Screening, Assays & High-Content Analysis

A selection of articles from European Pharmaceutical Review covering screening, assays & high-content analysis:

Screening In-depth Focus 2015

Screening: In-depth Focus 2015

6 January 2016  •  Caroline Richards

In this Screening In-depth Focus: High-content screening accelerates discovery rates in the life sciences; Phenotypic screening using 3D tissue culture and whole animal assays; Screening Roundtable...

Screening Supplement

Screening: In-depth focus 2014

5 January 2015  •  Prabir Basu, João A. Lopes

This free-to-view Screening In-Depth Focus is packed with articles that will give you a greater understanding of interesting topics such as the identification of compounds that protect against free radicals, advancements in sandwich immunoassay techniques and hit validation and optimisation...

Recombinant protein

A history of recombinant protein technology in small molecule drug discovery

28 October 2014  •  Rick Davies, Associate Director, AstraZeneca / Ian Hardern, Senior Research Scientist, AstraZeneca / Ross Overman, Associate Principal Scientist, AstraZeneca

Recombinant protein production is a prerequisite and essential component of most modern small molecule drug discovery programs. Target proteins are required to underpin screening, structural and mechanistic studies providing data that drives chemical design. From the initial establishment of recombinant protein production in the pharmaceutical industry in the 1980s, systems and technologies have evolved in step with developments in other areas to enable rapid production of many different target proteins, and their variants, specifically designed for their end use. This review describes the evolution of recombinant protein production over the past 30 years, tracking changes in technologies and working practices in relation to landmark changes in drug discovery strategies over that period...

Pharmaceutical Salts

Pharmaceutical salts of small molecule drugs: opportunities and challenges

28 October 2014  •  Axel Becker, Scientist, Merck KGaA

Over the past decades, pharmaceutical drug development has undergone some significant changes, a prominent example for this being the emergence of biomolecular drugs (New Biological Entities, NBEs) such as antibodies and peptides. However, classical small molecule drugs (New Chemical Entities, NCEs) are far from being a dying species, and in fact the number of NCE drugs approved by the US Food and Drug Administration (FDA) in the past few years has been higher with increasing trend compared to NBE drugs, which appear to stagnate at lower level in terms of new drug approvals...

Screening: In-depth focus 2013

16 December 2013  •  Roger Clark, Paul Harper and Mark Wigglesworth (AstraZeneca Global High Throughput Centre), Rob Jepras and Steve Ludbrook (GlaxoSmithKline)

Technological and sociological advances in HTS: evolution and revolution?
Flow cytometry as a drug screening platform
Show Preview: High-Content Analysis 2014

Figure 1: Principle of protein fragment complementation. Two interacting proteins are fused to N and C terminal fragments of a fluorescent or luminescent reporter protein (generic depicted in yellow). Upon interaction, the activity of the reporter is reconstituted and can be read out as fluorescence upon excitation (2) or as luminescence upon substrate addition (3)

Cell-based assays for protein-protein interactions

22 October 2013  •  Mark Wade, Center for Genomic Science of [email protected]

Protein-protein interactions (PPI) form the backbone of all cellular signalling networks, and aberrant PPI contribute to the pathology of several diseases. Thus, strategies to identify PPI modulators are expected to be therapeutically beneficial. However, there are very few examples of clinically approved PPI modulators, reflecting the difficulties of identifying effective compounds for this target class. This perspective reviews the challenges associated with targeting PPI, and summarises the major strategies used to detect and disrupt PPI, with a particular focus on cell-based assays for PPI.

Screening: In-depth focus 2013

30 July 2013  •  Anthony Mitchell Davies, Alice Vajda, Sarah Louise, Laure Marignol, Sheraz Gul, Emma J. Shanks

New approaches to cell based assays for high content screening and analysis.
Reduce, reuse, recycle: how drug repositioning is finding its niche in drug discovery.
Workshop Review: Biochemical assays for screening.
Screening roundtable.

What is label-free screening and why use it in drug discovery?

What is label-free screening and why use it in drug discovery?

18 December 2012  •  Matthew A. Cooper and Reena Halai, Institute for Molecular Bioscience, the University of Queensland

In the journey of a molecule from its origins in a compound library to candidate drug status, a large variety of profiling must occur to define activity, selectivity, potency, adverse effects, pharmacology and in vivo efficacy. Advances in biophysical methods that can analyse drug interactions with a molecular target, a whole cell, or even ex vivo tissue have enabled many of these studies to be carried out without the need for reporter-based or ‘labelled’ assays. Label-free screening in high-throughput mode can be used as a pathway independent screening tool with whole cells, or in low-throughput mode with individual receptors to define interaction kinetics and thermodynamics. We highlight advances in optical and impedance-based biosensors, and examine their utility and suitability for various stages of the drug discovery process. In the early to mid 20th century, drug discovery was a far more productive industry, and more drugs were launched per Pharma employee than today. The regulatory pathway that pre-empted the launch of a new drug was concise and easy to understand, and applications were dealt with expeditiously with a fraction of the supporting data required today. The process of discovery was also very different; it was driven largely by individuals in small teams who were prepared for serendipity, or by individuals with a very clear, defined hypothesis who drove rational drug design. Screening technologies could be summed up on one or two pages of a review; a dozen or so primary assays, some basic biochemistry to define ligand mode of action, perhaps some live cell work and a proof of concept demonstration in vivo...

Drug discovery assays for the histone deacetylase class of enzymes

Drug discovery assays for the histone deacetylase class of enzymes

18 December 2012  •  Sheraz Gul and Gesa Witt, European ScreeningPort GmbH

The histone deacetylase (HDAC) class of enzyme are a group of conserved enzymes known for their ability to remove acetyl groups from lysine residues on histone tails. Since aberrant HDAC enzyme expression is observed in various diseases, there is increasing interest in finding small molecules which function as HDAC enzyme inhibitors. This article reviews the various biochemical assays available for monitoring HDAC enzyme activity that have been validated for use in High Throughput Screening. The assays referred to are compatible with standard microtitre plates (96 and 384 well format) and make use of absorbance, luminescence and fluorescence detection methods. The histone deacetylase class of enzymes: The histone deacetylase (HDAC) class of enzymes are involved in many biological pathways and one of their best known properties is their ability to remove acetyl groups from lysine residues on amino-terminal histone tails. Thus far, 18 HDAC enzymes have been identified which are divided into zinc dependent and NAD dependent enzymes. The Class I HDAC enzymes include the zinc dependent HDACs 1, 2, 3, and 8 and consist of 350-500 amino acid residues. The Class II HDAC enzymes are also zinc dependent but are larger, consist of about 1,000 amino acid residues and are subdivided into Class IIa (HDAC4, 5, 7, and 9) and Class IIb (HDAC6 and HDAC10) enzymes. The Class I and Class II HDAC enzymes can be inhibited by trichostatin A (TSA) and this inhibitor is often used as a reference to bench-mark their assays. The Class III HDAC enzymes are the sirtuin enzymes (SIRT1-7) and are NAD-dependent. This class of enzymes is not sensitive to TSA but can be inhibited for example by nicotinamide.

Figure 2: Showing 96 and 384 well micro-plates which have has gel based bioreactor system incorporated within the body of the plate in regions in between and surrounding the wells

A reduction to practise for siRNA screening utilising high conent analysis (HCA) technologies

10 July 2012  •  Anthony Mitchell Davies & Anne Marie Byrne, Department of Clinical Medicine Trinity College Dublin; Holger Erfle, BIOQUANT-Zentrum Ruprecht-Karls-Universität Heidelberg; Graham Donnelly, Rita Murray & Peadar MacGabhann, Biocroi Ltd

One of the major limitations of performing large-scale High Content Analysis (HCA) screens is reagent cost, indeed this fact has been a key driver in the development of assay size reduction strategies here at The Irish National Centre for High Content Screening and Analysis at Trinity College’s Department of Medicine. As well as the obvious financial advantages of reducing assay volumes, we have also identified other key benefits to this approach, namely: Higher throughput; Improved signal to noise; Suited for the use of valuable cells, e.g. primary cells; Reduced storage and research space; Improved mixing of reagents. The practicalities of performing cell based assays at the nano-litre scale: Despite the clear benefits to adopting miniaturisation, there are several significant barriers that must be overcome before these methods can be utilised. These are sample delivery / handling and environmental stability.

RedHill Biopharma has last patient visit in phase IIa study with RHB-104 for multiple sclerosis

Discovery and validation of biomarkers for multiple sclerosis

13 December 2011  •  Ole Pless and Sheraz Gul, European ScreeningPort GmbH

Multiple Sclerosis (MS) is an autoimmune disease leading to a chronic inflammation and degeneration of the central nervous system. It is one of the major neurological diseases with approximately 2.5 million suffering patients worldwide. Until now, the underlying mechanisms have not been fully elucidated, but the cause of the disease can be modulated to limit progression and severity. Currently, there are no validated biomarkers available to predict the progression of MS or response to a clinical intervention apart from MRI. In order to identify protein biomarkers for MS as well as other diseases, significant infrastructure is required and this is discussed. The term ‘biomarker’ has been defined as a “characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”. The measurement of normal and dysfunctional biological processes and their changes in response to therapeutic intervention forms the basis of biomarkers. The advances in genetics and molecular biology leading to the sequencing of the human genome has resulted in the identification of a variety of novel targets implicated in different disease states. Further technological developments including high throughput profiling of various samples using genomics, transcriptomics and proteomics has led to the identification of gene and protein based markers that characterise disease states for a number of indications including breast cancer, colorectal cancer and cardiovascular diseases. Additional initiatives that have led to the identification of biomarkers with minimal invasive methods such as proteomics technologies and systems biology have proven extremely effective for discovering potential biomarkers and drug targets. These technologies tend to provide large data sets that can be difficult to deconvolute for biomarker discovery. This bottleneck can be reduced by using several strategies. The first is to constrict the number of potential biomarkers and drug targets by dividing the proteome into smaller, more biologically significant segments. The second is to widen the bottleneck with higheroutput and higher-throughput screening technologies. The third is to incorporate more preliminary validation into the discovery process. New and emerging technologies provide promise for each of these strategies.

Figure 1 High content screening analysis of HepG2 cells stained under control (A,C,E,G), or 24 (B,D,F) and 72 (H) h incubations with 10-6 / 10-5 M doxorubicin with different fluorescent probes, i.e.. Hoechst 33342, Fluo-4, TOTO-3 and TMRM

High Content Screening for in vitro toxicity testing

20 June 2011  •  Willem G.E.J. Schoonen, Walter M.A. Westerink, Femke M. van de Water and G. Jean Horbach, Department of Toxicology & Drug Disposition, Merck Sharp & Dohme

The application of High Content Screening for in vitro toxicity testing is a relatively new approach in the preclinical research phase of drug development. A battery of tests have been developed for screening on general parameters such as cytotoxicity, while more dedicated assays are available with respect to the identification of genotoxicity, phospholipidosis, steatosis and cholestasis. All these tests are very beneficial within the pharmaceutical industry for the selection of appropriate candidates for drug development as well as for reduction of the attrition rate. High content screening (HCS) is quickly growing in popularity within the field of in vitro toxicity testing. The maturity of HCS equipment and software has made HCS accessible for many technicians and scientists working in the area of cellular and molecular biology. Although this technique was introduced in the mid 1990’s, the simplification in the use of the software programs, the growth in computer storage capacities as well as the improved qualities of resolution of the digital microscopic cameras has largely increased the accessibility of this equipment. At the start of HCS technology, many scientists were sceptical about this technique as image-based mathematical algorithms had to be written for the analysis.

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