Concepts and Case Studies in Chemical Biology

Herbert Waldmann, Petra Janning

• Publisher: Wiley-VCH
• Year: 2014
• Print ISBN: 9783527336111
• eBook ISBN: 9783527676002
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

Retaining the proven didactic concept of the successful "Chemical Biology - Learning through Case Studies", this sequel features 27 new case studies, reflecting the rapid growth in this interdisciplinary topic over the past few years.
Edited by two of the world's leading researchers in the field, this textbook introduces students and researchers to the modern approaches in chemical biology, as well as important results, and the techniques and methods applied. Each chapter presents a different biological problem taken from everyday lab work, elucidated by an international team of renowned scientists.
With its broad coverage, this is a valuable source of information for students, graduate students, and researchers working on the borderline between chemistry, biology, and biochemistry.

1
Real-Time and Continuous Sensors of Protein Kinase Activity Utilizing Chelation-Enhanced Fluorescence

Laura B. Peterson and Barbara Imperiali

1.1 Introduction

Protein kinases, the enzymes responsible for phosphoryl transfer from a chemical donor such as adenosine triphosphate (ATP) to a peptide or a protein acceptor, are integral enzymes in signaling cascades, play crucial roles in numerous cellular processes, and are of fundamental importance in systems biology. In addition, aberrant kinase activities are commonly associated with disease states, making kinases important therapeutic targets in current drug development initiatives. Therefore, understanding kinase activation dynamics is of utmost biological and clinical importance. Accurate and physiologically relevant methods to quantify kinase activities are needed to understand the intricate dynamics of kinase activation and inactivation. This chapter describes the design, evolution, and application of fluorescent-based Ser/Thr/Tyr kinase activity sensors that take advantage of chelation-enhanced fluorescence (CHEF). These sensors are compatible with physiological conditions, are selective for specific protein kinases, and provide real-time kinetic information regarding kinase activity.

1.2 The Biological Problem

Phosphorylation, or the attachment of a phosphate group to amino acid side chains, is one of the most abundant posttranslational modifications (PTMs) of proteins. Phosphorylation reactions are mediated by phosphotransferase enzymes, termed kinases, with ATP as the typical source of the transferred phosphoryl group. Ser, Thr, and Tyr are the most commonly phosphorylated residues in eukaryotes, while His and Asp phosphorylation has also been observed, predominantly in prokaryotes. Protein activity, localization, and structure as well as protein–protein interactions are all affected by protein phosphorylation [1, 2]. As kinases play integral roles in cellular signaling, dysregulated kinase function has emerged as a driver for many different disease states, including cancer, neurodegenerative diseases, and metabolic disorders [3, 4]. Accordingly, much effort has been put forth toward understanding kinase structure, function, and activity as well as toward the clinical development of kinase inhibitors for the treatment of human disease.

Of considerable value to the scientific community are methods to study kinase activity, providing a means to evaluate kinase activity dynamics, inhibitor activities, and roles in cell signaling. Traditional assays for monitoring kinase activity utilize antibodies specific for the phosphorylated (activated) kinase, which is a common proxy for kinase activation, or rely on radioactivity-based measurements by monitoring the transfer of the radioactive γ-phosphoryl group from [γ-³²P]ATP to a substrate protein or peptide. Although the use of phosphopeptide/protein-specific antibodies is widely accepted as a useful detection method of kinase activity, antibodies may not take into account other factors affecting kinase activity, including kinase or substrate localization or additional PTMs that may also modulate activity. Radioactivity-based assays are limited in throughput, are inherently noncontinuous, and radioactive reagents require special handling. Mass spectrometry-based methods have also been developed and rely on the detection of phosphopeptides after enzymatic degradation. Fluorescence-based approaches represent valuable alternative methods for monitoring kinase activity.

Many strategies using fluorescence have been employed to detect kinase activity. Generally, these kinase sensors manifest increased fluorescence emission upon phosphorylation, while both dual fluorophore (fluorescence resonance energy transfer, FRET, Box 1) sensors and single fluorophore-containing sensors have been developed.

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Box 1.1

Förster Resonance Energy Transfer (FRET)

FRET is the process by which one fluorophore, “the donor,” transfers energy to a second fluorophore, “the acceptor.” When both chromophores are fluorescent, FRET occurs. In the case of FRET between fluorophores, the emission spectrum of the donor fluorophore must overlap with the absorption spectrum of the acceptor. In this case, the emission from the donor excites the acceptor causing it to emit light (fluoresce). The efficiency of FRET depends on the distance between the two fluorophores, the spectral overlap, and the relative orientations between the donor emission dipole and the acceptor absorption dipole.

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FRET-based sensors rely on conformational changes that often accompany phosphorylation, which alter the FRET efficiency. Many FRET sensors are plagued by small changes in fluorescence upon phosphorylation, rely on the bulky Aequorea victoria fluorescent proteins (AFP), are not compatible with high-throughput methods, and/or require genetic manipulation to incorporate the sensor into the system of choice [5]. Therefore, peptide-based fluorescent sensors provide an alternative approach to kinase sensing. Ideal kinase activity sensors should manifest high fluorescence changes upon phosphorylation, should provide a quantitative measure of catalytic activity, and be amenable to the establishment of continuous assays, ideally in a high-throughput format. They should also be selective for the kinase of interest, readily prepared, and the design should be generalizable to the diverse families of kinases that comprise the kinome. In addition, the sensors should be operationally compatible with endogenous concentrations of the ATP cosubstrate. This chapter describes the design, development, and application of fluorescent sensors for kinase activity that are based on the principle of CHEF using 8-hydroxyquinoline fluorophores.

1.3 The Chemical Approach

Kinase substrate peptides represent ideal platforms for sensor design. Peptides are readily prepared by solid-phase peptide synthesis (SPPS), can be chemically modified with fluorescent probes or other small molecules, and retain recognition elements contained w...

Table of contents

• Cover
• Related Titles
• Title Page
• Copyright
• List of Contributors
• Introduction and Preface
• Abbreviations
• Chapter 1: Real-Time and Continuous Sensors of Protein Kinase Activity Utilizing Chelation-Enhanced Fluorescence
• Chapter 2: FLiK and FLiP: Direct Binding Assays for the Identification of Stabilizers of Inactive Kinase and Phosphatase Conformations
• Chapter 3: Strategies for Designing Specific Protein Tyrosine Phosphatase Inhibitors and Their Intracellular Activation
• Chapter 4: Design and Application of Chemical Probes for Protein Serine/Threonine Phosphatase Activation
• Chapter 5: Autophagy: Assays and Small-Molecule Modulators
• Chapter 6: Elucidation of Protein Function by Chemical Modification
• Chapter 7: Inhibition of Oncogenic K-Ras Signaling by Targeting K-Ras–PDEδ Interaction
• Chapter 8: Development of Acyl Protein Thioesterase 1 (APT1) Inhibitor Palmostatin B That Revert Unregulated H/N-Ras Signaling
• Chapter 9: Functional Analysis of Host–Pathogen Posttranslational Modification Crosstalk of Rab Proteins
• Chapter 10: Chemical Biology Approach to Suppression of Statin-Induced Muscle Toxicity
• Chapter 11: A Target Identification System Based on MorphoBase, ChemProteoBase, and Photo-Cross-Linking Beads
• Chapter 12: Activity-Based Proteasome Profiling in Medicinal Chemistry and Chemical Biology
• Chapter 13: Rational Design of Activity-Based Retaining β-Exoglucosidase Probes
• Chapter 14: Modulation of ClpP Protease Activity: from Antibiotics to Antivirulence
• Chapter 15: Affinity-Based Isolation of Molecular Targets of Clinically Used Drugs
• Chapter 16: Identification of the Targets of Natural-Product-Inspired Mitotic Inhibitors
• Chapter 17: Finding a Needle in a Haystack. Identification of Tankyrase, a Novel Therapeutic Target of the Wnt Pathway Using Chemical Genetics
• Chapter 18: The Identification of the Molecular Receptor of the Plant Hormone Abscisic Acid
• Chapter 19: Chemical Biology in Plants: Finding New Connections between Pathways Using the Small Molecule Sortin1
• Chapter 20: Selective Targeting of Protein Interactions Mediated by BET Bromodomains
• Chapter 21: The Impact of Distant Polypharmacology in the Chemical Biology of PARPs
• Chapter 22: Splicing Inhibitors: From Small Molecule to RNA Metabolism
• Chapter 23: Photochemical Control of Gene Function in Zebrafish Embryos with Light-Activated Morpholinos
• Chapter 24: Life Cell Imaging of mRNA Using PNA FIT Probes
• Chapter 25: Targeting the Transcriptional Hub β-Catenin Using Stapled Peptides
• Chapter 26: Diversity-Oriented Synthesis: Developing New Chemical Tools to Probe and Modulate Biological Systems
• Chapter 27: Scaffold Diversity Synthesis with Branching Cascades Strategy
• Index
• End User License Agreement