Über die Autorin bzw. den Autor

ERNEST GIRALT, Department of Organic Chemistry, University of Barcelona and Institute for Research in Biomedicine, Barcelona, Spain

MARK PECZUH, Department of Chemistry, University of Connecticut, USA

XAVIER SALVATELLA, ICREA and Institute for Research in Biomedicine, Barcelona, Spain

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Protein Surface Recognition: Approaches for Drug Discovery

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 (PPIs). 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: Approaches for Drug Discovery presents a detailed treatment of this strategy. Starting with a survey of PPIs that are key players in human disease and biology and the potential for therapeutics derived from this new perspective, the book then examines 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 are given. Finally, the book surveys 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, and ends with two extended case studies - inhibitors of the MDM2-p53 PPI, and the discovery of potent LFA-1 antagonists.

Protein Surface Recognition: Approaches for Drug Discovery provides an intellectual ‘tool-kit' for investigators in medicinal and bioorganic chemistry looking to exploit this emerging paradigm in drug discovery.

Aus dem Klappentext

Protein Surface Recognition: Approaches for Drug Discovery

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 (PPIs). 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: Approaches for Drug Discovery presents a detailed treatment of this strategy. Starting with a survey of PPIs that are key players in human disease and biology and the potential for therapeutics derived from this new perspective, the book then examines 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 are given. Finally, the book surveys 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, and ends with two extended case studies - inhibitors of the MDM2-p53 PPI, and the discovery of potent LFA-1 antagonists.

Protein Surface Recognition: Approaches for Drug Discovery provides an intellectual ‘tool-kit' for investigators in medicinal and bioorganic chemistry looking to exploit this emerging paradigm in drug discovery.

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Protein Surface Recognition

John Wiley & Sons

Chapter One

C. W. Bertoncini, A. Higueruelo and X. Salvatella

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 growin media lacking specific nutrients.

One key advantage of the Y2H assay is its in vivo nature, that allows the investigation of PPIs under physiological conditions. Additional advantages of this method are its high sensitivity—it can detect very weak and therefore transient interactions, with Kd as low as 10^-7 M—its scalibility and its easy automation. The Y2H assay can also be used in a quantitative fashion to determine the strength of the interaction between the bait and prey proteins by monitoring the amount of reporter protein produced, for example by measuring, when using the lacZ reporter gene, the β-galactosidase activity. The main disadvantage of the Y2H approach to the identification of PPIs is the number of control experiments that it requires, that are mainly aimed at determining whether the bait and prey proteins have affinity for DNA and are indeed capable of self-activating the transcription of the reporter. An additonal concern when using this approach, one that is directly linked to the its in vivo nature, is the possibility that a third protein mediates, in the assay, the interaction between the pray and bait proteins; it is therefore important to validate all PPIs derived from this assay by other methods, including those discussed in this chapter. Other limitations of the Y2H assay are due to its use of yeast, as some post-translational modifications are different in yeast to those in other eukaryots, and to the localization of of interactions in the nucleus, where some target PPIs may experience an incorrect cellular environment. Membrane proteins are obviously not suitable for this assay, but interactions between the cytoplasmic domains of extracellular receptors can be screened, using this approach, to study signal transduction pathways. PPIs identified from genomic-scale Y2H analysis are expected to have a success rate of 50%, and bioinformatic analysis tools to refine the results with co-expression and co-localization analysis can very significantly increase the accuracy of the results.

1.2.2 Phage Display

This method was one of the earliest tools developed for screening PPIs, before the recent spread of mass spectrometry-assisted protein identification. Phages are bacteria-specific viruses which carry the viral DNA enclosed in an envelope of viral proteins. Phage particles are therefore unique in that they contain both DNA and protein copies of a given gene in a single entity. This singularity provided molecular biologist with a unique tool to isolate simultaneously both the protein displayed in the exterior of a phage particle and its DNA sequence.

The phage display technique involves the construction of a DNA library where the sequences that code for the proteins to be screened are fused to the sequence of a bacteriophage coat protein (P8 or P3) in a plasmid containing the rest of the components of the phage genome (6.5 Kbp for the filamentous bacteriophage M13). Upon infecting an E. coli host the phages display the chimeric proteins in their outer surface and bear inside the DNA sequences that correspond to such proteins. Phages are produced in E.coli individually, and the particles are then assayed for binding to the immobilized target protein in an ELISA (Enzyme-Linked Immunsorbent Assay) fashion. In order to reduce background bound phages are collected and re-amplified in E. coli and, after two to three rounds of binding, the DNA of phages strongly interacting with the target is isolated and sequenced, leading to the identification of the proteins interacting with the target protein. A concise recollection of several random peptide and genomic libraries constructed in different phage vectors, as well as different kind of proteins successfully displayed infilamentous phages was published in 1997 by Smith and Petrenko. Despite being slightly outdated, this survey highlights the range of proteins that withstand phage display that includes enzymes, hormones, receptors, cytokines and DNA binding proteins and account for more than 50...