Category: Assay Development and Screening
Miniaturised, automated Design of Experiment to accelerate assay development
Iterative, prior-knowledge-based assay development, though employed and respected throughout the pharmaceutical industry, can be a long, sometimes challenging and cumulatively expensive process. Additionally, new assay approaches, including increased use of human-biologically relevant material, brings new challenges, where in some cases, there is no prior knowledge or accepted “gold-standard” approach. Due to these challenges there is renewed interest in employing methodologies, such as Design of Experiments (DoE), to apply a more statistical, streamlined approach to reduce timelines and optimise new approaches. Automation and data handling have always been limiting factors in carrying out complex DoE experiments, in terms of data interpretation, speed of assay turnaround and relative cost. The limitations with existing methodologies and approaches has led to this approach not being used as widely and has led to a reliance on prior knowledge to develop and optimise new assays.
Here we present a study using two commonly used assay methodologies; fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET), to develop high throughput compatible assays for bromodomain binding interaction. Bromodomain-containing proteins are an increasingly important target class within drug discovery for a wide variety of diseases, including several forms of cancer, asthma and multiple sclerosis. The FP and TR-FRET detection methodologies applied to the bromodomain protein in this study utilised the same known small molecule binder labelled with a fluorophore to identify inhibitors via direct competition. DoE and a prototype liquid handling technology were employed to develop these assays in 1,536 well microplates in rapid time compared to traditional methods. This was achieved without the use of any manual pipetting and 384 well microplates, in addition to a significant reduction in reagent requirements due to low liquid handler dead volumes. The successful development and optimisation of these bromodomain assays in a miniaturised microplate format enabled us to utilise the same assay protocols to then screen in excess of 40,000 compounds, in both assay formats. This miniaturised, automated Design of Experiment approach demonstrates that it is possible to reduce assay development cycles times, in addition to removing well known assay bottlenecks associated with the transfer of subsequent assays into high throughput screening.
Peter Craggs– Investigator, The Francis Crick Institute, UCL, London, England, United Kingdom