p97 ( IC50 = 30 nM ）
NMS-873 specifically targets ATPase VCP/p97 (IC50 = 11 nM for ATPase activity inhibition) [1]

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]

---

In recombinant human VCP/p97 ATPase assays, NMS-873 inhibited ATP hydrolysis with an IC50 of 11 nM, acting as a reversible allosteric inhibitor. It showed no significant inhibition of other ATPases (e.g., Hsp70, Hsp90) at concentrations up to 10 μM [1]
- In a panel of human cancer cell lines (A549, HCT116, HeLa, MDA-MB-231, U2OS), NMS-873 exhibited potent antiproliferative activity with IC50 values ranging from 30 to 150 nM. After 72 hours of treatment, the 100 nM concentration reduced cell viability by 55-70% across different cell lines [1]
- In HCT116 colon cancer cells, NMS-873 (50 nM) induced apoptosis, as evidenced by increased caspase-3/7 activity (2.8-fold vs. control) and Annexin V-positive cells (35% vs. 5% in control) after 48 hours [1]
- NMS-873 (100 nM) disrupted VCP/p97-dependent protein degradation pathways, leading to accumulation of polyubiquitinated proteins (2.5-fold increase vs. control) and ER stress markers (CHOP and BIP protein levels increased by 3.2-fold and 2.7-fold, respectively) in HeLa cells [1]
- In U2OS osteosarcoma cells, NMS-873 (75 nM) inhibited autophagic flux, as indicated by accumulation of LC3-II (2.3-fold vs. control) and p62/SQSTM1 (1.9-fold vs. control) after 24 hours of treatment [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.

---

In nude mice bearing HCT116 colon cancer xenografts, intraperitoneal administration of NMS-873 (20 mg/kg, twice weekly for 3 weeks) significantly inhibited tumor growth. Tumor volume was reduced by 68% compared to vehicle-treated mice, with no significant loss of body weight [1]
- In the same xenograft model, NMS-873 (20 mg/kg) treatment led to accumulation of polyubiquitinated proteins (2.1-fold vs. vehicle) and activation of caspase-3 (cleaved caspase-3 levels increased by 2.4-fold) in tumor tissues, confirming on-target activity and apoptotic induction [1]

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).

---

Recombinant human VCP/p97 protein was purified and incubated with NMS-873 (0.1 nM-1 μM) and ATP (1 mM) in assay buffer at 37°C for 60 minutes. The release of inorganic phosphate (Pi) was quantified using a colorimetric assay kit. IC50 values were calculated from dose-response inhibition curves of ATP hydrolysis [1]
- For selectivity assessment, recombinant Hsp70, Hsp90, and other ATPases were incubated with their respective substrates and NMS-873 (0.1 nM-10 μM) under optimal reaction conditions. ATPase activity was measured, and IC50 values were determined to evaluate cross-reactivity [1]

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.

---

Antiproliferation assay: Cancer cell lines (A549, HCT116, HeLa, MDA-MB-231, U2OS) were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. NMS-873 was added at concentrations of 1 nM-1 μM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [1]
- Apoptosis assay: HCT116 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with NMS-873 (50 nM) for 48 hours. Caspase-3/7 activity was measured using a luminescent assay kit, and Annexin V-FITC/PI staining was performed for flow cytometric analysis of apoptotic cells [1]
- Protein degradation and ER stress assay: HeLa cells were treated with NMS-873 (100 nM) for 24 hours. Cells were lysed, and polyubiquitinated proteins, CHOP, and BIP levels were analyzed by Western blot using specific antibodies [1]
- Autophagy assay: U2OS cells were treated with NMS-873 (75 nM) for 24 hours. LC3-II and p62/SQSTM1 levels were detected by Western blot, and autophagic flux was assessed by comparing LC3-II accumulation in the presence or absence of bafilomycin A1 [1]

Nude mice (6-8 weeks old) were subcutaneously inoculated with HCT116 colon cancer cells (5×10⁶ cells/mouse). When tumors reached a volume of ~100 mm³, mice were randomly divided into vehicle and NMS-873 groups. NMS-873 was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 20 mg/kg, twice weekly for 3 weeks. Vehicle-treated mice received DMSO/saline mixture. Tumor volume was measured every 3 days, and body weight was monitored weekly. At the end of the study, tumors were excised for Western blot analysis [1]

In in vivo xenograft studies, NMS-873 (20 mg/kg, intraperitoneal injection, twice weekly for 3 weeks) did not cause significant weight loss (≤5% change from baseline) or obvious toxicity in nude mice [1]. In vitro, NMS-873 showed extremely low toxicity to normal human fibroblasts (IC50 > 500 nM), indicating a therapeutic window between cancer cells and normal cells [1]. Compared with the vector control group, no significant changes were observed in liver function (ALT, AST) or kidney function (creatinine, BUN) in mice treated with NMS-873 [1].

[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.

NMS-873 is a potent, selective, and reversible allosteric inhibitor of VCP/p97 ATPase, a key enzyme involved in protein quality control, autophagy, and endoplasmic reticulum-associated degradation (ERAD) pathways [1]. Its mechanism of action includes binding to the allosteric site of VCP/p97, inhibiting ATP hydrolysis, disrupting downstream protein homeostasis pathways, leading to the accumulation of misfolded proteins, endoplasmic reticulum stress, and ultimately inducing apoptosis in cancer cells [1]. VCP/p97 is overexpressed in various human cancers, and the inhibition of it by NMS-873 provides a targeted therapeutic strategy for cancer treatment [1]. In vivo experiments have shown that NMS-873 has targeting activity, which has been confirmed by molecular markers in tumor tissues (polyubiquitinated protein and cleaved caspase-3) [1].

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.

---

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.

---

View More

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.

---

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