Practical Workshop – Biochemical Assays for Screening

Recent years have witnessed an expansion in the disciplines encompassing drug discovery outside the pharmaceutical industry. This is most notable with a significant number of universities worldwide that now host infrastructure such as compound libraries and automated screening centres[1-3]. An archetypal small molecule drug discovery project will aim to identify chemical starting points that modify the functions of genes, cells or biochemical pathways. In some, but not all instances, these functions may be linked to disease processes, and an opportunity will exist to further develop the probes into novel therapeutic agents. In small molecule drug discovery, the ultimate aim is to identify new therapeutics, an activity which for reasons of high risk and cost has historically been conducted within the commercial sectors[4].

The expansion of small molecule drug discovery outside the pharmaceutical industry has coincided with increasing numbers of exploratory molecular targets and mechanisms, both therapeutic and non-therapeutic in origin[5]. Screening using miniaturised microtitre plate formats remains the most widely utilised methodology for identifying novel chemical starting points that are capable of modulating target function in a meaningful, biologically relevant manner[6]. The first practical steps are the selection synthesis, management and subsequent screening of molecular libraries. The second practical step is the selection and development of and their use in screening campaigns to identify primary hits, followed with their validation. In the context of drug discovery projects, the activities of selected primary hits would be further evaluated in biophysical assays such as surface plamson resonance and isothermal titration calorimetry. This effort would typically lead to the identification of validated hits and some of these would be further selected for optimisation using multiple criteria including structure activity relationships, selectivity, physicochemical properties and liability[7,8]. Due to the increase in drug discovery activities outside the pharmaceutical industry, a partnership was established between European Pharmaceutical Review and the European ScreeningPort in order to set up a unique Practical Workshop – Biochemical Assays for Screening.

Participant profile, learning objectives and contents

The Practical Workshop – Biochemical Assays for Screening was designed for scientists at all levels (undergraduates, postgraduates and laboratory based scientists within academic and industrial research organisations) engaged in early stage drug discovery and who have an interest in the development, validation and utilisation of biochemical assays for screening against small molecule libraries. The Practical Workshop – Biochemical Assays for Screening was equally well suited to technically focused staff from core facilities or contract research organisations who may wish to extend their expertise. In light of this, the Practical Workshop – Biochemical Assays for Screening attracted scientists predominantly from academic and biotechnology sectors. The evening dinner on the first day offered the opportunity for the participants to network and establish relationships that would be mutually beneficial.

The main objectives of the Practical Workshop – Biochemical Assays for Screening were to examine by way of practical sessions and lectures the design and application of assays for small molecule screening campaigns in drug discovery. All participants took part in the practical sessions that involved the development of screening compatible assays, primary screening using a small molecule library and the profiling of compounds in doseresponse experiments. Important aspects of the course were to 1) discuss and demonstrate practically the appropriate steps in selecting suitable assays in light of the fact that a multitude of assay technologies are currently available for a given target[9]; 2) how to select an appropriate technology; which criteria should be examined during the process and 3) whether a generic, flexible set of assay methodologies or customised solutions should be applied to the targets being investigated in small molecule biochemical screening campaigns.

The Practical Workshop – Biochemical Assays for Screening included the following laboratory sessions for a kinase, protease or histone deacetylase target:

1. Kinetic characterisation of the enzymes that included enzyme titration, Km determ – ination for substrates, IC50 determination for inhibitor, signal stability, choice of liquid handling and Z′ calculation
2. Screening of validated assays against a small molecule library (proof-of-concept screen)
3. Potential to confirm activities of selected hits in suitable secondary assays.

The vendors who sponsored the Practical Workshop – Biochemical Assays for Screening included BioTek, Cisbio, GE Healthcare, PerkinElmer, Promega and Thermo Fisher Scientific. These vendors provided the following hardware and reagents:

• Synergy NEO, MultiFlo™ Microplate Dispenser
• Synergy™ NEO HTS Multi-Mode Microplate reader
• HTRF® KinEASE™ kinase assay system
• EnVision® Multilabel Reader, EnSpire® Multimode Plate Reader, AlphaScreen® PhosphoSensor assay system, AlphaLISA® HDAC assay system
• Biacore™ T200 system
• HDAC-Glo Class I/II, ADP-Glo™ Kinase Assay systems
• Thermo ScientificTM MultidropTM
• Combireagent dispenser

The Practical Workshop – Biochemical Assays for Screening included the following lectures:

1. Introduction to drug discovery and the design and development of biochemical assays for drug discovery purposes – what can be achieved and learnings from past successes and failures
2. Screening jargon and terms
3. Selection of assays which will ensure translation of hits between formats
4. Data analysis and reduction – going beyond the Z′. Discussion of methods for analysing in vitro biological assays data including false positive/negative rates, dose-response curve fitting and correlations
5. Integrating your research program, design of project critical paths which integrate invitro, in-vivo and in-silico elements.

Outcomes and participant feedback

It was envisaged that upon completion of the course, participants would have gained an insight into the key parameters to be considered when developing biochemical assays and performing small molecule screening campaigns, associated data analysis and validation of hits in suitable secondary assays.

Upcoming courses in Germany in 2013

1. Second Practical Workshop – Biochemical Assays for Screening
2. First Practical Workshop – Cell-Based Assays for Screening
3. First Chemical Biology Workshop – Cell- Based Assays for Screening

The Practical Workshop – Biochemical Assays for Screening was approved by the Society of Biology for purposes of Continu ing Professional Development (CPD) and may be counted as 72 CPD credits if registered on the Society of Biology CPD Scheme.

References

1. Frearson JA and Collie IT. HTS and hit finding in academia – from chemical genomics to drug discovery. Drug Discov Today 2009;14:1150
2. Frye S, Crosby M, Edwards T and Juliano R. US academic drug discovery. Drug Discov Today 2011;10:409
3. Baker M. Academic screening goes highthroughput. Nat Meth 2010;7:787
4. Goodman M. Market watch: Pharma industry performance metrics: 2007-2012E. Nature Rev Drug Discov 2008;7:795
5. Overington JP, Al-Lazikani B and Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5:993
6. Macarron R, Banks MN, Bojanic D, Burns DJ, Cirovic DA, Garyantes T, Green DVS, Hertzberg RP, Janzen WP, Paslay JW, Schopfer U and Sittampalam GS. Impact of high-throughput screening in biomedical research. Nat Rev Drug Discov 2011;10:188
7. Bleicher KH, Bohm HJ, Muller K, Alanine AI. Hit and lead generation: beyond high-throughput screening. Nat Rev Drug Discov 2003;2:369
8. Gul S. Overview of the gene-to-Lead phase in drug discovery. Eur Pharm Rev Digital 2009;6:3
9. Ma H, Deacon S and Horiuchi K. The challenge of selecting protein kinase assays for lead discovery optimization. Expert Opin Drug Discov 2008;3:607

Biography

Sheraz Gul is Head of Biology at European ScreeningPort, Hamburg, Germany where he manages the assay development and screening of academic targets. Prior to this, he worked for GlaxoSmithKline for seven years where he developed biochemical and cellular assays for High Throughput Screening as well as hit characterisation. In addition, he has worked in academia for five years on proteases and kinases. He is the co-authored of the Enzyme Assays: Essential Data Handbook. He is involved in many European Initiatives involving government, the pharmaceutical industry and academia (e.g. EU Framework 7 and IMI). His research interests are directed towards maximising the impact of HTS for drug discovery.