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Screening, Assays & High-Content Analysis

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

Figure 1The autophagy pathway. Autophagy proceeds in three steps: initiation by protein kinase complexes

Molecular drug targets in autophagy

19 April 2011  •  Robin Ketteler, MRC LMCB, University College London

Autophagy is a cellular stress response to diverse stimuli such as starvation, infection and DNA damage. Autophagy plays important roles in the progression of various diseases including cancer, neurodegenerative diseases and Crohn’s disease. Despite recent advances in our understanding of the autophagy machinery, surprisingly little effort has been undertaken towards utilising this knowledge in drug discovery processes. Several phenotypic screens have been undertaken to identify drug candidates that modulate this process. Current highthroughput screening approaches assay the formation of the autophagosome and very little effort is made towards the identification of compounds that inhibit specific autophagy components. Here, I give an overview about potential molecular drug targets in the autophagy pathway and review the current status of targeted drug discovery towards identifying autophagy gene-specific drugs.

Figure 1 High Content Imaging Technology. The leading fluorescent high content imaging platforms established within the pharmaceutical and biotechnology industry. Represented are; A. Fully automated high throughput high content imaging systems (ImageXpress, InCell, BD Pathway, ScanR, Opera and the Arrayscan). B. Live cell kinetic imaging systems (IncuCyteFLR and Cell-IQ) and C. Fluorescent tissue slide imaging platform (Scanscope FL)

Advancing high content analysis towards improving clinical efficacy

16 February 2011  •  Neil Carragher, Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh

High-content analysis is primed to play a prominent role in a new era of drug discovery research that places greater emphasis on clinical translation at all stages of the discovery process from target identification to proof-of-concept testing. High content analysis provides a technical bridge between reductionist targetdirected drug discovery approaches and new technologies that embrace the biological diversity of human disease.The drug discovery industry is evolving rapidly, this evolution is stimulated by two key factors; (i) increased accessibility of new technologies such as next generation sequencing, systems biology and imaging that enhance our ability to interrogate complex biological systems and; (ii) the perceived failure of the widely adopted target directed drug discovery operating model to deliver novel medicines. Thus, high content imaging technologies provide a timely, pragmatic solution that enhances the effectiveness of conventional target-directed chemical approaches and provides the necessary biological context for understanding proteomic or genetic signatures. However, the future success of high-content analysis in improving the clinical success rates of drug discovery projects is entirely dependent upon the physiological relevance of the biological models under evaluation.

Figure 1 Typical critical path for small molecule Drug Discovery programs. Drug Discovery involves identification of a Target of interest for which an assay is developed. This assay is then adapted for screening purposes and utilised in a High Throughput Screening campaign against small molecule libraries. The High Throughput Screening campaign will usually yield many Hit molecules. Confirmation, Counter and Selectivity Screening will provide a final list of Validated Hits.

Establishing assays and small molecule screening facilities for Drug discovery programs

16 February 2011  •  Sheraz Gul, Vice President & Head of Biology, European ScreeningPort GmbH

Although many of the marketed small molecule drugs have been discovered by research and development efforts within the pharmaceutical industry, there has been a paradigm shift with external sources increasingly being relied upon to fill their pipelines. This trend is likely to increase and the key pre-clinical activities carried out by organisations outside the pharmaceutical industry include target validation, assay development and their use in High Throughput Screening campaigns, validation of the Hit molecules, Hit-to-Lead and Lead-to-Candidate screening/chemistry. In order to perform these activities, adequate know-how and technical expertise is essential so that the processes meet appropriate industry standards. This article discusses some of the challenges associated with assay development and the automation of High Throughput Screening.

Figure 1 Signal generation on an Optical Resonance Grating Biosensor. The Reflected Light Wavelength (RLW) variates depending on the index of refraction bound at the sensor surface. The magnitude of the RLW shift to the left is proportional to the mass increase to the material bound to within about 150 nm of the sensor surface. RLW is usually express as picometres wavelength or PWV.

Evaluation of cellular response to nicotinic compounds using optical label-free technology

16 February 2011  •  Isabel Coma & Julio J. Martin, Screening and Compound Profiling, GlaxoSmithKline R&D Pharmaceuticals

Cell signalling circuits are likely to have a key role in the future of pharmacological discovery and medical treatment. There is consensus about the importance of understanding cell components and their function, not at the level of genes, but at a higher level of abstraction, involving their pathways and circuits. The optical label-free detection is a powerful approach to study molecular interactions by measuring the cellular refraction index. The phenotypic kinetic responses or signatures elicited by a compound are mechanistically informative and directly offer experimental models to abstract cell functioning. This characteristic is a practical advantage over the so-called high content techniques or the pure mathematical modelling approaches where the integration of high level information about cell functioning requires mathematical algorithms that need validation and consensus.

Figure 1 Schematic illustration of the recombinant antibody microarray set-up

Developing and applying recombinant antibody microarrays for high-throughput disease proteomics

16 December 2010  •  Carl A.K. Borrebaeck and Christer Wingren, Department of Immunotechnology and CREATE Health, Lund University

Deciphering crude proteomes in the quest for candidate biomarker signatures for disease diagnostics, prognostics and classifications has proven to be challenging using conventional proteomic technologies. In this context, affinity protein microarrays, and in particular recombinant antibody microarrays, have recently been established as a promising approach within high-throughput (disease) proteomics1-3. The technology will provide miniaturised set-ups capable of profiling numerous protein analytes in a sensitive, selective and multiplexed manner.

Figure 1 Principle of 2D cell migration assays. (A) A confluent monolayer of cells is mechanically wounded (‘scratch assay’), usually with a sterile pipette tip, leaving two wound edges (dashed lines) separated by a void. Cells at the leading edges quickly assume a polarised morphology and form broad lamellae pointing into the direction of the void. Over time, cells migrate into the void and, eventually, completely close the wound (B). Cell migration is qualitatively assessed by visual inspection and can be quantitated by measuring gap width or by enumeration of cells populating the wound. Images show T98G human glioblastoma cells immediately after wounding (A) or after 24 hours of migration (B). Distance between lines is one millimetre

Advances in two-dimensional cell migration assay technologies

1 November 2010  •  Andreas Vogt, Department of Pharmacology and Chemical Biology and the University of Pittsburgh Drug Discovery Institute, University of Pittsburgh

Cell motility plays an important role in many human diseases and normal cellular processes. Cell migration is critical for wound healing as cells of the inflammatory system and fibroblasts populate the wound and initiate re-epithelialisation1. On the other hand, unregulated cell migration contributes to cancer cell invasion and metastasis2. Agents that affect cell motility, either positively or negatively, could therefore find applications as promoters of wound healing or as antimetastatic drugs. Cell migration in a biological context is an extremely complex process and the understanding of genetic and biochemical determinants remains incomplete.

Figure 1 Packing diagrams showing the different molecular arrangements in polymorphs A (left) and B (right) of the antipsychotic drug risperidone (centre). Unit cell parameters for each polymorph are shown.

Polymorph screening in pharmaceutical development

19 August 2010  •  Professor Alastair J. Florence, Solid-State Research Group, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde

The majority of active pharmaceutical ingredients (APIs) are produced by crystallisation and so the phenomenon of polymorphism, whereby an organic molecule can adopt more than one crystalline form (Figure 1 opposite), is of considerable importance when trying to achieve consistent product quality during the manufacture of pharmaceutical solids and solid dosage forms. Although morphology and particle size-distribution are important solid-state characteristics, the uncontrolled occurrence of multiple physical forms (polymorphs, solvates, salts, co-crystals or amorphous) of an API can have significant effects on the performance of the material during processing, manufacture, storage and administration. For example, the solubility difference between some polymorphs has been shown to be over four times that of the least soluble form1 and can vary by significantly more for amorphous forms2.

Figure 1 openBIS is a software framework for organising and annotating data and metadata from biological experiments, providing query and display functionality, integrating it into data pipelines and sharing it with other researchers

Towards a comprehensive open source platform for management and analysis of High Content Screening data

19 August 2010  •  Karol Kozak, Angela Bauch, Gabor Csucs,Tomasz Pylak & Bernd Rinn, ETH Zurich

As High Content Screening (HCS) has moved into the mainstream for biological and pharmaceutical investigations, a lag of well integrated pipelines for automated acquisition, management and analysis of HCS results turns out to be a bottleneck for fully leveraging the wealth of information contained in a screen and moving to higher throughput. For many applications, monolithic pipelines cannot deliver the flexibility and versatility needed. Laboratories and scientific service providers instead usually look into integrating components from both the open source world and the commercial software world into best-of-breed data pipelines. In this article, we will present two open source components that can be used as flexible and powerful building blocks for such a pipeline.

Figure 1 ASSET™ plan and detail Copyright: The Automation Partnership

High content cell based primary screening for oncology targets – a perspective

25 June 2010  •  Peter Alcock, Colin Bath, Carolyn Blackett & Peter B. Simpson, Screening & Assay Sciences, Cancer Bioscience, AstraZeneca Alderley Park

Over the last 15 years, vendors have offered microscope-based instruments capable of producing images of fluorescent labelled components of cells grown in microtitre plates. These instruments are typically bundled with analysis software capable of defining the relative distribution of several fluorescent markers on a cell by cell basis1,2. As the readers have improved and image acquisition and analysis times have reduced, the potential for screening larger compound libraries has presented itself. High Content Screening (HCS) i.e. the generation of multiparameter data from a single well, has thus become an important tool in the High-Throughput Screening (HTS) laboratory.

drug discovery & drug development

Implementation of appropriate assays and HTS technologies in drug discovery

9 May 2010  •  Sheraz Gul, Vice President, European ScreeningPort GmbH

In this article, an overview regarding advances in assay formats for specific target classes and options that should be considered when considering hardware will be given. There has been a significant growth in the assay and automation technologies that are available for compound screening activities and it is essential to evaluate a variety of these before beginning a drug discovery program, the aims of these being to ensure the most relevant assay formats that are available are adopted.

Cancer Biology

Cancer biology where do we go next?

22 February 2010  •  

The World Cancer Report (2008) predicts a 50% worldwide increase in cancer incidence by 2030, predicting 75 million people living within a five year diagnosis of cancer1. This increase is partially fuelled by significant medical advances in developed countries ensuring people live longer. However, it is also attributable to developing countries adopting habits linked to cancer risk such as increased uptake of smoking and the acquisition of western diets. In 2007, cancer caused approximately 7.6 million or 13% of all human deaths2. Cancers associated with the greatest mortalities are lung, stomach, colorectal, liver and breast cancer respectively. There are modifiable risk factors common to many malignancies, including tobacco, overweight or obesity, poor physical activity, dietary factors, alcohol, sunlight exposure and chronic infection. Effective prevention will reduce the risk of cancer, and efficient screening will enable many to be successfully treated for their disease.


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