Alphaplate 384

Assays were performed with minimal modifications from the manufacturer’s protocol (PerkinElmer, USA). Briefly, CRBN-DDB1 (50 nM), Ni-coated Acceptor Beads (20 μg/ml), and biotinylated-pomalidomide (15 nM) were incubated with 100 nL of compound in 384-well plates (AlphaPlate-384, PerkinElmer, USA) using a Janus Workstation (PerkinElmer, USA). Streptavidin-coated donor beads (20 μg/ml) were added, incubated room temperature for 1 hour, and read on an Envision 2104 (PerkinElmer, USA), per the manufacturer’s protocol.

All analogs were tested in duplicate. BRD4-1 and BRDT-1 AlphaScreen^{[46 (link)]} assays were performed with minimal modifications from the manufacturer’s protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween 20, pH 7.5 and allowed to equilibrate to rt prior to addition to plates. After the addition of Alpha beads to the master solutions, all subsequent steps were performed under low light conditions. A 2x solution of components with final concentrations of His-BRD4 or His-BRDT at 40 nM, Ni-coated Acceptor Bead at 25 μg/mL, and 20 nM biotinylated-JQ1(S) was added in 10 μL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Plates were spun down at 150×g, after which 100 nL of compound in DMSO from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (25 μg/mL final) were added in the same manner as the previous solution, in a 2x solution of 10 μL volume. Following this addition, plates were sealed with foil to prevent light exposure and evaporation. The plates were spun down again at 150×g. Plates were incubated at rt for 1 h and then read on an Envision 2104 (PerkinElmer, USA) plate reader using the manufacturer’s protocol.

Compound potency was assessed by relative IC₅₀ potency determined by an AlphaScreen biotin-JQ1 competition assay as reported previously by our group.⁴¹ All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5 and allowed to equilibrate to room temperature prior to the addition to plates. After addition of Alpha beads to master solutions, all subsequent steps were performed in low light conditions. A 2x solution of components with final concentrations of BRD at 40 nM, Ni-coated Acceptor Bead at 25 μg/mL, and 20 nM biotin-JQ1 was added in 10 μL to 384-well plates (AlphaPlate − 384, PerkinElmer, USA). After a 1 min 1000 rpm spin-down, 100 nL of compounds in DMSO from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The 2x, 10 μL streptavidin-coated donor beads (25 μg/ml) were added to the previous solution. Following this addition, the plates were sealed with foil to block light exposure and to prevent evaporation. The plates were spun down again at 1000 rpm for 1 min. Next, the plates were incubated at room temperature with the plate reader (for temperature equilibration) for 1 h prior to reading the assay. Signal is stable for up to 3 h after donor bead addition. AlphaScreen measurements were performed on an Envision 2104 (PerkinElmer, USA) utilizing the manufacturer’s protocol.

BRD4 (BD1) assays were performed with minimal modifications from the manufacturer’s protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5 and allowed to equilibrate to room temperature prior to addition to plates. After the addition of Alpha beads to master solutions, all subsequent steps were performed under low light conditions. A 2x solution of components with final concentrations of His-BRD4 at 40 nM, Ni-coated Acceptor Beads at 25 μg/ml, and 20 nM biotinylated-JQ1(S) was added in 10 μL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Plates were spun down at 150g, after which 100 nL of compound in DMSO from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (25 μg/ml final) were added in the same manner as the previous solution, in a 2x solution of 10 μL volume. Following this addition, plates were sealed with foil to prevent light exposure and evaporation. The plates were spun down again at 150g. Plates were incubated at room temperature for 1 hour and then read on an Envision 2104 (PerkinElmer, USA) using the manufacturer’s protocol.