B2168-5mg | NMS-873 [1418013-75-8] Clinisciences

Background

NMS-873 is a selective inhibitor of VCP with IC50 value of 30nM [1].

NMS-873 is the most potent and specific VCP inhibitor. It shows similar potency against both wild-type VCP and VCPC522T without affecting the oligomeric state of VCP. NMS-873 is selective against all of the AAA ATPases, HSP90 or some other kinases. The inhibition effect of NMS-873 is potent and somehow selective in a panel of tumor cell lines when its concentration ranges from 0.08μM to 2μM. NMS-873 suppresses cell proliferation in HCT-116 cell line through inducing accumulation of poly-Ub proteins and stabilization of cyclin E and Mcl-1 dose-dependently. Additionally, NMS-873 causes distribution of HCT116 cells, leading a dose and time-dependent increase in the G2 population and in the sub-G1 fraction. Further, NMS-873 shows cell killing activity in hematological tumors as well as a wide variety of solid tumors with IC50 range between 0.08μM and 2μM [1].

References:
[1] Magnaghi P, D'Alessio R, Valsasina B, Avanzi N, Rizzi S, Asa D, Gasparri F, Cozzi L, Cucchi U, Orrenius C, Polucci P, Ballinari D, Perrera C, Leone A, Cervi G, Casale E, Xiao Y, Wong C, Anderson DJ, Galvani A, Donati D, O'Brien T, Jackson PK, Isacchi A. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat Chem Biol. 2013 Sep;9(9):548-56. doi: 10.1038/nchembio.1313. Epub 2013 Jul 28.

Product Citation

1. Rycenga HB, Wolfe KB, et al. "Uncoupling of p97 ATPase activity has a dominant negative effect on protein extraction." Sci Rep. 2019 Jul 17;9(1):10329. PMID:31316150
2. McLelland GL, Goiran T, et al. "Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy." Elife. 2018 Apr 20;7. pii: e32866. PMID:29676259
3. Yeo SK, French R, et al. "Opposing roles of Nfkb2 gene products p100 and p52 in the regulation of breast cancer stem cells." Breast Cancer Res Treat. 2017 Apr;162(3):465-477. PMID:28190248
4. Fullbright G, Rycenga HB, et al. "p97 Promotes a Conserved Mechanism of Helicase Unloading during DNA Cross-Link Repair." Mol Cell Biol. 2016 Nov 14;36(23):2983-2994. PMID:27644328

Chemical Properties

Physical Appearance	A solid
Storage	Store at -20°C
M.Wt	520.67
Cas No.	1418013-75-8
Formula	C₂₇H₂₈N₄O₃S₂
Solubility	insoluble in H2O; ≥17.1 mg/mL in DMSO; ≥2.54 mg/mL in EtOH with gentle warming and ultrasonic
Chemical Name	3-[3-cyclopentylsulfanyl-5-[[3-methyl-4-(4-methylsulfonylphenyl)phenoxy]methyl]-1,2,4-triazol-4-yl]pyridine
SDF	Download SDF
Canonical SMILES	CC1=C(C=CC(=C1)OCC2=NN=C(N2C3=CN=CC=C3)SC4CCCC4)C5=CC=C(C=C5)S(=O)(=O)C
Shipping Condition	Small Molecules with Blue Ice, Modified Nucleotides with Dry Ice.
General tips	We do not recommend long-term storage for the solution, please use it up soon.

Protocol

Kinase experiment [1]:
Biochemical assay development and HTS	The 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 experiment [1]:
Cell lines	HCT116, HeLa cells
Preparation method	The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.
Reacting condition	0.5, 2.5 μM for 6h
Applications	NMS-873 activated the unfolded protein response, interfered with autophagy and induced cancer cell death. NMS-873 mediated stabilization of Mcl-1 in live cells. NMS-873 inhibited the proliferative activity in HCT116 and HeLa cells with IC50 values of 0.4 and 0.7 μM, respectively.
References: 1Magnaghi, P., D'Alessio, R., Valsasina, B., Avanzi, N., Rizzi, S., Asa, D., Gasparri, F., Cozzi, L., Cucchi, U., Orrenius, C., Polucci, P., Ballinari, D., Perrera, C., Leone, A., Cervi, G., Casale, E., Xiao, Y., Wong, C., Anderson, D. J., Galvani, A., Donati, D., O'Brien, T., Jackson, P. K. and Isacchi, A. (2013) Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat Chem Biol. 9, 548-556

Biological Activity

Description	NMS-873 is an allosteric and specific inhibitor of p97 with an IC50 value of 30 nM.
Targets	p97
IC50	30 nM

Quality Control

Purity = 99.72%

Chemical structure

NMS-873 [1418013-75-8]

Référence B2168-5mg

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Background

Product Citation

Chemical Properties

Protocol

Biological Activity

Quality Control

Chemical structure

Related Biological Data

Related Biological Data

Related Biological Data

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