In vitro activity:VLX1570 is an analogue of b-AP15 that shows higher potency and improved solubility. VLX1570 preferentially inhibits proteasomal DUB activity while not inhibiting the activities of a panel of non-proteasomal DUBs. VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Treatment of multiple myeloma cells with VLX1570 induces the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response.

Kinase Assay: Preparations of 26S proteasomes (1 nM) are pretreated with DMSO, VLX1570, or b-AP15 for 2 min in assay buffer (25 mM Tris, 5 mM MgCl2, 10% glycerol, 0.05 mg/mL BSA, 2 mM ATP, and 1 mM DTT) before addition of Ubrhodamine. Fluorescence is monitored at 37°C using Ex/Em = 490 nm/520 nm to read data every 10 second for 30 min using a TECAN infinite 200 instrument. For UbVS labeling of KMS 11 cells, cell pellets are lysed from control or treated cells with buffer (50 mM HEPES pH 7.4, 250 mM sucrose, 10 mM MgCl2, 2 mM ATP, 1 mM DTT) on ice for 30 min and removed debris by centrifugation. Twenty five μg of protein is labeled with 1 μM UbVS for 30 min at 37°C. Samples are resolved by SDS-PAGE and subjected to immunoblotting. For UbVS labeling of proteasomes, purified 19S proteasomes (50 nM) are pretreated with DMSO, VLX1570, or b-AP15 (50 μM) for 10 min at room temperature, followed by labeling with 1 μM HA-UbVS for 30 min at 37°C and by immunoblotting.

Cell Assay:OPM-2 MM cells are exposed to 0.5 μM VLX1570 for 3 hours and 25 μg whole cell lysates are subsequently labeled with Ub-VS (1 μM), followed by SDS gel electrophoresis and immunoblotting with USP14 or UCHL5 antibodies.