High-Throughput Screening in Drug Discovery (Methods and Principles in Medicinal Chemistry) - Hardcover

Hardcover

ISBN 10: 3527312838 ISBN 13: 9783527312832

Publisher: Wiley-VCH, 2006

Synopsis

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 pharma and biotech industries working on small molecule screening.

About this title

About the Author

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

From the Back Cover

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.

From the Inside Flap

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.

Excerpt. © Reprinted by permission. All rights reserved.

High-Throughput Screening in Drug Discovery

John Wiley & Sons

Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All right reserved.
ISBN: 978-3-527-31283-2

Chapter One

Chemical Genetics: Use of High-throughput Screening to Identify Small-molecule Modulators of Proteins Involved in Cellular Pathways with the Aim of Uncovering Protein Function

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 I[C.sub.50] of ~5 M and BTS also inhibited the activity of skeletal muscle myosin in a gliding-filament assay. Subsequent studies demonstrated that BTS inhibits the binding of myosin-ADP to actin and also affects various properties of rabbit and frog muscle preparations.

A similar reverse chemical genetic screen identified an inhibitor (blebbistatin) of nonmuscle myosin II. Blebbistatin was found to inhibit a variety of activities in whole vertebrate cells, including directed cell migration and cytokinesis. The use of blebbistatin and additional drugs (including monastrol) to manipulate the mitotic process in whole cells led to the discovery that ubiquitin-dependent proteolysis is required for exit from the cytokinetic phase of the cell cycle. Blebbistatin is thus another example of how small molecules identified via HTS can be used to study the function of both individual proteins and major cellular processes.

The above points are summarized in Table 1.1.

1.3 Identifying Bioactive Molecules

Biologically active small molecules have often been discovered by testing a single compound at a time, but such an approach is obviously highly inefficient and cost-intensive in terms of both reagents and personnel time. As a result, HTS technologies have been developed to screen large numbers of compounds simultaneously, typically through the miniaturization and automation of assay protocols. What constitutes high throughput will vary depending on technical considerations and the screener's economic situation, but generally, the number of compounds involved may range from tens of thousands to several million. With current technologies, throughput is high enough that screening this number of compounds can be accomplished within several weeks, if not within several days.

A large number of screening technologies are available today for identifying bioactive small molecules; in fact, there are too many methods to be discussed adequately in this chapter. A very general discussion is provided here, but the reader is referred to other chapters in this volume for greater detail.

High-throughput screening assays can be divided into two main classes: "pure protein" and "cell-based". Pure protein screens generally have optical assay readouts that monitor enzymatic or binding activity. For instance, fluorescence polarization or FRET techniques are commonly used to screen for compounds that affect binding between protein partners. In pure protein assays, every compound screened should have equal access to the target. However, the membrane permeability characteristics of any active molecules that are identified may subsequently pose a major concern if the target is intracellular.

Readouts for cell-based screens may rely on reporter gene systems (e.g. luciferase, -lactamase), cell density, cell viability or cell morphology. Screen readouts can also be divided into two broad classes: uniform well readout acquired via plate readers and images acquired via automated microscopy. Screens involving image-based readouts are usually technically more difficult and their computational analysis more challenging than plate-reader screens. However, image-based readouts can provide a vast amount of information and microscopy screens are therefore often referred to as high-content screens. The development of equipment and data analysis techniques for automated imaging screens is an area of active research, but application of early technologies has already yielded promising results.

1.4 Target Identification

When biologically active small molecules are identified through chemical screens, particularly forward genetic screens, a substantial amount of work is often required to identify the molecular target. Ignorance of the target does not preclude clinical or research use of the molecule; indeed, many clinical agents have been used effectively even when their targets were not known (e.g. aspirin, nitroglycerin, fumagillin, epoxomycin). Nevertheless, defining the molecular mechanism of action is vital to understanding the biological principles involved and also for potentially creating more potent or specific molecules through synthetic chemistry approaches.

Target identification is currently a major area of research in chemical genetics and Tochtrop and King have recently provided an excellent discussion of this topic. This section will therefore emphasize HTS-related examples and also discuss some recent work not included in that review.

1.4.1 Hypothesis-driven Target Identification

For well-characterized cellular pathways, it is sometimes possible to deduce the target of a small molecule by comparing data from across the field. For instance, characteristic phenotypes induced by the small molecule may permit the assignment of the target to a previously identified complementation group. Subsequent hypothesis-driven testing of potential targets can then be undertaken.

This deductive approach was used successfully to define the target of monastrol, a small-molecule inhibitor of mitotic spindle bipolarity. As noted above, monastrol was identified in a forward chemical genetic screen for compounds that affect the structure of the mitotic spindle. Monastrol induces the formation of a monoastral microtubule array and, since previous studies had noted that antibody inhibition of the Eg5 kinesin also causes monoaster formation, it was postulated that Eg5 might be a target of monastrol. Subsequent work demonstrated that monastrol reversibly inhibits Eg5-driven microtubule motility in vitro but is not a general inhibitor of motor proteins.

Hypothesis-driven target identification was also used in a chemical screen for inhibitors of SARS coronavirus (SARS-CoV) replication. In this study, a library of 50 240 compounds was screened by imaging for compounds that inhibit the cytopathic effect (CPE) of SARS-CoV towards Vero cells. A total of 104 compounds demonstrating effective inhibition of CPE and viral plaque formation were then tested in vitro against two protein targets known to affect SARS-CoV replication ([M.sup.pro] protease and the NTPase/helicase), and also in a pseudotype virus assay for inhibition of S protein-ACE2-mediated entry of SARS-CoV into 293T cells. Two inhibitors of [M.sup.pro], seven inhibitors of Hel and 18 inhibitors of viral entry were identified and each of these three classes contained at least one inhibitor active in the low micromolar range. The authors subsequently assayed the 104 compounds against other common RNA viruses and found that most were specifically active against SARS-CoV, and approximately 3% were active against all viruses tested.

1.4.2 Affinity-based Target Identification

Affinity-based methods such as affinity labeling, affinity chromatography and crosslinking are also commonly used target identification strategies. Since these approaches often require synthetic modification of the molecule (e.g. addition of linkers or immunoreactive epitopes), care must be taken that the modifications do not interfere with the molecule-target interaction. Nonspecific interactions may also complicate the analysis and controls must be designed to address this issue. Despite such limitations, however, these strategies are of tremendous utility in target identification.

With affinity labeling, molecules may be radioactively or chemically labeled; they may be synthetically modified with reactive groups to promote covalent attachment to the target or tagged with specific moieties to facilitate detection. Standard protein fractionation and detection techniques can then be used to identify proteins from crude extracts that are specifically labeled by the molecule. Examples of drugs whose cellular targets were determined by such methods include acetylcholine, the anti-angiogenic agent fumagillin and the antifungal lipopetide echinocandin.

Affinity purification of putative targets from cellular extracts is accomplished with molecules immobilized on a solid support used either as a column matrix or as a bead slurry. Small molecules for which cellular targets were identified through affinity purification include the immunosuppressant FK506, the kinase inhibitor purvalanol and the anti-inflammatory compound SB 203580. Additional studies involving affinity chromatography were instrumental in further characterizing the biochemistry of FK506 and purifying the target of the structurally related compound rapamycin.

A natural technological development of affinity-based target identification involves probing proteome chips with small molecules. A recent application of this approach is discussed in Section 1.4.4. Conversely, it is also possible to probe small-molecule microarrays with a known protein to identify specific interactions.

(Continues...)

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Edition details

• Publisher: Wiley-VCH
• Publication date: 2006
• ISBN 10: 3527312838
• ISBN 13: 9783527312832
• Binding: Hardcover
• Language: English
• Edition number: 1
• Number of pages: 362
• Editor: Hüser Jörg