In vitro activity: In HeLa cells, VR23 induces ubiquitinated proteins accumulation. In RPMI 8226 and KAS 6 cells, VR23 inhibits cell growth with IC50 of 2.94 and 1.46 μM, respectively. VR23 is also equally effective on both bortezomib (BTZ)-sensitive and -resistant RPMI 8226 and ANBL6 cells cells. When used in combination of bortezomib in the cells above, VR23 shows synergistic effects on cell growth inhibition. In addition, VR23 selectively induces cancer cell apoptosis by causing the accumulation of ubiquitinated cyclin E.

Kinase Assay: Exponentially growing cells on a 96-well clustered plate are treated with different concentrations of drugs or left untreated (control) for 6 hours. Proteasomes extracted with 0.5% NP40 buffer are mixed with equal amounts of samples in 100 μL total volume, and then incubated with 25 μmol/L of fluorogenic substrates (LRR- specific for trypsin-like activity, LLE-specific for caspase-like activity, and SUVY-specific for chymotrypsin-like activity) in black-bottom 96-well plates at 37°C. Fluorescence is monitored every 5 minutes at the wavelength of 360 nm (excitation) and 480 nm (emission).

Cell Assay: Treatment with VR23 made cancer cells undergo an abnormal centrosome amplification cycle. This cycle was caused by the accumulation of ubiquitinated cyclin E. Bortezomib is clinically approved as a chymotrypsin-like proteasome inhibitor. VR23 in combinations with bortezomib, caused a synergistic effect in killing multiple myeloma cells. This synergistic effect was also effective to myeloma cells resistant to bortezomib.

Cancer Res. 2015 Oct 1;75(19):4164-75