association under ambient pressure conditions will beconsidered. However, extension to higher order association involves no additionalprinciples, and extension to high pressure by inclusion of the appropriate pressure–volume work term is straightforward. In terms of the binding reaction above, theassociation anddissociation constants are defined asK¼ [AB]/[A][B] andKD¼ [A][B]/[AB] respectively, where [] indicates concentration. Either K or KD is the primaryexperimental observable measured in binding reactions. KD is sometimes obtainedindirectly by inhibition of binding of a different ligand as aKi. Froma thermodynamicperspective, the information content from K, KD, and Ki is the same.

1.2The Binding Constant and Free Energy

To connect the experimental observable K to thermodynamics, one often finds in theliterature the relationship

DGbind ¼ �kT ln K; ð1:1Þwhere k is the Boltzmann constant, T is the absolute temperature, and DGbind is theabsolute or standard binding free energy. Several comments are given to avoidmisuse of this expression. First, one cannot properly take the logarithm of a quantitywith units such as K, so Eq. (1.1) is implicitly

DGbind ¼ �kT ln KVref ; ð1:2Þ

j3

Protein-Ligand Interactions, First Edition. Edited by Holger Gohlke.� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

whereVref is the reference volume in units consistent with the units of concentrationinK, that is, 1 l/mol or about 1660A

� 3/molecule formolarity units. The choice ofVref isoften referred to as the standard state problem. Equivalently, one says thatDGbind isthe free energy change when reactants A and B and the product AB are all at thereference concentration. Second, although the units of concentration used in K arealmost always moles/liter, this is entirely a convention, so the actual numerical valuefor DGbind obtained from Eq. (1.2) is arbitrary. Put another way, any method forcalculating the free energy of bindingmust explicitly account for a particular choice ofVref before it can meaningfully be compared with experimental values of DGbindobtained using Eq. (1.2). Furthermore, ligand efficiency-type measures, such asDGbind/nwhere n is the number of heavy atoms in a ligand or themolecular weight ofa ligand [1], can change radically with (arbitrary) choice of concentration units. Ofcourse, differences inDGbind can be sensibly compared provided the same referencestate concentration is used. Finally, in Eq. (1.2), the free energy actually depends onthe ratio of activities of reactants and products, not on concentrations. For neutralligands andmolecules of low charge density at less thanmicromolar concentrations,the activity and concentration are nearly equal and little error is introduced.However,this is not true for high charge density molecules such as nucleic acids and many ofthe ligands and proteins that bind to nucleic acids.Here, the activity coefficient can besubstantially different from unity even at infinitely low concentration. Indeed, muchof the salt dependence of ligand–DNAbinding can be treated as an activity coefficienteffect [2–4]. The issue of standard state concentrations, the formal relationshipbetween the binding constant and the free energy, and the effect of activitycoefficients are all treatable by a consistent statistical mechanical treatment ofbinding, as described in Section 1.3.

1.3A Statistical Mechanical Treatment of Binding

Derivation of a general expression for the binding constant follows closely theapproach of Luo and Sharp [5], although somewhat different treatmentsusing chemical potentials, which provide the same final result, are given else-where [6–8]. It is a statistical mechanical principle that any equilibrium observablecan be obtained as an ensemble, or Boltzmann weighted average, of the appropriatequantity. Here, the binding constant K¼ [AB]/[A][B] is the required observable.Consider a single molecule each of A and B in some volume V (Figure 1.1) and forconvenience define a coordinate system centered on B (the target) in a fixedorientation. Over time, the ligand (A) will explore different positions and orientations(poses) relative to B, where r andV represent the three position and three orientationcoordinates of A with respect to B. Now A and B interact with each other with anenergy that depends not only on their relative position (r,V) but in general also on theconformations of A, B, and the surrounding solvent. If na, nb, ns are the number ofatoms in A, B, and solvent, then the energy is a function of 3naþ 3nbþ 3ns� 6coordinates. In principle, one could keep all these degrees of freedomexplicit. Froma

4j 1 Statistical Thermodynamics of Binding and Molecular Recognition Models

practical standpoint, this would be a complicated and expensive function to evaluate.However, one may integrate over the solvent coordinates and the (3na� 6)þ(3nb� 6) internal coordinates so that the interaction between A and B for a given(r, V) is described by an interaction potential of mean force (pmf) v(r, V). If onedefines the pmf between A and B at infinite separation in their equilibriumconformations to be 0, then v(r, V) is the thermodynamic work of bringing A andB from far apart to some mutual pose (r,V), accounting for both solvent effects andinternal degrees of freedom of A and B. A will sample each pose (r, V) with aprobability given by the Boltzmann factor of the pmf:

pðr;VÞ / e�bvðr;VÞ; ð1:3Þwhereb¼ 1/kT. Indeed, onemay consider the pmf to be definedby this equation. Thebinding constant will then be given by the fraction of time A is in the bound state, fab,relative to that in the free state, ff:

K ¼ ½AB�½A�½B� ¼fab=V

ðff=VÞðff=VÞ �!V !¥ fabV ; ð1:4Þ

where in the dilute limit ff ! 1. It is convenient to introduce a functionH(r,V) thattakes a value of 1 for poses where A is bound and a value of 0when it is free. Then, thefraction of the time A is bound is given by the ensemble average of H:

fab ¼ðdr dVHðr;VÞe�bvðr;VÞ=

ðdr dVe�bvðr;VÞ: ð1:5Þ

The integrals are taken over all orientations and over the entire volume of thesolution, so the denominator gives 8p2V. Substituting into Eq. (1.4), the finalexpression for the association constant is

Figure 1.1 Schematic illustration of two molecules A and B interacting through solvent with apotential of mean force v as a function of their relative position r and orientation V.

1.3 A Statistical Mechanical Treatment of Binding j5

K ¼ 18p2

ðdr dVHðr;VÞe�bvðr;VÞ: ð1:6Þ

One may then convert this to an absolute binding free energy using Eq. (1.2):

DGbind ¼ kT lnð8p2Vref Þ � kT lnðdr dVHðr;VÞe�bvðr;VÞ: ð1:7Þ

. Equation 1.6 is a general and exact expression for the association constant. Theintegral depends explicitly on just six variables describing the pose of A withrespect to B. The other degrees of freedom are included implicitly, but exactlythrough the thermodynamic quantity v(r, V), the potential of mean force.

. The different treatment of coordinates for translation/orientation versus theothers is a formal one: Any subset of coordinatesmay in principle be kept explicit,with the appropriate pmf being used for the rest. For example, one may keep theinternal coordinates of A and B explicit, making the solvent coordinates implicit.The choice here is designed to highlight the translation/rotation contribution tobinding that has been widely discussed, with some disagreement, in the litera-ture [5, 6, 9–13]. It also reflects the practical fact that in many docking andscreening applications, a particular pose is generated explicitly, that is, (r, V) isspecified, and then the pose is scored in some way. The pmf also provides anatural way to introduce approximations necessary for any practical calculation ofK in biological systems, for example, in the treatment of solvent.

. The integral has the correct units of volume, with the length scale for thetranslation coordinates being determined by the units of concentration used inK. The first term in Eq. (1.7) is the contribution of the rotation/translation (R/T)entropy in the unbound state, which depends on the reference concentration. Theintegral term in Eq. (1.6) is the Boltzmann phase volume of the bound state.

. Through H(r, V), there is explicit consideration of what constitutes the boundcomplex, in terms of the relative position and orientation of A with respect to B.For example, if B has more than one binding site for A, this would be taken intoaccount in the specification of where H¼ 1.

. Either Cartesian coordinates or the bond length, bond angle, and dihedral anglecoordinates may be used. The trend now is toward the latter, as they lendthemselves more naturally to the analysis of different internal motions of themolecules and their contribution to binding.

The meaning of Eq. (1.6) is illustrated by two simple examples.

1.3.1Binding in a Square Well Potential

Let the pmf be approximated by a simple, three-dimensional square well potential ofdepth e and width b in each of the x, y, z directions and the bound complex be theregion in the well only. From Eq. (1.6), the association constant is


K ¼ b3ebe ð1:8Þand Eq. (1.2) yields

DGbind ¼ �eþ kT lnðVref=b3Þ: ð1:9ÞThe first term, the well depth, makes a direct, linear contribution to the binding

free energy. The second term is positive and comes from the restriction of the ligandto the square well. It is the translation entropy penalty for binding, and it depends onthe ratio of the volumes available to the ligand in the free state at say 1M (the entirevolume Vref) versus that in the bound state. In this simple example, there is norotational entropy penalty because in the bound state the ligand can rotate freely in8p2 of orientation phase volume, just as in the free state. However, restriction inrotation in the bound state will add another positive term to DGbind, the rotationentropy penalty, with a similar form: kT ln(8p2/VV), where VV< 8p

2 is the orien-tation phase volume in the bound state. We can see even from this simple examplethat for any meaningful degree of binding, the translational and rotational phasevolumes available to a ligand in the bound state must be less than Vref and 8p

2,respectively, so there is always a R/T entropy penalty to be overcome for binding tooccur. The question is how much is it in specific cases. A related point is that eventhough the depth of the well may be known, for example, from some calculation (inthe parlance of the field, from a single point energy determination), this cannot bedirectly compared with DGbind because the second term is not included. Thenumerical value of the binding free energy depends on the reference concentration,which is nowhere in the single point calculation.Oneway or another, the residual R/Tentropy of A in the bound state must be accounted for.

1.3.2Binding in a Harmonic Potential

If one is starting from a known complex structure derived from, for example, X-ray,NMR, or molecular mechanics minimization, one is presumably close to theminimum energy (pmf) configuration. The pmf in this region may be close toharmonic or at least expandable in a Taylor expansion, which to second order isharmonic. It is, therefore, instructive to consider binding in a harmonic potential,although this is a simplified model of the real situation. Let the potential well be athree-dimensional harmonic potential of the form

vðrÞ ¼ e rb

� �2� 1

� �ðr < bÞ; vðrÞ ¼ 0ðr � bÞ; ð1:10Þ

where e is the depth of the well at the minimum, r is the radial distance from theminimum, and b defines the width so that for r� b, v¼ 0 (Figure 1.2). Again, thebound complex is defined to be the region in thewell only. Substituting Eq. (1.10) intoEq. (1.6) and integrating, the association constant for this truncated harmonicpotential is

1.3 A Statistical Mechanical Treatment of Binding j7

K ¼ b3ebe 4pX¥n¼0

�1nðbeÞnð2nþ 3Þn!

!� b3ebe p

be

� �3=2: ð1:11Þ

The approximate equality comes from using an untruncated harmonic potential(i.e., the potential goes to infinity as the complex is dissociated), which for this casegives a binding free energy of

DGbind ¼ �eþ kT lnðVref=b3Þ � 3=2kT lnðp=beÞ: ð1:12ÞComparing the square well and harmonic potential models, one sees that the

depth and volume factors, ebe and b3, contribute in the same way to thebinding constant, the difference being a well shape factor. We see from the formof the expression for the association constant that the lower the pmf, the more thecontribution to the integral by that region, so most of the contribution to bindingshould come from the near minimum energy configuration. This is illustrated inFigure 1.2, using a well half-width of 2 A

�and a depth of 19.6kT. These parameters

are chosen to give a moderate affinity of 10 mM – typical of the compounds studiedby virtual screening and docking calculations in early lead identification – with areasonable degree of motion in the binding pocket. It can be seen that almost allthe contribution to the binding constant comes significantly before reaching thewell boundary. Thus, the problem of giving the exact definition of the complex in

Figure 1.2 Contributions to the binding-phaseintegral. Dotted line: Value of the integrand ofEq. (1.6) at r. Solid line: Value of the resultingintegral from 0 to r. Both are expressed as apercentage of the total association constant.

Contributions were calculated for a truncatedthree-dimensional harmonicwell potential, half-width 2 A

�, and depth 19.6kT (inset) that has

Kd¼ 10mM.


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