Targeting Enzymes for Pharmaceutical Development

                  Targeting Enzymes for Pharmaceutical Development

Drug development is the process of bringing a new pharmaceutical drug to the market once a lead compound has been identified through the process of drug discovery. Enzymes offer unique opportunities for drug discovery, as they are one of the most important groups of drug targets. Many of the significant biochemical processes in the cell are enzyme-catalyzed reactions (biosynthesis and catabolism) or specific cellular signaling pathways that enzymes are involved with. Several important diseases are often associated with the elevation or repression of certain enzymes. The attractiveness of enzymes as targets for drug discovery stems from their high levels of disease association (target validation) and druggability (target tractability), which typically characterize this class of proteins. In general, drug discovery starts by manipulating the target enzyme with a compound that usually leads to inhibition or activation of its catalytic activity. Today, there are seventy-one human, bacterial, viral, and fungal enzymes that have been used successfully for the development of currently marketed clinically utilized drugs. All these drugs act as reversible or irreversible enzyme inhibitors.

This volume contains methods and detailed experimental protocols on the evaluation of the effect of a compound or a mixture of compounds on the action of enzymes that are significant targets in the pharmaceutical industry. It examines the most reliable and robust methods for both bench and R&D scientists and sets a standard for best practices in the field. This volume consists of three different sections, each of which deals with different steps in the process from target selection and compound design to inhibitor evaluation. The first section presents biocomputing and bioinformatics protocols that have been developed recently. It underlines the progress in this area and points out the advantages that enzymologists and medicinal chemists can exploit for new target selection, druggability assessment, and structure-based design. The next section contains a selection of the state-of-the-art modern biophysical, electrophoretic, and chromatographic methods and high-throughput screening approaches that have been developed and are currently used for the assessment of enzyme/inhibitor interaction. The subsequent section provides detailed protocols and examples of the inhibition analysis and evaluation of selected enzymes. It contains critical information on enzyme structure-function relationships as well as mechanistic aspects on how these enzymes are inhibited.

This volume has been written by international scientists, who are active in biochemical and biomedical research, with expertise in chemistry, protein biochemistry, enzymology, molecular biology, and genetics. While it is not possible to detail and include every possible method and protocol related to enzyme inhibition, the present volume attempts to provide working tips with examples and analysis relevant to a wide range of more important enzyme targets and commonly available enzyme inhibition techniques and protocols.

The present book would definitely be an ideal source of scientific information for advanced students, junior researchers, and scientists involved in health sciences, cellular and molecular biology, biochemistry, biotechnology, cosmetology, and other related areas in academia. It is also aimed at professionals including academic faculty members, industrial scientists, and anyone working in the pharmaceutical, food, and cosmetics industries.

I sincerely hope that the reader will enjoy the information provided in this book and find its contents interesting and scientifically stimulating. I also hope that I have established a successful compilation of chapters within the exciting area of enzymes as drug targets. I would like to thank all the contributing authors for their enthusiasm and for the time they spent preparing the chapters for this book. I would also like to thank Dr. John Walker, the series editor, for his help and encouragement, and everybody at Springer for their helpful advice and support. I would especially like to thank my family for their understanding and patience during the editing and organization of the book chapters.

In Silico Laboratory: Tools for Similarity-Based Drug Discovery

Computational methods that predict and evaluate binding of ligands to receptors implicated in different pathologies have become crucial in modern drug design and discovery. Here, we describe protocols for using the recently developed package of computational tools for similarity-based drug discovery. The ProBiS stand-alone program and web server allow superimposition of protein structures against large protein databases and predict ligands based on detected binding site similarities. GenProBiS allows mapping of human somatic missense mutations related to cancer and non-synonymous single nucleotide polymorphisms and subsequent visual exploration of specific interactions in connection to these mutations. We describe protocols for using LiSiCA, a fast ligand-based virtual screening software that enables easy screening of large databases containing billions of small molecules. Finally, we show the use of BoBER, a web interface that enables user-friendly access to a large database of bioisosteric and scaffold hopping replacements.

The In Silico Fischer Lock-and-Key Model: The Combined Use of Molecular Descriptors and Docking Poses for the Repurposing of Old Drugs

Not always lead compound and/or derivatives are suitable for the specific biological target for which they are designed but, in some cases, discarded compounds proved to be good binders for other biological targets; therefore, drug repurposing constitute a valid alternative to avoid waste of human and financial resources. Our virtual lock-and-key methods, VLKA and Conf-VLKA, furnish a strong support to predict the efficacy of a designed drug a priori its biological evaluation, or the correct biological target for a set of the selected compounds, allowing thus the repurposing of known and unknown, active and inactive compounds.

Determination of Half-Maximal Inhibitory Concentration of an Enzyme Inhibitor

Drug development is the process of bringing a new pharmaceutical drug to the market once a lead compound has been identified through the process of drug discovery. Enzymes are one of the most important groups of drug targets; thus, enzyme inhibition is widely used for the treatment of certain disorders. The assessment of an inhibitor against an enzyme is predominantly based on two different parameters: the half-maximal inhibitory concentration (IC50) and the inhibition constant (Ki). This chapter describes an experimental procedure for the determination of the IC50 value of an enzyme inhibitor. The relationship between IC50 and Ki is also discussed.

Applications of Differential Scanning Fluorometry and Related Technologies in Characterization of Protein-Ligand Interactions

Differential scanning fluorometry (DSF) is an efficient and high-throughput method to analyze protein stability, as well as detect ligand interactions through perturbations of the protein’s melting temperature. The method monitors protein unfolding by observing the fluorescence changes of a sample, whether through an environmentally sensitive fluorophore or by intrinsic protein fluorescence, while a temperature gradient is applied. Here, we describe in detail how to develop and optimize DSF assays to identify protein–ligand interactions while exploring different buffer and additive conditions. Analysis of the data and further applications of the method are also discussed.

High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins

Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure-activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format.

Enzyme–Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism

CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of the protein-ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC.

This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints, and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand.

The highly collimated microbeam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules to measure good quality, artifact-free SRCD, and CD data will be described in this chapter in particular for the study of protein–ligand interactions and protein photostability.


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