In vitro activity: NMS-E973 shows a widespread antiproliferative activity with an average IC50 of 1.6 μM, and induces the degradation of client protein, such as Flt3, B-Raf, AKT, which further blocks tumor-related pathways, such as the Raf/MAPK, PI3K/AKT, and JAK/STAT pathways.

Kinase Assay: For competition experiments, a protein concentration of 5 nM for Hsp90 and of 200 nM for Trap1 are mixed with 0.5 nmol/L probe (final concentrations). After incubation, the dimethyl sulfoxide (DMSO) compound solution is added to the mixture. The plate is incubated for 18 hours at room temperature and then the fluorescence polarization signal was measured. Data are fitted with the program Dynafit version 3.28.039 or SigmaPlot (SSI) using the mathematical equation for competitive binding of 2 ligands to the receptor.

Cell Assay: NMS-E973 is a potent and selective Hsp90 inhibitor with DC50 of<10 nM for Hsp90 binding, no activiy against a panel of 52 diverse protein kinases. NMS-E973 exerts antiproliferation effects on A2780 tumor cell line and BT-474 breast cancer cell line with IC50 values of 69nM and 110nM, respectively. When treated with mice bearing A2780 xenografts, the intravenous administration of NMS-E973 significantly inhibits tumor growth with TGI value of 53% at dose of 30mg/kg.