Assays
References 1 Comley, J. Assay Interference a limiting factor in HTS? Drug Discovery World (2003): 4(3): 91-98. 2 Fluorescence Lifetime Trends 2007 Report, published by HTStec Limited, Cambridge, UK, March 2007. 3 FLT-Based Kinase Platform Trends 2009 Report, published by HTStec Limited, Cambridge UK, September 2009. 4 Riester, D et al, Non-isotopic dual parameter competition assay suitable for high- throughput screening of histone deacetylases. Bioorg. Med. Chem. Lett. (2009): 19:3651-3656. 5 Doering, K et al. A Fluorescence Lifetime-Based Assay for Inhibitor Profiling on Human Kallikrein 7. J.Biomol.Screening 2009: 14(1):1-9. 6 Hassiepen, U et al. Fluorescence Lifetime Assays – A Smart Solution for Inhibitor Profiling on Protease Panels. Screening – Trends in Drug Discovery 2009; 4:11-13. 7 Pritz, S et al. A Fluorescence Lifetime-Based Assay for Abelson Kinase. J Biomol. Screening (2010): Submitted for publication. 8 Ramage, R et al. Novel fluorescent dyes and uses thereof. PCT/GB2006/004015 2006. 9 Paterson, MJ et al. A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening. Anal. Biochem. (2010): 402(1): 54-64.
to be directly monitored in real time through a change in the average lifetime. The ability to devel- op individual substrates for the significant majori- ty of enzymes in this class indicates the potential of FLT for assay and screening of proteases. One of the clear advantages of employing FLT as a reading modality is its robustness. With Z’ factors consis- tently above 0.9, the FLEXYTE® platform clearly lends itself to large scale screening and profiling applications. In the development of the platform, Almac has sought to engage major screening cen- tres and following a successful evaluation of the FLEXYTE® platform, AstraZeneca’s scientists plan to submit their work for publication. With the focus expanding further in the near future to lipid kinases, Tyr kinases, and ligand binding assays, Almac’s FLEXYTE® assays offer to deliver on FLT’s long promised potential to circumvent assay interference and provide an effective assay plat- form for screening and profiling groups alike.
Discussion FLT largely failed to make a significant inroad as a screening technology when it was first introduced nearly a decade ago, despite the fact that intrinsi- cally it has a lot going for it, particularly in dealing with assay interferences1. Market studies undertak- en by HTStec in the intervening period have consis- tently uncovered a latent appetite within drug dis- covery for FLT assays2,3. Up until recently this appetite has for the most part gone unmet. However, recent published work at Novartis6-8 and unpublished work at other major pharmas have successfully addressed target class applicability (in the protease and kinase areas), assay development and validation, and assay reagent availability issues using FLT. The marketing of the Puretime dyes from AssayMetrics and the Almac’s 9AA dye, plus the possibility of new dyes under development, has brought new life to this field and lead to the devel- opment of a new generation of off-the-shelf assay kits based on lifetime (FLEXYTE®). The FLT reader situation has also changed over the past eight years. Tecan no longer offers an FLT option on it current generation of Infinite microplate readers, yet it still has customers using FLT on the ULTRA Evolution. CyBio withdrew the CyBi®-NanoScan HT after a change of strategic direction within the company. Most Evotec Mk1 to Mk3 screening systems able to offer lifetime as a component of a multi-parameter fluorescence read- out have largely ceased routine operation. The IOM MicroScan reader which originally offered Microsecond FLT has now evolved into the LF 502 NanoScan multimode fluorescence microplate
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reader from Berthold Detection Systems. Edinburgh Instruments’ NanoTaurus dedicated FLT plate reader is a main new addition that has already proven itself able to generate a large signal window with excellent precision in Almac FLEXYTE® assays. However, other yet to be commercialised FLT reading possibilities also exist, and may become available in the near future. Fluorescent Innovations developed a lifetime reader several years ago that so far has been mainly used internal- ly. This reader does not utilise the better charac- terised TCSPC approach, but uses digitisers to enable direct recording methods resulting in extremely fast read times, in low assay volumes and in 1536 – all key requirements for some potential Pharma customers. AssayMetrics is also now devel- oping its own dedicated fluorescence lifetime read- er, focusing on short read times and better automa- tion while keeping it highly flexible for a wide vari- ety of applications. Furthermore, it is rumoured that one additional instrumentation company will also enter the market with a new FLT reader later this year. It is not clear whether this additional investment in new instrumentation was triggered by the availability of new lifetime dyes and kits, but there is an undoubted sense that the momentum around FLT is picking up. Clearly the advances in FLT highlighted in this article when viewed collec- tively make FLT a much more attractive option today than in 2002. The question is will this trans- late into sufficient probe, assay kit and instrumen- tation sales to fund future investment in those new and emerging areas where FLT has even greater potential to impact, for example, epigenetic enzyme assays and some cell-based targets.
Acknowledgement
The assistance of Klaus Döring in the preparation of ‘Fluorescence lifetime assays – the basics’ info box is gratefully acknowledged.
DDW
Dr John Comley is Managing Director of HTStec Limited, an independent market research consul- tancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid han- dling, laboratory automation, detection instrumen- tation and assay reagent technologies) to drug dis- covery and the life sciences. Since its formation seven years ago, HTStec has published more than 50 market reports on drug discovery technologies and Dr Comley has authored more than 30 review articles in Drug Discovery World. Please contact
info@htstec.com for more information.
Drug Discovery World Summer 2010
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