BI-9321

Potency assay (off target): BI-9321 was profiled against 14 other purified PWWP domains using DSF. No stabilization ≥2 °C was observed for the other domains, suggesting selectivity. The closest family members NSD2-PWWP1 and NSD3-PWWP2 were additionally tested by ITC Kd determination and binding was not detected. Both BI-9321 and BI-9466 were tested at a concentration of 500 µM in a diverse set of methyl-lysine binding domains with differential static light scattering (DSLS) and no significant Tagg shift was observed. Inhibition of 35 protein, DNA and RNA methyltransferases was tested and no liabilities at 10 µM were observed for BI-9321 and BI-9466. No kinase inhibition was detected in a selection of 31 diverse kinases for BI-9321 at 10 µM. Inhibition of KDM1B and antagonism of BRD4(1) bromodomain was also tested in enzymatic and AlphaLisa assays respectively, revealing no significant inhibition (KDM1B IC50 > 100 µM; BRD4(1) IC50 ≥ 1 mM). BI-9321 was tested in a broad panel of 48 bromodomains using a thermal shift assay revealing no binding, indicated by no stabilization >2 °C.

Potency assay, off target (cells): Selectivity profiling by quantitative chemical proteomics revealed NSD3 as interactor of immobilized BI-9321 in high-salt chromatin extracts of Cal-120 cells, expressing high levels of NSD3 due to an amplification of the locus. No specific interactors of BI-9321 were identified at 10 µM in low-salt whole-cell extract.