Application Notes

4 A P P L I C A T I O N N O T E Traditionally, target based high throughput screening methods use either a biochemical assay where there is a colorimetric or fluorescence-based readout of activity or, coupled assays (e.g. luciferase-based ATP activity assays) [8], [9]. Oen experimental screening methodologies can have disadvantages, such as displacement-based assays where binding sites need to be known and probes have to be constructed; or with FRET based assays, specific antibodies need to be generated to produce a signal [10]. Compared to FRET and TR-FRET assays, Spectral Shi assays offer a simpler and more direct readout of binding events, reducing the potential for interference from autofluorescence via robust data analysis and fitting algorithms, and non-specific detection probe binding. Spectral shi-based assays overcome displacement-based assay limitations by being binding site agnostic and requiring only the target to be labelled. The assays are 'mix and measure', and do not require multiple mixing or washing steps. Previously Spectral Shi technology was launched in a capillary based format (Monolith X) and 384 well based format (Dianthus). In these instrument platforms, the measurement time is not fast enough to be considered ultra-high- throughput [11]. With the development of Dianthus uHTS, particular focus was placed upon reaching a measurement throughput of greater than 100,000 datapoints per day. This was achieved by changing the measurement format to a 1536 well plate, and the re-engineering of the measurement technology to allow on the fly measurement of plates, while keeping the required signal to noise for robust assay development. This study aims to evaluate the performance of the Dianthus uHTS instrument integrated within an automated screening platform to screen high diversity small molecule library (5112 total molecules) against the model target system, MEK1. MEK1 is a key kinase in the MAPK/ERK signaling pathway, playing a pivotal role in regulating cell proliferation, differentiation, and survival. Dysregulation is associated with various malignancies, including melanoma and non-small cell lung cancer, making it a critical target for therapeutic intervention. As a dual-specificity kinase, MEK1 phosphorylates and activates ERK1/2, driving oncogenic signaling pathways that promote tumorigenesis and resistance to targeted therapies [12]. MEK1 small molecule binding can also be monitored using thermal shi and ATPase activity assays, allowing for orthogonal characterization of hits. uHTS Screening

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