Application Notes

1 7 A P P L I C A T I O N N O T E DISCUSSION This study highlights the efficacy of integrating biophysical screening approaches, such as the Dian- thus uHTS platform into automated screening workflows. This was demonstrated here in the context of identifying both active site and allosteric binders of MEK1, in both single dose and confirmatory dose response formats. Integration into Automated Platforms. Simple integration is pivotal for the adoption of a new in- strument within an automated laboratory. The successful incorporation of the Dianthus uHTS into our automated screening platform, without the assistance or support of ThermoFisher, underscores its adaptability and compatibility with existing robotic infrastructures. Our integration was performed on the ThermoFisher Momentum product, but the intuitive design of the Dianthus platform allows for simple integration into any state-of-the-art automation solution, for example the Automator LINQ platform, and the PAA Overlord soware, as presented by representatives of Amgen at the SLAS con- ference 2025 [17]. The Dianthus uHTS straightforward API, based on the gRPC framework, facilitated the rapid development of a Python-based command-line driver. This allowed for efficient control over gate and plate handling, and measurement initiation. The speed of measurement (ca. 7 mins or less per plate) allows for the generation of over 250 000 data points per day, within the realm of ultra high throughput instrumentation. Efficient Assay Setup and Protein Utilization. The mix-and-measure assay format of the Dianthus uHTS is advantageous in minimizing protein consumption. In our screening of 5,112 compounds (conducted in duplicate) and subsequent dose-response assays, only 28 µg of MEK1 was utilized, averaging 1.4 ng per data point. In contrast, traditional ADP Glo assays required approximately 12.3 ng of MEK1 per data point, a nine-fold increase in protein usage. This significant reduction not only conserves valuable resources but also enhances the feasibility of large-scale screens where protein availability may be limited. Comparing Spectral Shi with traditional techniques such as Sypro dye- based Differential Scanning Fluorimetry where typical protein concentrations are in the 1 – 5 µM range, Spectral Shi has significant (up to 1000-fold less) protein requirements. An orthogonal ther- mal shi assay with SpS.DSF in an Andromeda X allows rapid hit confirmation via T m shis, from the same sample as measured on Dianthus, albeit in a non-automated manner.

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