BI-9564, a specific cell-permeable BRD9 BD inhibitor. The Kd for BRD9 is 5.9 nM, and IC50 for BET family is > 100 μM.

BRD9:Kd: 20 nM

EOL-1, human acute myeloid eosinophilic leukemia cell line, was antiproliferative byBI-9564 with EC50 of 800 nM.BRD7 implied as a tumor suppressor and is down-regulated in cancer cells, BI-9564 shows Kd of 73 nM for it, and is >10-fold more selective for BRD9 over the high homologues bromodomain. BI-9564 (<5 μM) shows no activity against 324 kinases, when BI-9564 at 10 μM, an inhibition >40% is observed for only 2 out of 55 GPCRs.

In vivo proof-of-concept studies, BI-9564 (180 mg/kg, p.o.) shows attractive ADME/PK profiles. Compared to survival of the control group in a xenograft model of human AML, BI-9564 results in a modest but significant additional survival benefit of 2 days.

To 750,000 MV-4-11 cells in 250 μl growth medium (in IMDM, 10 % FBS, GlutaMAX, 25 mM HEPES and 0,1% 2-Mercaptoethanol) per well compound is added at the desired concentration from a 10 mM stock solution using the HP D300 Digital Dispenser. After 2 hours of incubation with the compound, cells are collected by centrifugation, washed in ice cold PBS and lysed in 15 μl of cell extraction buffer. After 30 minutes on ice, nucleic acids are disrupted by sonication. cMYC levels are measured using the human c-Myc (Total) ELISA Kit. (Only for Reference)

Female CIEA-NOG mice engrafted intravenously with 1×107 EOL-1 AML cells, which express stably luciferase and GFP, were administered orally on day 5 with BI-9564(10 mL/kg body weight), which is formulated with 0.5% Natrosol.