BI-9564

BI-9564 was discovered by fragment-based screening and optimized by structure-based design. BI-9564 has a higher affinity for BRD9 (ITC Kd =14) than BRD7 (ITC Kd = 239 nM), and the affinities for BET bromodomains are reported >100 μM (by AlphaScreen). It displays cellular activity in a FRAP assay (1 μM). Although it displays some off-target binding to CECR2 (ITC Kd = 258 nM), no cellular inhibition of CECR2 was observed in the CECR2 FRAP assay at 1 μM, and the off-target binding effect does not translate into other types of cellular experiments.The use of BI-9564 in murine leukemia cells with a BRD4/BRD9 domain-swap has shown that the BRD9 bromodomain is responsible for mediating the antiproliferative effects of BI-9564. However, the compound only displays an IC50 of 800 nM (cellular proliferation assay) in the most sensitive cell line in vitro to BRD9 inhibition (EOL-1). Some off-target effects were observed when used at concentrations >5 uM. A disseminated mouse model of AML was then used to assess the efficacy of the compound, using the human acute myeloid eosinophilic leukemia cell line EOL-1. The compound can be used to evaluate the effects of the in vivo modulation of BRD9 activity.

This is a pharma probe optimized by SBD from a fragment screen. More potent BRD9 (1-80 nM) vs. BRD7 (200-4000 nM), and selective (>1000-fold vs. BRD2, BRD4, BET). The probe hits one BRD protein outside BRD 9/7, namely, CECR2. However, CECR2 binding did not translate in cells to functional consequence (no CECR2 inhibition at 1 µM in FRAP; also not cytotoxic).

This probe appears better for both in vitro and cell work than the companion probe in the paper (BI-7273). The selectivity in vitro appears quite good for BRD9 even versus the closely related BRD7. In cells, the response to this probe also seems to be better than for the companion probe, though with lower fold selectivity. In mice, the probe was extensively tested only at one dose, though lower doses were used in some of the PK studies.