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High-throughput screening

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Cole Black Kenneth New Unlisted York Profiling

Chapter 9) within large compound libraries. The decision as to whether a particular hit is worth pursuing as a chemical lead in a drug discovery project depends on several factors, important ones being its chemical characteristics and its pharmacodynamic and pharmacokinetic properties. These aspects, broadly covered by the term ‘compound profiling’, are discussed in detail in the next three chapters.

The technology involved in miniaturization, automation and assay readouts required for HTS has developed rapidly and continues to do so. As this technology evolves, the laboratory set-ups installed in HTS facilities are steadily broadening their capabilities beyond their primary function of identifying hits to apply HTS techniques to more diverse compound profiling assays relating not only to the target selectivity of compound libraries, but also to their pharmacokinetic characteristics. Increasingly, therefore, early compound profiling tasks on ‘hit’ compounds are being carried out in HTS laboratories where the necessary technological expertise is concentrated. Such assays are also very helpful in the ‘lead identification’ stage of a project, where focused synthetic compound libraries based on the initial hits need to be assessed. As this work generally involves testing small compound libraries, usually fewer than 1000 compounds at a time, in several different assays, small dedicated robotic workstations are needed, rather than the fast but inflexible factory-style robotic assemblies used for large-scale HTS.

In vitro pharmacokinetic assays (see Chapter 10), which are not generally project specific and can be automated to run in medium-throughput fashion, are very suitable for running in this environment. This extension of the work of HTS laboratories beyond the primary task of finding hits is a clear and continuing trend, for which the term ‘high-throughput profiling’ (HTP) has been coined. It brings the work of HTS laboratories into a close and healthy relationship with drug discovery teams. The highly disciplined approach to assay formats and data logging that is essential for HTS, but not second nature to many laboratory scientists, brings the advantage that profiling data collected over a wide range of projects and drug targets is logged in standard database formats, and is therefore a valuable company-wide tool for analysing structure–activity relationships. This necessity to handle and visualize such data has driven the development software packages such as Spotfire (spotfire.tibco.com).

In summary, it is clear that pharmacological profiling will be an increasing activity of HTS units in the future, and will help to add further value in the drug discovery chain.

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Forensic Science

Hans H. Maurer, in Handbook of Analytical Separations, 2008

12.7 Concluding summary and perspectives

High-throughput drug screening procedures in analytical toxicology mean, that thousands of relevant toxicants can simultaneously be screened for, so-called systematic toxicological analysis (STA). GC-MS, especially in the EI full-scan mode, is still the reference method for comprehensive screening and reliable library-assisted identification, because universal sample work-up and GC separation as well as huge libraries of reference EI mass spectra are available. They allow identifying unknown compounds even in absence of reference substances, if certain prerequisites are fulfilled. However, LC-MS has shown to be an ideal supplement, especially for detection of more polar, unstable or low-dosed drugs, especially in blood plasma.

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Volume II

Jacques E. Dumont, ... Pierre P. Roger, in Endocrinology: Adult and Pediatric (Seventh Edition), 2016

Black York Cole Unlisted New Kenneth Activation/Inhibition by Low Molecular Agonists/Antagonists

Oral administration of the agonist to mice stimulated thyroid, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Apart from their interest to unravel the mechanism of activation of the receptor, these molecules constitute leads for development of drugs to use in place of recombinant human TSH (e.g., in patients with thyroid cancer).

Similarly, low molecular weight antagonists have been identified, showing promising therapeutic potential in Graves’ disease, but also in toxic adenomas. Indeed these compounds display inverse agonist characteristics on constitutive TSH receptor mutants, which means that they could be effective in counteracting tissue autonomy in toxic adenomas.

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Fluorescence-Based Biosensors

Pauline Scholler, ... Julie Kniazeff, in Progress in Molecular Biology and Translational Science, 2013

Abstract

High-throughput screening requires easy-to-monitor, rapid, robust, reliable, and miniaturized methods to test thousands of compounds on a target in a short period, in order to find active drugs. Only a few methods have been proved to fulfill all these requirements. New screening approaches based on fluorescence and especially on the principle of resonance energy transfer are being developed to study one of the main targets in the pharmaceutical industry, namely, the G protein-coupled receptors (GPCRs). Two types of approaches are clearly defined: generic approaches that are immediately applicable to a lot of targets such as second messenger kits or kinase kits; target-specific approaches that sense the receptor itself such as fluorescent ligands or fluorescent partners. This chapter focuses on sensors and approaches using the time-resolved Förster resonance energy transfer and homogeneous time-resolved fluorescence principle, their use, and their prospective applications for screening drugs acting on GPCRs.

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Measuring Biological Responses with Automated Microscopy

Christine C. Hudson, ... Carson R. Loomis, in Methods in Enzymology, 2006

Meeting the Demand of High‐Throughput Screening with Transfluor

High‐throughput screening assays need to be predictive of the pharmacology of a test compound on its target, simple and easy to perform, robust, and automatable. The arrestinGFP translocation assay has been shown to meet all of these requirements. First, it has been used successfully to determine the pharmacology of known agonists and antagonists for several GPCRs (). Next, the Transfluor assay is performed easily because it involves a few, basic steps (Fig. 6). In contrast to other high‐content screening assays, which require primary and secondary antibody incubations and washes after cell fixation, no wash steps are necessary with Transfluor. In addition, all steps in the assay are automated easily by liquid‐handling robotics such as MultiDrops and MiniTraks, and detection of assay results has been validated on multiple, automated fluorescent imaging platforms (). Furthermore, the assay is very reproducible and gives excellent screening statistics, including Z prime values above 0.5, ensuring an optimal screening assay window.

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Drug Discovery Technologies

R. Flaumenhaft, in Noble Leather Wedge Sandal Clarks Zia Black Women's B0PwFwEqZ, 2007

3.07.3.2.2 Assays using multicellular organisms

An advantage of this approach is that the small molecule can be applied at any time during development and for a duration determined by the investigator. For example, a compound that inhibits zebrafish otolith development only when incubated with embryos between 14 and 26 h postfertilization was identified. This result showed that the compound acted on a developmentally regulated gene product. In a second zebrafish model, mutant zebrafish were used to identify small molecules capable of reversing phenotypic abnormalities. The zebrafish mutant gridlock exhibits an aortic abnormality that mimics aortic coarctation. Fluorescence microangiography was used to screen a 5000-compound library for small molecules that reversed the defect. The screen identified two small molecules that suppressed the gridlock phenotype. Both compounds were subsequently found to enhance vascular endothelial growth factor (VEGF) expression. In addition, overexpression of VEGF in gridlock zebrafish suppressed the aortic abnormality. These results raise the possibility that chemical genetics can be used in model organisms to study complex phenotypes and identify novel therapeutic strategies for inherited diseases.

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Neuropeptide Receptors – Drug Development

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Nonpeptide Antagonists to Neuropeptide Receptors

Unlisted Cole New Black Kenneth York 2+ release from intracellular store or to the production of cyclic adenosine monophosphate (cAMP) and to cAMP responsive element-binding protein (CREB)-dependent transcription of luciferase. Such advances and the availability of additional reporter systems led to the screening of large chemical libraries for ligands to neuropeptide receptors.

Thus, nonpeptide antagonists and allosteric ligands of agonist and antagonist type have been found for many receptors that were screened, although some screens may have been unsuccessful, in spite of the large size of the libraries used. Of course, the chemical space that has been explored so far is very limited, especially given the nature of some of the targets investigated.

However, even when the HTS has identified hits, the subsequent development of drugs by pharmaceutical chemists follows the same slow and difficult path of engineering selectivity for receptor subtypes and for other relevant targets, proper safety, and determination of pharmacokinetic properties to reach the clinical candidate.

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Drug Discovery Technologies

W. Jahnke, in Unlisted York Cole New Black Kenneth Comprehensive Medicinal Chemistry II, 2007

3.41.2.5 Fragment-Based Ligand Design Makes ‘Undrugable’ Targets Drugable

and Bcl-xL. These case studies suggest that FBLD can be successful in identifying high-affinity ligands even for difficult targets for which HTS has failed, be it because the corporate archive did not contain the proper chemotypes, or that those targets are indeed poorly drugable.

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Morikatsu Yoshida, ... Kenji Kangawa, in Methods in Enzymology, 2012

4 Recombinant Expression Systems for Orphan GPCRs

Koller et al., 1997). Another approach has been to generate the target receptor as a fusion protein with green fluorescent protein (Kallal and Benovic, 2000). Coupled with fluorescence-activated cell sorting, confocal fluorescent microscopy, or immunocytochemistry, both of these approaches have enabled verification of the site of target protein expression.

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THE DISCOVERY OF ELTROMBOPAG, AN ORALLY BIOAVAILABLE TpoR AGONIST

KEVIN J. DUFFY Ph.D., CONNIE L. ERICKSON-MILLER Ph.D., in Winter Boot N997 Angkor Brown El Naturalista Women's xgnA6C, 2007

High-throughput screening (HTS) of small-molecule compound collections is a well-validated and efficient methodology for discovering new lead molecules for drug discovery. The successful application of such an approach to the discovery of hematopoeitic cytokine agonists, however, is a daunting task because of the complicated and extensive nature of a cytokine-cytokine receptor interaction and the myriad downstream signaling pathways involved in transcriptional activation and cellular survival. Nevertheless, HTS for agonists of thrombopoietin receptor (TpoR)-activated signaling pathways, utilizing hematopoetic cell lines co-transfected with the human TpoR, a luciferase gene, and several requisite signal-transduction responsive elements, successfully identified several small-molecule leads. These leads were shown to activate TpoR-mediated signaling pathways in cell lines and, ultimately, to stimulate the formation of megakaryocyte progenitor cells ex vivo in human-bone-marrow tissue samples. The data from these assays were critical in the optimization of the molecular leads and in the discovery of a pharmacophore for TpoR activation. Ultimately, this understanding of the structure-activity relationships (SAR) for TpoR activation culminated in the design of SB-497115 (Eltrombopag), a potent, orally bioavailable TpoR agonist. The progression of Eltrombopag into clinical trials provides the final validation of our approach, wherein a dose-dependant stimulation of platelet production is observed in humans.

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