Protein Surface Recognition E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 639

Veröffentlichungsjahr: 2011

Beschreibung

A new perspective on the design of molecular therapeutics is emerging. This new strategy emphasizes the rational complementation of functionality along extended patches of a protein surface with the aim of inhibiting protein/protein interactions. The successful development of compounds able to inhibit these interactions offers a unique chance to selectively intervene in a large number of key cellular processes related to human disease. Protein Surface Recognition presents a detailed treatment of this strategy, with topics including: * an extended survey of protein-protein interactions that are key players in human disease and biology and the potential for therapeutics derived from this new perspective * the fundamental physical issues that surround protein-protein interactions that must be considered when designing ligands for protein surfaces * examples of protein surface-small molecule interactions, including treatments of protein-natural product interactions, protein-interface peptides, and rational approaches to protein surface recognition from model to biological systems * a survey of techniques that will be integral to the discovery of new small molecule protein surface binders, from high throughput synthesis and screening techniques to in silico and in vitro methods for the discovery of novel protein ligands. Protein Surface Recognition provides an intellectual "tool-kit" for investigators in medicinal and bioorganic chemistry looking to exploit this emerging paradigm in drug discovery.

Leseprobe

Contents

Preface

List of Contributors

Part I Principles

1 The Discovery and Characterization of Protein-Protein Interactions

1.1 Introduction

1.2 Techniques to Identify Protein-Protein Interactions

1.3 Techniques to Characterize Protein-Protein Interactions

1.4 Structure and Dynamics of Protein Complexes

1.5 Protein-Protein Complexes as Therapeutic Targets

1.6 Conclusions

2 Biophysics of Protein-Protein Interactions

2.1 Introduction

2.2 Intermolecular Forces in Protein Recognition

2.3 Basic Binding Thermodynamics

2.4 Thermodynamically Driven Drug Design

2.5 Measurement of Binding Energetics

2.6 Structure-based Calculation of Protein Binding Energetics

2.7 Interfacial Water Molecules in Protein Recognition

2.8 The Linkage Between Binding and Conformational Equilibrium in Proteins

Part II Approaches

3 On the Logic of Natural Product Binding in Protein-Protein Interactivity

3.1 Introduction

3.2 Structural Logic

3.3 Functional Logic

3.4 The Need for Programmers

3.5 Compiling the NPPI Mapper

4 Interface Peptides

4.1 Interface Peptides Defined

4.2 Unmodified Peptides

4.3 Modified Peptides

4.4 Summary/Perspective

5 Inhibition of Protein-Protein Interactions by Peptide Mimics

5.1 Introduction

5.2 Inhibition of Calmodulin

5.3 Inhibition of HIV-1 Fusion

5.4 Inhibition of the Nuclear Estrogen Receptor

5.5 Inhibition of the Bcl-xL/Bak Interaction

5.6 Inhibition of the p53/MDM2 Interaction

5.7 Miscellaneous Protein Targets

5.8 Conclusion

6 Discovery of Inhibitors of Protein-Protein Interactions by Screening Chemical Libraries

6.1 Introduction

6.2 Screening Strategies to Identify and Develop Antagonists of Protein-Protein Interactions

6.3 Mimetics of Common Protein Structure Motifs and Structure-based Design of Peptidomimetics

6.4 Conclusions and Outlook

Part III Techniques

7 High-throughput Methods of Chemical Synthesis Applied to the Preparation of Inhibitors of Protein-Protein Interactions

7.1 Introduction

7.2 Survey of High-throughput Organic Synthesis

7.3 Synthesis of 'Peptide-Inspired' Compounds and Libraries

7.4 Synthesis of 'Natural Product-Inspired' Compounds and Libraries

7.5 Diversity Oriented Synthesis (DOS) in the Discovery of PPI Inhibitors

7.6 Summary and Outlook

8 In Silico Screening

8.1 Introduction

8.2 Methods for Virtual Ligand Screening

8.3 Binding Site Characterization

8.4 Case Studies

8.5 Outlook and Conclusions

9.1 In Vitro Screening: Screening by Nuclear Magnetic Resonance

9.1.1 Saturation Transfer Difference (STD)

9.1.2 STD in Fragment-based Drug Design

9.1.3 Chemical Shift Perturbation (CSP)

9.1.4 19F-NMR in Molecular Recognition Studies

9.2 In Vitro Screening: Methods of High-throughput Screening

9.2.1 Introduction

9.2.2 Statistical Evaluation of the HTS Assay Performance

9.2.3 Biochemical Assays

9.2.4 Cell-based Assays

9.2.5 Conclusion

Part IV Case Studies

10 Case Study: Inhibitors of the MDM2-p53 Protein-Protein Interaction

10.1 MDM2-p53 Protein-Protein Interaction: A Case Study

10.2 Regulation of p53 by the MDM2-p53 Protein-Protein Interaction

10.3 Structural Basis of the MDM2-p53 Interaction

10.4 Design of p53-based Peptides

10.5 Design of Nonpeptidic Small-Molecule Inhibitors of the MDM2-p53 Interaction

10.6 Challenges in the Design of Small Molecule Inhibitors of the MDM2-p53 Interaction

10.7 Reactivation of p53 by Inhibitors of the MDM2-p53 Interaction

10.8 Development of MDM2 Inhibitors as New Anticancer Drugs

10.9 Concluding Remarks

11 Case Study: The Discovery of Potent LFA-1 Antagonists

11.1 Introduction

11.2 Structural, Molecular and Cellular Biologies of LFA-1

11.3 The Search for Small Molecule LFA-1 Antagonists

11.4 Screening Assays

11.5 Lead Identification and Optimization

11.6 Protein and Small Molecule Structure Activity Relationships (PSAR) in the LFA-1/ICAM-1 Interaction

11.7 Summary

Colour plates

Index

This edition first published 2011

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Library of Congress Cataloging-in-Publication Data

Protein surface recognition: approaches for drug discovery/editors, Ernest Giralt, Mark Peczuh, Xavier Salvatella.

p.; cm.

Includes bibliographical references and index.

ISBN 978–0-470–05905-0 (cloth)

1. Drugs–Design. 2. Protein-protein interactions 3. Proteins–Inhibitors. 4. High throughput screening (Drug development) I. Giralt, Ernest. II. Peczuh, Mark. III. Salvatella, Xavier.

[DNLM: 1. Proteins–metabolism. 2. Drug Discovery–methods. 3. Enzyme Inhibitorspharmacology. 4. Protein Binding. 5. Surface Properties. QU 55 P9689 2010]

RS420.P76 2010

615′.19–dc22

2010018781

Preface

By and large, current drugs fall into two broad categories: small molecules and protein therapeutics (biologics). While specific notions of ‘small molecule’ may vary, they can generally be characterized by their low (<1 kD) molecular weight, high functional group density, and often the presence of heterocycles as part of the core structure. As such, small molecules may be derived from, or be inspired by, natural products or they may be the product of organic synthesis. Such ‘synthetic drugs’ are at the origin of the pharmaceutical industry itself.

From a financial perspective, small molecules are presently the bread and butter of the industry with worldwide annual sales in the hundreds of billions (USD). Biologics, however, are themselves a multibillion dollar annual market and are seen by some as having a high potential for growth. The success of biologics has been mainly the result of advances in biotechnology that have facilitated the identification and subsequent expression of the appropriately tailored proteins. To be active, both small molecules and protein therapeutics must bind to a target biomolecule. It is how each of these types of molecules binds its partner that further differentiates them. Small molecules usually bind at an interior active site whereas proteins are involved in protein-protein interaction (PPI) that involve the exterior surfaces of proteins.

An example of each is illustrative of this point. Atorvastatin (Lipitor), a second generation statin derived from the related fungal metabolite pravastatin (Prevachol) is the number one small molecule therapeutic in the US as determined by 2009 retail sales. It is a competitive inhibitor of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. This reductase focuses functional groups of the peptide backbone and side chains in a convergent manner inward toward the drug. In contrast, binding to the protein therapeutic Trastuzumab (Herceptin ca 1.3b USD/yr) by the HER2 cellular receptor covers a sizeable exterior surface area (ca. 1600 Å2). Functional group presentation by the two binding partners in the Herceptin-Her2 interaction is more divergent in nature, especially when compared to the interaction between Lipitor and HMG-CoA reductase.

A growing body of evidence suggests that a middle ground - using small molecules to bind the exterior protein surface and inhibit PPIs - can be a powerful strategy for the development of new tools for chemical biology and medicinal chemistry. A small molecule with these properties, a binder of Bcl-XL and other anti-apoptotic Bcl proteins, is already in Phase 2a clinical trials (ABT-263); notably, the molecule is a result of a fragment-based drug discovery effort. It binds the Bcl proteins in a groove usually occupied by an α-helix of the native protein binding partners. Molecules such as ABT-263 can combine the mode of action of protein therapeutics with the synthetic accessibility, ease of administration, bioavailability, and robustness of traditional small molecule drugs. The development of protein surface binders by structure-based drug design or similar approaches should be contrasted with the discovery of small molecules that inhibit a given PPI by an allosteric mechanism, which have largely arisen via serendipity and are testament to our still crude understanding of the physico-chemical principles that govern the interactions between proteins.

In between the contrasting worlds of small molecules and therapeutic proteins, peptides are likely to play an important role as therapeutic agents that modulate protein-protein interactions. Compared to protein therapeutics, the most important advantages offered by small molecules are their relatively straightforward synthetic accessibility and bioavailability. Protein therapeutics, by contrast, have specificity as a key asset, which arises from their ability to establish a large number of noncovalent interactions with the surface of the target. Nowadays, peptides combine the advantages of therapeutic proteins with those of small molecules due, among other developments, to recent progress in their modification to improve their bioavailability profile.

This book provides both a context and a guidepost for the development of molecules that alter protein function by inhibiting protein-protein interactions (PPIs) as opposed to conventional active site inhibition. The subject material has been broken into four broad sections: principles, approaches, techniques, and case studies. The principles section provides a general description of the biophysical properties of PPIs with an emphasis on those that are relevant to drug design; in Chapter 1, ‘The Discovery and Characterization of Protein-Protein Interactions’, we provide an overview of the methods used for identifying and characterizing PPIs, a survey of the main structural and dynamical properties of proteinprotein complexes and a discussion of the challenges and opportunities inherent to inhibiting their formations whereas in Chapter 2, ‘Biophysics of Protein-Protein Interactions’, we provide, instead, a detailed account of the noncovalent interactions that provide the driving force for complex formation and of the thermodynamics and kinetics of the process.

Following this overview, the approaches section reviews established strategies for the inhibition of PPIs in terms of the small molecule inhibitor. Chapter 3, ‘On the Logic of Natural Product Binding in Protein-Protein Interactivity’ presents a rationale on how natural products bind protein surfaces and the functional consequences of the interactions. Chapters 4 and 5, ‘Interface Peptides’, and ‘Inhibition of Protein-Protein Interactions by Peptide Mimics’, detail the progression of a strategy whereby peptide sequences from the protein-protein interface are used as inhibitors and then subsequently serve as models for the development of peptide mimics with the same activity. Secondary structures such as turns and α-helices are common in the collection of interface peptides. As such, mimicry of these elements has received significant attention. Chapter 6, ‘Discovery of Inhibitors of Protein-Protein Interactions by Screening Chemical Libraries’, collects examples of small molecule inhibitors of protein-protein interactions that have come about via screening efforts.

A review of technologies that enable the evaluation of protein surface binding constitutes the next section of the book. This details aspects that range from organic synthesis to screening methods. Chapter 7 ‘High-throughput Methods of Chemical Synthesis Applied to the Preparation of Inhibitors of Protein-Protein Interactions’, describes methods for the preparation of small molecule inhibitors of PPIs and the strategies behind their synthesis. Chapters 8, ‘In Silico Screening’, and 9.1 ‘In Vitro Screening: Screening by Nuclear Magnetic Resonance’, provide accounts of how computational tools and Nuclear Magnetic Resonance can provide key information, while aspects of high throughput screening in terms of in vitro and cell-based assays are outlined in Chapter 9.2, ‘In Vitro Screening: Methods of High-throughput Screening’.

Finally, the integration of the previous concepts is illustrated through two case studies in the final section of the book. These case studies include ‘Inhibitors of the MDM2-p53 Protein-Protein Interaction’ (Chapter 10) and ‘The Discovery of Potent LFA-1 Antagonists’ (Chapter 11).

We trust that the readers of the book will find it a source of valuable information in addressing the challenges and potential rewards associated with the inhibition of proteinprotein interactions and we wish to thank all our co-workers and co-authors for their enthusiastic contributions in making the book possible. We specifically would like to thank Brendan Orner and David Bolstad for very valuable discussions and Paul Deards for initiating the project.

Ernest Giralt, Mark W. Peczuh and Xavier Salvatella

List of Contributors

Xavier Barril, ICREA and University of Barcelona, Barcelona, Spain

Jorge Becerril, Department of Chemistry, Yale University, New Haven, CT, USA

Denzil Bernard, Comprehensive Cancer Center and Departments of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

C. W. Bertoncini, Department of Chemistry, University of Cambridge, UK

Richard T. Desmond, Department of Chemistry, University of Connecticut, Storrs, CT, USA

Annaliese K. Franz, Department of Chemistry, University of California at Davis, Davis, CA, USA

Tom Gadek, SARcode Corporation, San Francisco, CA, USA

Carlos García-Echeverría, Novartis Institutes for Biomedical Research, Basel, Switzerland

Ernest Giralt, Department of Organic Chemistry, University of Barcelona and Institute for Research in Biomedicine, Barcelona, Spain

Andrew D. Hamilton, Department of Chemistry, Yale University, New Haven, CT, USA

A. Higueruelo, Department of Biochemistry, University of Cambridge, UK

James J. La Clair, Xenobe Research Institute, San Diego, CA, USA

Qing Lin, Department of Chemistry, State University of New York at Buffalo, Buffalo, NY, USA

Francisco Javier Luque, Department of Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Irene Luque, Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences, University of Granada, Granada, Spain

Mark W. Peczuh, Department of Chemistry, University of Connecticut, Storrs, CT, USA

Johanna M. Rodriguez, Department of Chemistry, Yale University, New Haven, CT, USA

Xavier Salvatella, ICREA and Institute for Research in Biomedicine, Barcelona, Spain

Sanjeev Shangary, Comprehensive Cancer Center and Departments of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

Jared T. Shaw, Department of Chemistry, University of California at Davis, Davis, CA, USA

Wenjiao Song, Department of Chemistry, State University of New York at Buffalo, Buffalo, NY, USA

Yuchen Tang, Department of Chemistry, University of California at Davis, Davis, CA, USA

Shaomeng Wang, Comprehensive Cancer Center and Departments of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

Pauline N. Wyrembak, Department of Chemistry, Yale University, New Haven, CT, USA

Part I

Principles

1

The Discovery and Characterization of Protein-Protein Interactions

C. W. Bertoncini1, A. Higueruelo2 and X. Salvatella3

1Department of Chemistry, University of Cambridge, UK

2Department of Biochemistry, University of Cambridge, UK

3ICREA and Institute for Research in Biomedicine, Barcelona, Spain

1.1 Introduction

The regulation of protein-protein interactions (PPIs) is fundamental for cellular function because PPIs are involved in virtually all biological processes. A complete and detailed description of the interaction map for proteins, known as interactome, is therefore one of the most important challenges in molecular biology, one that will provide great opportunities for therapeutic intervention in the complex diseases that challenge the biomedical community and the pharmaceutical industry. In this chapter we provide an overview of the different techniques that are currently available for the discovery and structural and thermodynamic analysis of PPIs as well as a survey of the general structural and dynamical properties of proteins and protein complexes that affect drug design. Rather than a comprehensive survey of the technical literature on methods to screen and characterize PPIs we present here a general discussion of these tools and refer the reader to the reviews and examples of application that we cite to identify the primary literature.

1.2 Techniques to Identify Protein-Protein Interactions

Many methods have been developed for the isolation and characterization of protein complexes, both in vitro and in vivo. Among them five methodologies are particularly suitable for high-throughput, and account for the majority of proteome-wide studies.

1.2.1 The Yeast Two Hybrid Assay (Y2H)

This system exploits the formation of a stable complex between interacting proteins to bring together two modules of a cis-acting transcriptional promoter, stimulating the expression of a reporter gene. It requires the construction of two hybrid genes, one encoding the DNA-binding domain (BD) of the transcription factor fused to a target protein (the bait) and a second encoding its transcription-activation domain (AD) fused to a different protein (the prey). If the prey and bait proteins interact through a PPI the two modules of the transcription factor (BD and AD) are brought together to reconstitute the transcription activity. Provided that the interaction between the prey and bait proteins is sufficiently strong, the now functional transcription factor will bind to the promoter sequence in the proximity of the reporter gene, via its DNA binding domain (BD), and recruit the transcriptional machinery, via its transcription-activation domain (AD, Figure 1.1A). The most commonly employed DNA-binding domains are derived from the yeast Gal4 and LexA transcription factors, while activating domains come also from Gal4 or from the viral activator VP16. Expression of the reporter gene gives the yeast a unique characteristic which allows identification of a successful PPI interaction between the bait and prey proteins. Reporter genes commonly employed are lacZ, that codifies for the enzyme β-galactosidase, that metabolizes X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) to give a distinctive blue color, or auxotrophic genes such as HIS3, LEU2 or URA3, which confer positive colonies the ability to grow in media lacking specific nutrients [1].

Figure 1.1 Methods to study protein-protein interactions I. (A) Yeast two hybrid. The formation of a complex between the bait (X) and prey (Y) proteins brings together the binding domain (BD) and the activation domain (AD) of the transcription factor, which stimulates the expression of a reporter gene (adapted from Shoemaker and Panchenko [4]) (B) Phage display. The genome of a bacteria-specific virus, a phage, is engineered to carry the DNA of an exogenous protein. This protein is also displayed on the outer envelope of the phage, making the phage particle a unique carrier of both the genetic information and functional polypeptide for a given gene. Positive interaction partners are isolated by incubating the phages with the target protein immobilized on a solid support (adapted from [5] with permission from Elsevier)