In vitro activity: NMS-873 reduces p97 sensitivity to trypsin digestion, preventing degradation of the linker-D2 domain. NMS-873, as a p97 inhibitor, produces antiproliferative activity in a variety of hematological and solid tumor lines. The mechanism study indicates that NMS-873 activates the unfolded protein response, interfers with autophagy and thus induces cancer cell death.

Kinase Assay: ATPase activity and the kinetic parameters of recombinant wild-type VCP and its mutants were evaluated by monitoring ADP formation in the reaction, using a modified NADH-coupled assay. As ADP and NADH are ATP-competitive inhibitors of VCP ATPase activity, the standard protocol for the NADH-coupled assay was modified into a two-step procedure. In the first part, an ATP-regenerating system (40 U/ml pyruvate kinase and 3 mM phosphoenolpyruvate) recycles the ADP produced by VCP activity, keeps the substrate concentration constant (thus preventing product inhibition) and accumulates a stoichiometric amount of pyruvate. In the second part, the VCP enzymatic reaction is quenched with 30 mM EDTA and 250 μM NADH and stoichiometrically oxidized by 40 U/ml lactic dehydrogenase to reduce accumulated pyruvate. The decrease of NADH concentration was measured at 340 nm using a Tecan Safire 2 reader plate. The assay was performed in 96- or 384-well UV plates in a reaction buffer with 50 mM Hepes, pH 7.5, 0.2 mg/ml BSA, 10 mM MgCl2 and 2 mM DTT. Experimental data were fitted with a cooperative equation obtaining a Ks* of about 60 μM and a Hill coefficient (n) of 2.0 ± 0.1.The HTS campaign was performed against a 1-million-compound library using a miniaturized assay in 1,536-well format and a more sensitive ADP detection system, Transcreener ADP FP. A 20-min preincubation of 10 nM VCP and 10 μM inhibitor was performed, after which 10 μM ATP was added to the reaction, which was allowed to proceed for 90 min before quenching. The average Z′ of the screening was 0.58, and the hit rate using 3× s.d. (38% inhibition) as cutoff was 1.7%. Primary hits with>60% inhibition at 10-μM concentration were pruned using physicochemical and structural filters to leave 7,516 compounds. At the end, reconfirmation was performed in duplicate on 3,988 primary hits, and 500 compounds were selected for a dose-response evaluation using the previously described NADH-modified coupled assay.The potency of the most interesting HTS hits was measured against both wild-type VCP and the C522T mutant. ATP concentrations that yielded the half-maximal velocity (Ks*) for each enzyme, corresponding to 60 μM and 130 μM for the wild type and C522T mutant, respectively, were used in the assay. To explore the dependency of reversible inhibitors from substrate concentration, their potency was evaluated also at saturating ATP concentration (1 mM) and compared to the potency of a standard ATP competitive inhibitor (AMP-PNP).

Cell Assay: Cells are seeded at 1,600 cells per well in 384-well white clear-bottom plates. Twenty-four hours after seeding, cells are treated with the compounds (eight dilution points, in duplicate, for each compound) and incubated for an additional 72 h at 37 °C under a 5% CO2 atmosphere. Cells are then lysed, and the ATP content in each well is determined using a thermostable firefly luciferase–based assay as a measure of cell viability. IC50 values are calculated using the percentage of growth of treated cells versus the untreated control.

Nat Chem Biol. 2013 Sep;9(9):548-56