| Assay Method Information | |
| | ALPHA Screen Assay with TLX LBD |
| Description: | Assays were performed according to the manufacturer's protocol (PerkinElmer, USA), with the following modifications: Ligands were tested for TLX-coactivator binding in 50 mM phosphate buffer, 150 mM NaCl, pH 7, 1 mM DTT and 100 μM CHAPS. Ligands were dissolved and diluted in DMSO as stocks and were prepared in assay buffer from the original stock in the 100% DMSO and added to the assay plate. Biotinylated SRC peptide and polyhistidine tagged TLX LBD were diluted in assay buffer, subsequently added to respective ALPHA beads, under low light. The plates were spun down, mixed with a shaker and incubated for 120 minutes and read on a TECAN Plate reader using the manufacturer's protocol. The effective concentration that provokes 50% signal change (EC50) was calculated by nonlinear regression fit using Prism 7 software. Additional piperazine compounds were prepared and tested for TLX-coactivator binding according to the methods described above. Table 3 shows the dose response of piperazine-based compounds 31-96 that were also tested for TLX LBD targeting and coactivator recruitment using the ALPHA screen assay. |
| Affinity data for this assay | |
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