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Effective Drug Discovery Begins with the Right Assay

By The Lab Man

(AKA SLAS Director of Education Steve Hamilton)

"The amount of time assay development can take is often not appreciated," says Nathan Coussens, U.S. National Institutes of Health (NIH) research scientist. "The results from multiple assays are needed to interpret the results of any one assay, which multiplies the amount of work. Rigor in developing the best possible assays with well-separated high and low controls is very important."

 

The Lab Man appreciates Nathan Coussens' comments. Developing assays – properly – is the cornerstone for life sciences R&D. Those new to high-throughput screening (HTS) and lead discovery may struggle to know where to start. They lean on their more experienced colleagues who have learned the intricacies of effective assay development over the years. Practical how-to information from experienced colleagues is one of the most valuable ways to transfer information in science, and it builds upon knowledge gained from reading peer-reviewed journals, browsing other publications like the SLAS LabAutopedia wiki, listening to conference presentations and referencing the NIH Assay Guidance Manual.

NIH Assay Guidance Manual

The well-respected Assay Guidance Manual is an e-book available for download from NIH. This document began life within Eli Lilly during the early 1990s and grew with contributions from over 100 scientists into a valuable training manual to help people develop good primary screening assays and to analyze them correctly. In 2004-2005 it was transferred as a free community resource to the NIH Chemical Genomics Center (NCGC) for ongoing public development, distribution and maintenance; first on the NCGC website and now as an e-book on the National Library of Medicine/National Center for Biotechnology Information bookshelf. In its first year online, the Assay Guidance Manual had more than 53,000 unique visitors.

Assay Guidance Manual sections include:

• Introduction (2 chapters)
• Data and Compound Management (1 chapter)
• In Vitro Biochemical Enzyme Assays (5 chapters)
• In Vitro Biochemical Binding Assays (7 chapters)
• In Vitro Cell Based Assays (6 chapters)
• Image Based Assays (4 chapters)
• Assay Artifacts and Interferences (1 chapter)
• Validation (3 chapters)
• Instrumentation (3 chapters)
• In Vivo Assays (1 chapter)
• Pharmacokinetics and Drug Metabolism (1 chapter)
• Glossary of Quantitative Biology Terms (1 Chapter)

Chapter authors are a veritable who's who of experienced scientists from multiple disciplines working in drug discovery and development worldwide. Among them, you'll recognize names of many active SLAS volunteers, conference presenters, journal authors, webinar presenters and Special Interest Group leaders.

The manual is targeted for the beginner and loaded with meaningful, basic information. As noted in the Preface:

"This manual is intended to provide guidance in the area of biological assay development, screening and compound evaluation. In this regard, an assay is defined by a set of reagents that produce a detectable signal allowing a biological process to be quantified. In general, the quality of an assay is defined by the robustness and reproducibility of this signal in the absence of any test compounds or in the presence of inactive compounds. This robustness will depend on the type of signal measured (absorbance, fluorescence, radioactivity, etc.), reagents, reaction conditions and analytical and automation instrumentation employed. The quality of the HTS is then defined by the behavior of this assay system when screened against a collection of compounds. These two general concepts, assay quality and screen quality, are discussed with specific examples in the chapters of this manual."

New chapters are added to the manual as needed to address emerging technologies, detail best practices and stay current with the rapidly changing landscape in drug discovery and development.

SLAS2016 Assay Guidance Workshop for High-Throughput Screening and Lead Discovery

Editors of the Assay Guidance Manual are the perfect instructors for a highly interactive full-day workshop at SLAS2016 on Saturday, Jan. 23. Designed for individuals with little or no experience in assay development for high-throughput screening and lead optimization, this is an excellent opportunity for small group tutoring about assay development from real experts who have learned much from the school of hard knocks. If you're new to the HTS and lead discovery field, plan to attend and bring your questions and problems for discussion.

Workshop includes:

• Overview of the Assay Guidance Manual as an important resource for detailed information about the development of a variety of robust assay methods and best practices in quantitative biology.
• Practical approaches for developing robust biochemical and cell-based assays as well as selection and development of optimal assay reagents.
• Overview of common sources of assay artifacts and strategies to identify artifacts through the development and implementation of counter assays.
• Biophysical approaches for validating hits from a primary screen
• Discussion of important statistical and data analysis concepts with an emphasis on using these concepts to collect the best possible data and make go/no go decisions based on experimental results.
• Development and optimization of in vitro assays for testing compound toxicity in a qHTS platform and assessing ADME properties of lead molecules
• Small group discussions to share experiences and seek practical advice about individual research concerns.

And, there's more. For instance, one of the less enjoyable aspects of assay development, one not generally learned in academic research laboratories, is rigorous assay validation in a biologically and statistically valid manner. This is necessary to characterize assay reliability, assure reproducible assay results and provide a baseline of assay behavior if it's ever necessary to move the assay to a new laboratory, new personnel, new equipment or reagents. Workshop instructors walk attendees through all aspects of this important topic, including:

• Reagent stability and storage
• Reaction stability during projected assay time
• Solvent compatibility (DMSO)
• Evaluation of plate uniformity and signal variability
• Assay plate formatting
• Inter-plate and inter-day variability
• How to deal with assay variability
• Bridging studies

Coussens, Assay Guidance Manual editor and SLAS2016 workshop instructor, says workshop participation will help demystify those aspects of assay development and validation that surprise most novices.

"High quality data is needed to identify the weak hits that are usually the more interesting compounds, whereas strong hits are often found to be assay artifacts," Coussens says. "Frequently great scaffolds do not show strong responses from a primary screen and can be easily missed with noisy data or from data containing a high rate of false positive hits due to assay interferences. Another critical aspect that requires careful consideration is the development of biologically and pharmacologically relevant assays for the target of interest. For example, the use of appropriate cell types, enzyme preparations, physiologically relevant substrates, detection labels and assay conditions can significantly impact the type of hits and leads that are identified."

Coussens notes the Z' factor and signal window (SW) are important statistical concepts that those new to HTS must learn.

"These concepts are both excellent and widely used statistics to evaluate assay performance by indicating the amount of variability in an assay," he states. "They reflect the separation in mean values for the high and low controls while taking into consideration the variability within each group. However these need to be calculated from mock runs using full plates (DMSO blanks) to get an accurate assessment of the assay performance and the ability of HTS to identify truly active compounds. For concentration-response screening formats where IC50/EC50/AC50 are used as primary endpoints, we use another statistical metric called the Minimum Significant Ratio (MSR), which helps determine the ability of the screening assay to differentiate and rank order compounds as well as the assay robustness and replicability."

Screening assays have evolved a great deal over the 20 years since the first draft of the Assay Guidance Manual was written. In addition to classic biochemical enzyme assays and binding assays, the SLAS2016 workshop discusses cell-based assays and image-based assays. High-content screening (HCS) involves multiparametric analyses, which extract large data sets from multiple targets in thousands or millions of cells in microplates. Thus HCS can produce an enormous amount of morphometric and kinetic outputs. Careful consideration must be made regarding cell type, to which conditions and reagents are then tailored. Optimal conditions must be determined to yield the largest separation between positive and negative results. This includes choosing a relevant substrate, incubation times and immunostaining protocols. Cell culture methodology, cell density and cell plating methods, plate washing techniques and permeabilization are all factors that can affect the assay and so must be understood and optimized. Such assays are not simple by any stretch of the imagination and when run in a high-throughput mode can quickly generate massive amounts of irrelevant data if not well developed and well controlled. Experts, such as Coussens and his colleagues, are available at the workshop to discuss your cellular assay questions and issues.

"Choosing the right cell type is one of the key decisions as well as determining if one needs primary cells or an engineered cell line," Coussens says. "It is often the case that the assay statistics are less favorable for high-content assays than for biochemical assays, which require rigorous optimizations to both the assay and data analysis procedures. Depending on the biology under investigation, a number of factors might be considered. In all cases, knowledge of cell biology and the mechanisms that might affect the readout are critical for establishing and improving both the assay design and analysis of results. During the course of an assay, it is crucial to monitor the cells closely and utilize good tissue culture practices. Understanding how to develop image analysis algorithms and the ability to work with large volumes of data are important. It is absolutely critical to ensure the authenticity of the cell types used. In many cases, cell lines have turned out to differ from what was assumed, or are contaminated with mycoplasma or microorganisms."

Why SLAS2016?

SLAS delivers practical information for life sciences R&D and technology professionals in ways that also help them connect to, and continue learning from, one another to move the field forward. At SLAS2016, meaningful, in-depth workshops like the Assay Guidance Workshop for High-Throughput Screening and Lead Discovery are a mainstay.

See you in San Diego January 23-27.

About the Author

The Lab Man is SLAS Education Director Steve Hamilton, Ph.D. He is a creative change maker, delivering the fresh thinking and energy that has helped make SLAS the go-to resource for those in life sciences R&D and technology. After years in the drug discovery world, heading many leading-edge automation projects for companies such as Eli Lilly, Scitec and Amgen, Hamilton joined the SLAS professional team in 2010. He received his Ph.D. in analytical chemistry from Purdue University and a B.S. in chemistry from Southeast Missouri State University.

December 7, 2015