Echo acoustic liquid handler

The primary assay was performed in a black non-binding 1536-well plate (Corning 3728) and sealed with universal lids (Corning 3098). First, 10 nL of test compounds (4 mM) or 100% DMSO was dispensed into the 1536-well assay plates using an Echo acoustic liquid handler (Beckman Coulter, Brea, CA, USA). Then, 2 μL of protein working solution (5 × 10⁻⁴ mg/mL) was dispensed to the negative control (min effect) and compound wells (final well concentration 2.5 × 10⁻⁴ mg/mL) using a Certus Flex liquid dispenser (Fritz Gyger AG, Thun, Switzerland) and 2 μL assay buffer to the positive control wells (max effect) using a FLEXdrop (PerkinElmer, Waltham, MA, USA). Next, 2 μL of 75 μM substrate working solution was dispensed into all wells using the FLEXdrop (final well concentration 37.5 μM). The assay plates were then centrifuged at 187.5× g for 30 s and incubated in the dark at RT for 60 min and finally read on an Envision plate reader (PerkinElmer) (Ex. 355 nm/Em. 460 nm, LANCE/DELFIA mirror, 3 flashes per second). During the dose–response curve analysis in the primary and deselection assay setup, 40 nL of compound (range 2.00 × 10⁻³ M–2.74 × 10⁻⁶ M) or 100% DMSO was dispensed into the assay plates using an Echo acoustic liquid handler (Beckman Coulter) with a final well concentration range of 20 μM–27.4 nM compound and 1% DMSO.

The orthogonal assay was performed in a black non-binding 1536-well plate (Corning 3728) and sealed with universal lids (Corning 3098). First, 20 nL of test compounds (4 mM) or 100% DMSO was dispensed into the 1536-well assay plates using an Echo acoustic liquid handler (Beckman Coulter). Then, 4 μL of protein working solution (5 × 10⁻⁴ mg/mL) was dispensed to the negative control (min effect) and compound wells (final well concentration 2.5 × 10⁻⁴ mg/mL) using the Certus Flex liquid dispenser (Fritz Gyger AG) and 4 μL assay buffer to the positive control wells (max effect) using the FLEXdrop (PerkinElmer). Next, 4 μL of 2 mM substrate working solution was dispensed into all wells using the FLEXdrop (final well concentration 1 mM). The assay plates were then centrifuged at 187.5× g for 30 s, incubated in the dark at RT for 120 min, and finally read on an Envision plate reader (PerkinElmer) (absorbance at 405 nm, 10 flashes per second). During the dose–response curve analysis in the orthogonal assay, 80 nL of compound (range 2.00 × 10⁻³ M–2.74 × 10⁻⁶ M) or 100% DMSO was dispensed into the assay plates using an Echo acoustic liquid handler (Beckman Coulter) with final well concentration range of 20 μM–27.4 nM compound and 1% DMSO.

The ReFRAME library is composed of 11,948 small molecules that have reached clinical development or have undergone significant preclinical profiling (8 (link), 9 (link)). The compounds were prespotted into 1,536-well assay plates with the Echo Acoustic Liquid Handler (Beckman Coulter) to achieve a final concentration of 5 μM in a final assay volume of 8 μL per well. The KLF4 reporter SW1353 cells were dispensed into the assay plates with 100 cells per well and were allowed to grow for 24 hours. Quantitation of luminescence activity was performed Nano-Glo HiBiT Lytic Detection System, and the raw signal intensities were normalized across each plate using Z scores. Compounds with Z scores above 2 SDs were replated in triplicate for confirmation assays and evaluated in 10-point dose-response concentration curves. EC₅₀ for each compound was calculated from the dose-response curves, and EC₅₀ < 1 μM was adopted as a criterion for the validated hit selection.