Home Ubiquitin p97 inhibitor NMS-873 For research use only.

NMS-873

NMS-873 is an allosteric and specific p97 inhibitor with IC50 of 30 nM that demonstrates potent selectivity for VCP/p97 compared to a panel of other AAA ATPases, Hsp90, and 53 additional analyzed kinases (IC50s >10 μM).

NMS-873 Chemical Structure

NMS-873 Chemical Structure

CAS No. 1418013-75-8

Purity & Quality Control

Batch: S728501 DMSO]100 mg/mL]false]Water]Insoluble]false]Ethanol]Insoluble]false Purity: 99.91%
99.91

NMS-873 Related Products

Signaling Pathway

p97 Signaling Pathways

p97 Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
Hi5 cells Function assay 20 mins Inhibition of human recombinant His-GST tagged VCP expressed in baculovirus infected Hi5 cells assessed as ADP formation incubated for 20 mins proir to substrate addition measured after 90 mins by NADH coupled assay, IC50=0.024 μM 23245311
HCT116 cells Cytotoxic assay 72 h Cytotoxicity against human HCT116 cells after 72 hrs by luciferase reporter gene assay, IC50=0.38 μM 71521142
Click to View More Cell Line Experimental Data

Biological Activity

Description NMS-873 is an allosteric and specific p97 inhibitor with IC50 of 30 nM that demonstrates potent selectivity for VCP/p97 compared to a panel of other AAA ATPases, Hsp90, and 53 additional analyzed kinases (IC50s >10 μM).
Features The most potent and specific p97 inhibitor described to date.
Targets
p97 [1]
(Cell-free assay)
30 nM
In vitro
In vitro 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. [1]
Kinase Assay Biochemical assay development and HTS
The ATPase activity and the kinetic parameters of recombinant wild-type VCP and its mutants are 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 is 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 is measured at 340 nm using a Tecan Safire 2 reader plate. The assay is 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 are 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 is 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 is performed, after which 10 μM ATP is added to the reaction, which is allowed to proceed for 90 min before quenching. The average Z′ of the screening is 0.58, and the hit rate using 3× s.d. (38% inhibition) as cutoff is 1.7%. Primary hits with >60% inhibition at 10-μM concentration are pruned using physicochemical and structural filters to leave 7,516 compounds. At the end, reconfirmation is performed in duplicate on 3,988 primary hits, and 500 compounds are selected for a dose-response evaluation using the previously described NADH-modified coupled assay. The potency of the most interesting HTS hits is 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, are used in the assay. To explore the dependency of reversible inhibitors from substrate concentration, their potency is evaluated also at saturating ATP concentration (1 mM) and compared to the potency of a standard ATP competitive inhibitor (AMP-PNP).
Cell Research Cell lines A variety of hematological and solid tumor lines
Concentrations ~10 μM
Incubation Time 72 hours
Method

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.

Chemical Information & Solubility

Molecular Weight 520.67 Formula

C27H28N4O3S2
CAS No. 1418013-75-8 SDF Download NMS-873 SDF
Smiles CC1=C(C=CC(=C1)OCC2=NN=C(N2C3=CN=CC=C3)SC4CCCC4)C5=CC=C(C=C5)S(=O)(=O)C
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 100 mg/mL ( (192.06 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : Insoluble

Ethanol : Insoluble


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In vivo
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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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