Potency was also determined in Bromoscan assay and Alphascreen activity assays.
Bromoscan IC50s: BRPF1=7.9 nM, BRD1=48 nM, BRPF3=260 nM; Alpha assay IC50s: BRPF1=140 nM, BRD1=760 nM, BRPF3=2,000 nM.
PDB ID for probe-target interaction (3D structure):
5T4U, 5T4V
Structure-activity relationship:
Yes, SAR is available in both DSF and BROMOscan assays in the paper.
Potency (off target):
IC50
Off target protein and potency:
BRD9 310 nM; BRD4 4,500 nM; BRD7 82 nM
Potency assay (off target):
NI-42 was screened in Bromoscan assay and with differential scanning fluorimetry (DSF) against a panel of 48 BRDs and showed excellent selectivity. For the latter assay, all activity was confined to the class IV family of BRDs with ∆Tm < 1 oC stabilization for all non-class IV BRDs.
Probe Selectivity in Vitro:
NI-42 was tested for activity against 8 ion channels (Eurofins CardiacProfile: Nav1.5, Kv4.3/KChIP2, Cav1.2, hKv1.5, KCNQ1/mink, HCN4, Kiv2.1 and hERG) yiedling 45.6% inhibition of hERG current amplitude at 30 uM.
In cell validation
Potency assay (cells):
An indexed cell panel of 211 cancer cell lines (CLIMB panel) was treated with NI-42 for 72 h and observed modest and selective inhibition of proliferation of certain AML cell lines (OCI-AML2, Nomo-1, THP-1, KG-1, MV-4-11) with GI50s 1-10 uM.
Target engagement assay (cells):
Yes, in FRAP assays, NI-42 displaced BRPF1B-BRDx3 from chromatin at 1 µM using BRPF1B-GFP fusion protein.