Issue 5 2007

The expanding world of small RNA: from germ cells to cancer

Issue 5 2007 / 21 September 2007 / Eric A. Miska, The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK

Over the last ten years a small RNA revolution has swept biology. In 1998 RNA interference (RNAi) was discovered as an experimental tool by Andy Fire and Craig Mello₁, a finding that was awarded with the 2006 Nobel Prize for Physiology or Medicine. Although the biology of RNAi is still not understood, it has become a powerful experimental tool and is currently being developed for human gene therapy₂. During a similar time-frame and linked in some aspects to RNAi, microRNAs (miRNAs) were discovered as a new class of regulatory RNAs in animals, plants and viruses₃.

MicroRNAs are transcribed from endogenous genes as long, primary RNA transcripts and are processed to their mature form: a single-stranded RNA with a length of approximately 22 nucleotides, indistinguishable from a small-interfering RNA (siRNA), the mediator of RNAi (Figure 1). In animals these long RNA precursors (pri-miRNAs)₄ are processed in the nucleus by the RNase III enzyme Drosha and Pasha/DGCR8 to form the approximately 70-base pre-miRNAs₅. Pre-miRNAs are exported from the nucleus by Exportin-5₆, processed by the RNase III enzyme Dicer, and incorporated into an Argonaute-containing silencing complex (RISC)₇. MicroRNAs are thought to regulate gene expression post-transcriptionally by forming Watson-Crick base pairs with target miRNAs. Their mechanism of action is still under debate, but likely includes inhibition of translation and mRNA degradation₈. In animals, most miRNAs are thought to form imperfect base-pairs with their target mRNA(s) and these interaction sites are enriched in 3’ un-translated regions (3’UTRs)₃. As a consequence, miRNA target identification using computational approaches is non-trivial₉. The public database for miRNAs, miRBase release 9.2, currently lists 533 human microRNAs₁₀ and estimates for the total number of human microRNAs range from over one thousand₁₁ to tens of thousands₁₂. Although miRNAs have only been studied intensely for the last five years, important functions for miRNAs in animal development and, potentially, human disease, have already emerged₁₃. (more…)

Tagged with: Eric A. Miska, microRNA, RNAi, University of Cambridge

Proteomics – the frontiers and beyond

Issue 5 2007 / 21 September 2007 / Walter Kolch, The Beatson Institute for Cancer Research/ Institute for Biomedical and Life Sciences, University of Glasgow

Within a decade proteomics has evolved from a fledgling discipline reserved for specialised laboratories, to a firm fixture in our standard omics arsenal used routinely by the research community. This stunning progress is due to many factors; the finishing of the genome projects provided major intellectual motivation and the development of better and much easier to use mass spectrometry (MS) instruments were technological drivers. What is even more remarkable is that this progress has been made despite leaving some main issues in proteomics unsolved. Besides these known boundaries, it also has revealed new frontiers and new interesting glimpses into the world beyond. This essay discusses some selected issues, but cannot necessarily be exhaustive or free of personal opinion. (more…)

Tagged with: Proteomics, The Beatson Institute for Cancer Research, University of Glasgow, Walter Kolch

Statistical techniques for handling high content screening data

Issue 5 2007 / 21 September 2007 / Edward Ainscow, Research Scientist, AstraZeneca

One of the chief incentives for the use of high content screening (HCS) approaches is the data rich return one gets from an individual assay. However, conventional methods for hit selection and activity determination are not well suited to handling multi-parametric data. Tools borrowed from the genomics area have been applied to HCS data, but there are important differences between the two data types that are driving the development of novel statistical approaches for HCS data analysis. This article will describe the use of techniques such as principal component analysis, classification trees, neural networks and random forests, as well as recently published approaches for the identification and classification of compound profiles resulting from HCS assays.

High content screening (HCS) is becoming an increasingly popular format for the early identification of biologically active compounds. The attraction of HCS assays is that they give flexible and comprehensive data on compound effects in a biologically relevant setting, typically the cell. Diverse responses such as changes in protein expression, activation, ion fluxes and cytotoxicity can be monitored, in some cases simultaneously.

Commonly, but not exclusively, HCS is applied at the secondary screening or lead optimisation phase of pharmaceutical development. At this stage, obtaining a rapid and in depth assessment of the cellular effects of a compound is invaluable in selection of compounds or series for progression. HCS is ideally suited for this purpose since the application of automated imaging and analysis algorithms can report a multitude of on-target and off-target responses in a single assay. (more…)

Tagged with: AstraZeneca, Edward Ainscow, HCS (High Content Screening)

Maximising the efficiency and application of automated planar patch clamp electrophysiology

Issue 5 2007 / 21 September 2007 / Paul J Groot-Kormelink PhD, Pamela R Tranter PhD and Martin Gosling PhD, Novartis

The widespread expression of ion channels and their ability to significantly modulate cell function makes them attractive drug targets₁. Therapeutic agents which target ion channel proteins comprise the third best selling class of prescription drugs with US sales in 2002 estimated at $12 billion. Somewhat surprisingly the discovery (more…)

Tagged with: Cell line optimisation, Martin Gosling PhD, Novartis AG, Pamela R Tranter PhD, Paul J Groot-Kormelink PhD

The real-time reverse transcription polymerase chain reaction – treat with caution

Issue 5 2007 / 21 September 2007 / Stephen A. Bustin, Academic Department of Surgery, Institute of Cell and Molecular Science, Queen Mary’s School of Medicine and Dentistry, University of London

The real-time reverse transcription polymerase chain reaction (RT-qPCR) has become the enabling technology par excellence in every field of molecular research and development, including that of clinical drug development and discovery. Its ability to detect as well as quantify RNA biomarkers sensitively, specifically and speedily has made it an indispensable tool in translating the identification of complex biological processes into an understanding of their roles in disease pathology, response to therapy and associated pharmacological proof-of-concept drug efficacy and toxicity studies.

The principal advantages of the real-time polymerase chain reaction (qPCR) are its capacity to generate quantitative data over a wide dynamic range, coupled with high through-put, speed and convenience1, along with its potential for generating more reliable data2. The addition of a reverse transcription (RT) step3 has extended these benefits to the quantification of messenger, regulatory and genomic RNAs, making RT-qPCR to-day’s de facto standard for RNA analysis4,5, even in resource-limited settings6. RT-qPCR assays combine exquisite sensitivity and specificity; turn-around time from sample receipt to result can be less than two hours (the details of the assay5 and appropriate protocols7,8 are described elsewhere). They are performed in a closed system, thus minimising the risk of contamination and quantitative results are calculated using the threshold cycle (Ct), which is the cycle fraction when the real-time PCR instrument first detects the fluorescence generated during a successful amplification reaction. The Ct is obtained during the exponential part of the amplification process, when the most accurate measurement of accumulation is possible. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. Real-time monitoring permits not only accurate quantification of target copy numbers but also critical assessment of the reaction itself. The extensive choice of chemistries, enzymes and instrument platforms available for RT-qPCR is another reason for its immense appeal, albeit at the risk of substantial potential for the generation of discordant results9. (more…)

Tagged with: Polymerase Chain Reaction (PCR), qPCR, Stephen A. Bustin

Maintaining a spore-free environment in the cleanroom

Issue 5 2007 / 21 September 2007 / Karen Rossington, Marketing and Development Manager, Shield Medicare Ltd.

The manufacture of sterile pharmaceutical products is governed in the European Union by the requirements of EU Good Manufacturing Practice for Medicinal Products. The GMP guide gives very specific details on the environmental and microbial requirements for aseptic processing. However, little or no guidance is given on how to create and maintain the correct level of microbial contamination in the aseptic suite. This article focuses on two important issues – creating and maintaining a spore-free environment and preventing spore contamination that may result from the use of disinfectants.

As a disinfectant manufacturer, Shield Medicare offers a useful perspective on the selection and use of disinfectants, as it has an in-depth knowledge of the products and also operates its own cleanrooms to the same cGMP standards as pharmaceutical manufacturers. (more…)

Tagged with: Cleanrooms, Karen Rossington, Shield Medicare Ltd.

Protein crystallography in drug design: current bottlenecks

Issue 5 2007 / 21 September 2007 / Timothy Allison & Sanjeev Munshi, Department of Structural Biology, Merck, Westpoint, PA

Protein crystallography is an integral component of the structure-guided drug discovery process. Rapid access to structural information about drug targets as well as bound ligands has been pivotal in accelerating lead identification and optimisation processes. While automation and robotics have been employed at every stage along the gene-to-structure path, significant challenges remain in increasing successful outcomes and in reduction of timelines. Advances in high through-put technologies to automate protein expression and crystallisation, the two weakest links in the gene-to-structure process chain, are beginning to address these issues. This article will highlight the importance of rapid structure determination of protein-ligand complexes in lead optimisation, and describe recent developments towards overcoming these bottlenecks. (more…)

Tagged with: Drug discovery, Merck & Co., Protein crystallography, Sanjeev Munshi, Timothy Allison

A new era for microcalorimetry in drug development

Issue 5 2007 / 21 September 2007 / Dr Ernesto Freire, Faculty Professor, Johns Hopkins University, Baltimore

Drug development involves the identification and subsequent optimisation of low molecular weight compounds with a desired biological activity. Often, the initial binding affinity of those compounds towards their intended target needs to be improved by five or more orders of magnitude before they become viable drug candidates; a process that would be greatly facilitated if the different forces that contribute to binding were experimentally accessible. Isothermal titration calorimetry (ITC) provides such a tool.

Of all the techniques available to measure binding, ITC is the only one capable of measuring not only binding affinities, but also the different thermodynamic forces that determine the binding energy. In the past, however, ITC has been used retrospectively rather than as a guiding tool for lead optimisation. This situation is changing due to two factors: improved understanding of the relationships between thermodynamic forces (enthalpy, entropy and heat capacity) and chemical structure, and a new generation of instruments with reduced sample requirements and much faster throughputs. In this article these developments, which anticipate a new era in microcalorimetry, will be discussed.

Prevalent strategies in drug discovery rely heavily on the screening of large libraries of compounds. Most often, targets for drug development are enzymes and the screening is performed by implementing enzyme inhibition assays in a high throughput format that allows the rapid identification of compounds with inhibitory activity. Usually, compounds identified by screening ‘hits’ have binding affinities in the low to high micromolar range, requiring potency improvements of five or more orders of magnitude before they can become viable drug candidates. Nevertheless, these hits provide the starting material for further development. (more…)

Tagged with: Dr Ernesto Freire, Drug discovery, Johns Hopkins University, Thermal Analysis

An eight-step Six Sigma toll-gate approach to PAT implementation

Issue 5 2007 / 21 September 2007 / Bikash Chatterjee, President of Pharmatech Associates, Inc and Jeremy Green, Senior Consultant for Pharmatech Associates, Inc.

The FDA’s recent guidance regarding Process Analytical Technology (PAT) offers the pharmaceutical and biotech industries an unprecedented opportunity to leverage hard-won experience with scientific inquiry and innovation. However, the leap to PAT is significant for even the most rigorous development program. Many aspects of Six Sigma; including its use of statistical tools and its phase- or toll-gate approach to project management, can facilitate and accelerate a PAT initiative. Rather than advocating company-wide Six Sigma adoption as a prerequisite to effective PAT implementation, an eight-phase Design for Lean Six Sigma approach is recommended that can be used on a project-by-project basis. (more…)

Tagged with: Bikash Chatterjee, Jeremy Green, PAT, Pharmatech Associates

Roundtable: The future direction for clinical trials

Issue 5 2007 / 21 September 2007 / Participants: Sally Burtles, Affiliation: Cancer Research UK; Stephen Freestone, Affiliation: Charles River Clinical Services; Eddie Caffrey, Affiliation: Quintiles Transnational

Sally Burtles, Stephen Freestone and Eddie Caffrey discuss the future direction for clinical trials, covering the main challenges faced in the current application of Phase I clinical trials, changes the industry be implementing in order to further improve the efficiency of early clinical trials and how the Regulatory bodies’ guidance on safe dosages affects First-In-Man trials. (more…)

Tagged with: Cancer Research UK, Charles River Clinical Services, Clinical trials, Eddie Caffrey, Quintiles Transnational, Roundtables, Sally Burtles, Stephen Freestone