p97 ( IC50 = 30 nM ）

NMS-873 inhibits the degradation of the linker-D2 domain by decreasing the sensitivity of p97 to trypsin digestion. Several hematological and solid tumor lines exhibit antiproliferative activity in response to NMS-873, a p97 inhibitor. According to the mechanism study, NMS-873 increases the unfolded protein response, obstructs autophagy, and ultimately causes the death of cancer cells.[1]

NMS-873 exhibits cell-killing efficacy against a broad range of solid tumors with an IC50 range of 0.08μM to 2μM, in addition to hematological tumors.

A modified NADH-coupled assay is used to monitor ADP formation in the reaction in order to assess the ATPase activity and kinetic parameters of recombinant wild-type VCP and its mutants. Given that both ADP and NADH are ATP-competitive inhibitors of VCP ATPase activity, the standard protocol for the NADH-coupled assay has been altered to occur in two steps. In the initial phase, an ATP-regenerating system consisting of 3 mM phosphoenolpyruvate and 40 U/ml pyruvate kinase recycles the ADP generated by VCP activity, maintains a constant substrate concentration to avoid product inhibition, and builds up a stoichiometric amount of pyruvate. The second section involves quenching the VCP enzymatic reaction with 30 mM EDTA and 250 μM NADH, followed by a stoichiometric oxidation by 40 U/ml lactic dehydrogenase, which lowers the pyruvate accumulation. A Tecan Safire 2 reader plate is used to measure the drop in NADH concentration at 340 nm. The assay is run in 96-or 384-well UV plates in a reaction buffer containing 2 mM DTT, 10 mM MgCl₂, pH 7.5, 0.2 mg/mL BSA, and 50 mM Hepes. A cooperative equation is used to fit the experimental data, yielding a Ks* of roughly 60 μM and a Hill coefficient (n) of 2.0 ± 0.1. A more sensitive ADP detection system called Transcreener ADP FP is used in conjunction with a miniaturized assay in a 1,536-well format to conduct the HTS campaign against a library of one million compounds. After preincubating 10 μM inhibitor and 10 nM VCP for 20 minutes, 10 μM ATP is added to the reaction, which is then allowed to proceed for 90 minutes before quenching. With 3× s.d. (38% inhibition) as the cutoff, the screening average Z′ is 0.58, and the hit rate is 1.7%. Physicochemical and structural filters are used to remove primary hits that exhibit more than 60% inhibition at a concentration of 10 μM, leaving 7,516 compounds. Finally, 500 compounds are chosen for a dose-response analysis utilizing the previously mentioned NADH-modified coupled assay after reconfirmation is carried out in duplicate on 3,988 primary hits. The C522T mutant and wild-type VCP are used to gauge how potent the most intriguing HTS hits are. In the assay, ATP concentrations that produced the half-maximal velocity (Ks*) for each enzyme—60 μM for the wild type and 130 μM for the C522T mutant, respectively—are employed. In order to investigate the reversible inhibitors' dependence on substrate concentration, their potency is assessed at saturating ATP concentrations of 1 mM and juxtaposed with the potency of a conventional ATP competitive inhibitor (AMP-PNP).

In 384-well white clear-bottom plates, 1,600 cells are seeded per well of the plate. The compounds are added to the cells twenty-four hours after they are seeded (eight dilution points, in duplicate, for each compound), and they are then incubated for a further 72 hours at 37°C with 5% CO2 in the air. After that, the cells are lysed, and a thermostable firefly luciferase-based assay from Promega is used to measure the amount of ATP present in each well as a proxy for cell viability. The percentage of treated cell growth compared to the untreated control is used to calculate IC50 values.

[1]. Covalent and allosteric inhibitors of the ATPATP ase VCP/p97 induce cancer cell death. Nat Chem Biol. 2013 Jul 28. doi: 10.1038/nchembio.1313.

C27H28N4O3S2

520.67

520.16

C, 62.28; H, 5.42; N, 10.76; O, 9.22; S, 12.32

1418013-75-8

71521142

White solid powder

1.3±0.1 g/cm3

769.3±70.0 °C at 760 mmHg

419.0±35.7 °C

0.0±2.6 mmHg at 25°C

1.675

4.77

0

7

8

36

795

0

S(C1=NN=C(C([H])([H])OC2C([H])=C([H])C(C3C([H])=C([H])C(=C([H])C=3[H])S(C([H])([H])[H])(=O)=O)=C(C([H])([H])[H])C=2[H])N1C1=C([H])N=C([H])C([H])=C1[H])C1([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

UJGTUKMAJVCBIS-UHFFFAOYSA-N

InChI=1S/C27H28N4O3S2/c1-19-16-22(11-14-25(19)20-9-12-24(13-10-20)36(2,32)33)34-18-26-29-30-27(35-23-7-3-4-8-23)31(26)21-6-5-15-28-17-21/h5-6,9-17,23H,3-4,7-8,18H2,1-2H3

3-[3-cyclopentylsulfanyl-5-[[3-methyl-4-(4-methylsulfonylphenyl)phenoxy]methyl]-1,2,4-triazol-4-yl]pyridine

NMS-873; NMS873; NMS 873

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 20.5~100 mg/mL (9.4 ~192.1 mM）

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.80 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.80 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)