TR-FRET-binding assays and Ki calculation

N-terminal GST-tagged-Mcl-1 protein from Mcl-1 (E171-G327), N-terminal GST-tagged Bcl-xL protein from Bcl-xL (1-209), N-Terminal 6His-tagged Bfl-1 protein from Bfl-1 (M1-K151), N-Terminal 6His-tagged Bcl-w protein from Bcl-w (M1-R171), and C-Terminal 6His-tagged Bcl-2 protein from Bcl-2 (M1-F212) were expressed as a tagged fusion protein in E. coli and subsequently purified via Glutathione Sepharose-affinity or Ni-NTA resin purification, and size-exclusion chromatography.

For TR-FRET, tagged fusion proteins were incubated with a Europium-labeled antibody and a HyLite Fluor 647-labeled peptide, or a biotin-labeled peptide with a streptavidin-labeled ulight fluorophore allowing the assembly of donor and acceptor dye pairs for use in protein-binding assays.

The assay was performed in 384-well plates and IC₅₀ values were assessed from a 10-point, half-log₁₀ dilution starting at 100 or 10 µM of compound. The reaction final concentrations for Mcl-1 were 1.5 nM GST-Mcl-1, 0.5 nM LanthaScreen Eu tagged GST antibody (LifeTechnologies cat#PV5594), and 4 nM HyLite Fluor 647-labeled Bim peptide C (Hilyte647 C2 Maleimide)-WIAQELRRIGDEFN; for Bcl-xl were 2 nM GST-Bcl-xl, 2 nM LanthaScreen Eu-tagged GST antibody, and 10 nM HyLite Fluor 647-labeled BAK peptide C(Hilyte647 C2 Maleimide)-GGGQVGRQLAIIGDDINR; for Bfl-1 were 4 nM His-Bfl-1, 0.5 nM LanthaScreen Eu-tagged HIS antibody (PerkinElmer cat#AD0205), and 10 nM HyLite Fluor 647-labeled BIM peptide C(Hilyte647-C2-Maleimide)-WIAQELRRIGDEFN; for Bcl-2 and Bcl-w: 2 nM His-Bcl-2, or 2 nM His-Bcl-w, with 0.5 nM LanthaScreen Eu-tagged HIS antibody, 10 nM biotin-labeled Bim peptide (Biotin-lc-GGMRPEIWIANELRRIGDEFNA), and 2.4 nM a streptavidin labeled ULight Fluorophore (PerkinElmer cat#TRF0102-D). Reactions were incubated at 24 °C for 120–180 min before reading on a Tecan (InfiniteM1000 spectrofluorometer) with excitation at 340 nm and emission at 612 and 665 nm. Ratio of fluorescent emission intensity at 665–620 nm was calculated for each reaction (equation ¹). Percent inhibition was calculated based upon min (control compound) vs. max (DMSO) according to Eq. (²) and the IC₅₀ was derived the smart fit curve (Genedata screener) of % inhibition vs. concentration. K_i values were calculated from Eq. (³) and the parameters in Table 2.

TR-FRET assay-binding parameters of Bim or Bak peptides to Bcl-2 pro-survival proteins used in K_i calculations