Figure 6.

(A) Overall HTS workflow and instrumentation. Library compounds are spotted with an Echo 555 Acoustic Liquid Handler into 384-well PCR plates. STEP₄₆ and SYPRO Orange working solutions are added using a Multidrop Combi reagent dispenser. A ViiA 7 real-time PCR system is used to incrementally heat samples over a temperature gradient and measure fluorescence intensity. Protein Thermal Shift software is used to analyze the fluorescence raw data and calculate melting temperatures from the first derivative of the melting curve. After hit definition, hit compounds are cherry-picked from compound library plates using Echo, and triplicate single concentration followed by triplicate dose-response PTS hit confirmation assays are performed. (B) HTS results and statistics of our 50K small-molecule STEP₄₆ PTS primary screen, depicted as ΔT_mD (melting temperature calculated using the derivative method) distribution vs. plate well ID. The dashed horizontal lines define the hit threshold (ΔT_mD > ±1 °C).