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A P P L I C A T I O N N O T E
Data Interpretation and Hit Prioritization. The Spectral Shi fluorescent measurement is a bio-
physical readout, which requires a slightly modified approach to hit selection and prioritization. In
single-dose screening, our suggested strategy is to perform measurements in duplicate to assess hit
reproducibility and compare signals to the unbound DMSO reference. The magnitude of a Spectral-
Shi measurement (or the Z-score of a compound) is not directly related to binding strength. Instead,
hits are prioritized based on their deviation from the DMSO median—using a threshold of at least 4
Relative Standard Deviation Equivalents (RSDE) of the Median Absolute Deviation, depending on the
desired hit rate. Depending on the library size and hit rate, it may be feasible to proceed directly from
primary hits to secondary dose-response screening. However, if additional verification is needed
to reduce the number of follow-up experiments, hits—particularly those with low consistency—can
undergo a second round of single-dose screening (n = 3 or 4) to ensure only highly consistent profiles
are advanced for further analysis.
Alignment of results with alternative methods and the Identification of Active Site and Al-
losteric Inhibitors. A notable outcome of our study was the platform's ability to discern between
active site and allosteric inhibitors of MEK1. Among the prioritized hits, several compounds exhibited
binding without inhibiting MEK1's ATPase activity. This suggests potential allosteric binding sites,
where compounds bind to sites distinct from the ATP-binding pocket, potentially modulating in vivo
kinase activity indirectly. Such allosteric inhibitors are known to offer higher selectivity by targeting
unique regulatory mechanisms specific to the kinase, thereby reducing off-target effects. In addition,
the ability of Spectral Shi for binding site agnostic signals allows for the potential development of
novel therapeutic modalities such as molecular glues or bifuncational degraders (PROTACs).
