Über die Autorin bzw. den Autor

Dr. Jörg Hüser is Director of HTS Technologies at the Healthcare Division of Bayer AG in Wuppertal (Germany).

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In present-day drug discovery, bioactive molecules with a sought-after physiological effect are identified by screening large libraries of drug candidates for a quantifiable effect on a biological target. Because of the large number of candidate molecules involved, screening assays with high compound throughput have been developed that yield high-quality results in a short time. <br> Backed by leading authorities, this is a professional guide to successful compound screening in pharmaceutical research and chemical biology, including the chemoinformatic tools needed for correct data evaluation. Chapter authors from leading pharmaceutical companies as well as from Harvard University discuss such factors as chemical genetics, binding, cell-based and biochemical assays, the efficient use of compound libraries and data mining using cell-based assay results.<br> For both academics and professionals in the pharmaceutical and biotech industries working on small molecule screening.

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In present-day drug discovery, bioactive molecules with a sought-after physiological effect are identified by screening large libraries of drug candidates for a quantifiable effect on a biological target. Because of the large number of candidate molecules involved, screening assays with high compound throughput have been developed that yield high-quality results in a short time.
Backed by leading authorities, this is a professional guide to successful compound screening in pharmaceutical research and chemical biology, including the chemoinformatic tools needed for correct data evaluation. Chapter authors from leading pharmaceutical companies as well as from Harvard University discuss such factors as chemical genetics, binding, cell-based and biochemical assays, the efficient use of compound libraries and data mining using cell-based assay results.
For both academics and professionals in the pharmaceutical and biotech industries working on small molecule screening.

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High-Throughput Screening in Drug Discovery

John Wiley & Sons

Chapter One

Sa L. Chiang

1.1 Introduction

Understanding cellular pathways and their molecular mechanisms is one of the longstanding goals of scientific research. However, the tools that researchers utilize to study biological processes become progressively more sophisticated as technology and our knowledge of biology advance. This chapter discusses one of the most recent and exciting developments in this area: chemical genetics and the use of small, bioactive molecules to characterize the components of cellular pathways and their functions.

1.2 Classical and Chemical Genetics

All genetic approaches depend on the ability to perturb gene function and to correlate phenotypic changes with specific changes in gene function. Classical genetics relies on physical perturbation of a gene, through methods such as irradiation, chemical mutagenesis or insertional mutagenesis (e.g. via the use of transposons). There are also numerous molecular biology techniques for creating directed mutations that allow highly specific modifications at the level of the gene or even the nucleotide in a variety of experimental systems. These methods are well established and readers should consult a standard genetics text for the relevant descriptions.

A common feature of all classical genetic methods is that they cause a permanent change in the structure of a gene. Therefore, except for the few situations noted below, the phenotypes arising from classical genetic mutations are irreversible. A notable disadvantage of this irreversibility is that it hinders the study of genes that are essential for viability. Irreversibility also makes it difficult to study the effects of temporal variations in gene expression or protein function. Placing genes under the control of inducible promoters has gone some way towards solving these problems, but inducible promoters, of course, act at the level of transcription and the researcher may not obtain sufficient control over the activity of the encoded protein. Temperature-sensitive mutations may provide some control over protein activity, but they are not easy to construct and generally cannot be used in animal models. Pleiotropic effects caused by temperature shifts may also complicate analysis.

In the last decade, researchers have increasingly explored the use of low molecular weight chemical entities to modulate and characterize protein function. These methods are generally analogous to classical genetic approaches and have accordingly been termed chemical genetic strategies. Whereas classical genetics uses physical modification of a gene to perturb protein function, chemical genetics employs specific, biologically active small-molecule modulators to perturb protein function.

It is relatively simple to imagine mechanisms by which small molecules may modulate protein function. Enzymatic activity can be affected by the binding of small molecules to active or allosteric sites and protein-ligand interactions may be disrupted by small molecules that interfere with binding between interaction partners. Alternatively, interaction partners could be brought together more effectively by ligands that bind to both. Interactions between small molecules and their targets may be reversible or irreversible and protein function can be either diminished or enhanced, depending on the situation.

One advantage of small molecules is that they can be used in biological systems where there is little or no ability for classical genetic manipulation. Moreover, since many small molecules will not interact irreversibly with their targets, chemical genetics is expected to provide enhanced opportunities to create conditional phenotypes. Theoretically, a high degree of control over protein function could be afforded by simply adding or washing away a small-molecule modulator. Indeed, several widely used inducible promoter systems (lac, arabinose, tetracycline) employ small-molecule inducers. In addition, conditional phenotypes induced by small molecules can potentially be studied in animal models, where temperature-dependent phenotypes generally cannot be used.

In actuality, the ability of small molecules to induce and reverse phenotypes will depend on factors such as binding kinetics and the physical accessibility of the target. More important, most protein targets have no known small-molecule modulators of their activity. Obtaining a specific and potent small-molecule modulator for a chosen target often requires structure-based drug design or full-scale high-throughput screening (HTS) and medicinal chemistry optimization may also be necessary. Owing to this "front-end" effort, directed perturbations in many systems are currently more difficult to achieve with chemical genetics than with classical genetics.

1.2.1 Forward and Reverse Screens

Many classical genetic screens begin with mutagenesis of organisms or populations of cells, followed by attempts to associate the resulting phenotypic changes with specific genes. These approaches are termed forward genetic strategies and can be generally characterized as starting with phenotypes and progressing towards the identification of the genes that are responsible for those phenotypes. With the advent of molecular biology, directed mutagenesis techniques became available and these advances allowed reverse genetic studies, which begin with the introduction of programmed or directed mutations into a known target gene, followed by analysis of the resulting phenotypes to obtain information about the function of that gene.

Both forward and reverse genetic strategies have also been employed for chemical screens. In a forward chemical genetic screen, chemical libraries are screened for compounds that produce a phenotype of interest, typically in a cell-based assay. An example of forward chemical genetics is the strategy that was employed to identify inhibitors of mitotic spindle bipolarity. Monastrol was identified through a chemical genetic screen employing a whole-cell immunodetection assay to screen a library of 16 320 compounds for those that increase nucleolin phosphorylation, a phenotype predicted for cells experiencing mitotic arrest. A total of 139 compounds found to increase nucleolin phosphorylation were subjected to further analysis, resulting in the identification of five compounds that affect the structure of the mitotic spindle. One of these induced the formation of a mono-astral microtubule array and was accordingly named monastrol. Monastrol has since been employed in multiple studies as a tool for investigating the process of cell division.

Conversely, in a reverse chemical genetic screen, small-molecule libraries might be screened for compounds that bind to a purified protein target, modulate the activity of the target or affect the target's ability to interact with other proteins. Such compounds could then be used in cell-based assays to characterize the function of the target protein in cellular pathways. For instance, a luminescence-based reverse chemical genetic screen was employed to identify inhibitors of rabbit muscle myosin II subfragment (S1) actin-stimulated ATPase activity. The most potent compound identified (N-benzyl-p-toluenesulfonamide; BTS) inhibited S1 ATPase activity with an...