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NVS-ZP7-4 is a Zinc transporter SLC39A7 (ZIP7) inhibitor that is also the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
| Targets |
NVS-ZP7-4 targets zinc transporter ZIP7 (SLC39A7) (FRET-based zinc transport assay IC50 = 3.2 nM; SPR binding Ki = 2.8 nM) [1]
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|---|---|
| ln Vitro |
NVS-ZP7-4 raises zinc levels in the ER, suggesting that ZIP7 is functionally regulated [1].
1. ZIP7 zinc transport activity inhibition: - NVS-ZP7-4 potently inhibits ZIP7-mediated zinc transport across endoplasmic reticulum (ER) membranes, with an IC50 of 3.2 nM (FRET-based zinc transport assay) [1] - It shows no significant inhibition of other ZIP family transporters (ZIP1, ZIP3, ZIP6) at concentrations up to 20 μM, confirming ZIP7 selectivity [1] 2. Notch signaling pathway suppression: - NVS-ZP7-4 inhibits Notch pathway activation in Notch-dependent cell lines, with an IC50 of 4.5 nM in Notch luciferase reporter assay (HEK293T cells) [1] - It reduces mRNA levels of Notch target genes (HES1, HEY1) by 68% and 72% at 10 nM (RT-qPCR in Jurkat cells) [1] - Western blot analysis confirms 75% reduction in cleaved Notch1 (NICD) and 65% reduction in HES1 protein levels in Jurkat cells treated with 10 nM NVS-ZP7-4 for 24 hours [1] 3. Antiproliferative activity in Notch-dependent tumors: - NVS-ZP7-4 inhibits proliferation of Notch-dependent cancer cell lines in a concentration-dependent manner: Jurkat (IC50 = 5.1 nM), KOPN-8 (IC50 = 7.3 nM), T-ALL (IC50 = 6.8 nM) (72-hour CCK-8 assay) [1] - It exhibits minimal antiproliferative effect on Notch-independent cancer cells (HCT116, MCF-7) with IC50 > 10 μM [1] - Long-term clonogenic assay shows >90% inhibition of colony formation in Jurkat cells at 15 nM NVS-ZP7-4 [1] 4. Intracellular zinc homeostasis disruption: - NVS-ZP7-4 (5, 10 nM) increases ER zinc ion concentration and decreases cytoplasmic zinc level in Jurkat cells, as detected by ER-targeted and cytoplasmic zinc fluorescent probes [1] - The zinc homeostasis imbalance is reversed by ZIP7 overexpression, confirming ZIP7-dependent mechanism [1] 5. Apoptosis induction: - NVS-ZP7-4 (10 nM) induces apoptosis in Jurkat cells, with apoptotic rate increasing from 4.8% (control) to 35.2% (48 hours, Annexin V/PI staining) [1] - Western blot shows upregulation of pro-apoptotic proteins (cleaved caspase-3/9, Bax) and downregulation of anti-apoptotic protein Bcl-2 [1] |
| ln Vivo |
1. Antitumor activity in Jurkat T-ALL xenograft model:
- NVS-ZP7-4 (10, 30 mg/kg, p.o., twice daily for 21 days) inhibits Jurkat tumor growth in nude mice by 55% and 78%, respectively, compared to vehicle control [1] - The 30 mg/kg group shows significant reduction in tumor weight (from 0.76 g to 0.17 g) without obvious body weight loss or systemic toxicity [1] - Tumor tissue analysis reveals 70% reduction in NICD protein level and 65% reduction in HES1 mRNA level, along with increased apoptotic cells (TUNEL assay) [1] 2. Notch pathway suppression in vivo: - Immunohistochemical staining of tumor tissues from drug-treated mice shows decreased Notch1 activation (NICD) and reduced Ki-67 (proliferation marker) positive cells [1] - Serum levels of Notch pathway-related cytokines (IL-6, TNF-α) are reduced by 50-60% in the 30 mg/kg group [1] |
| Enzyme Assay |
1. FRET-based ZIP7 zinc transport assay:
- Recombinant human ZIP7 protein is expressed in HEK293T cells and enriched in ER membrane fractions [1] - ER membrane vesicles are loaded with a zinc-sensitive FRET probe, then mixed with NVS-ZP7-4 (0.1 nM to 50 nM) in transport buffer containing zinc ions [1] - FRET signal changes (excitation 430 nm, emission 480 nm and 530 nm) are monitored in real-time for 30 minutes to detect zinc transport across ER membranes [1] - IC50 is calculated by fitting the dose-response curve of FRET signal inhibition [1] 2. Surface Plasmon Resonance (SPR) binding assay: - Recombinant ZIP7 extracellular domain is immobilized on a CM5 sensor chip via amine coupling to a density of ~900 resonance units (RU) [1] - NVS-ZP7-4 is serially diluted (0.3 nM to 30 nM) in running buffer (PBS with 0.05% Tween-20) and injected over the chip at a flow rate of 30 μl/min [1] - Association (120 seconds) and dissociation (300 seconds) phases are monitored, and the chip is regenerated with 10 mM glycine-HCl (pH 2.3) [1] - Binding affinity (Ki) is calculated using a 1:1 Langmuir binding model with reference subtraction [1] |
| Cell Assay |
1. Cell proliferation assay (CCK-8):
- Notch-dependent (Jurkat, KOPN-8, T-ALL) and Notch-independent (HCT116, MCF-7) cell lines are seeded in 96-well plates at 3×10^3 cells per well and cultured overnight [1] - NVS-ZP7-4 is serially diluted (0.1 nM to 20 μM) and added to the cells, incubated at 37°C with 5% CO2 for 72 hours [1] - CCK-8 reagent is added to each well, incubated for 2 hours, and absorbance at 450 nm is measured to calculate cell viability and IC50 values [1] 2. Notch luciferase reporter assay: - HEK293T cells are co-transfected with Notch1 intracellular domain (NICD) expression plasmid and CSL-luciferase reporter plasmid [1] - Transfected cells are seeded in 96-well plates and treated with NVS-ZP7-4 (0.1 nM to 50 nM) for 24 hours [1] - Luciferase activity is measured using a luminometer, and IC50 is calculated by fitting the dose-response curve of activity inhibition [1] 3. Intracellular zinc concentration detection: - Jurkat cells are loaded with ER-targeted zinc fluorescent probe (Zinpyr-1-ER) or cytoplasmic zinc probe (FluoZin-3 AM) for 30 minutes at 37°C [1] - Cells are treated with NVS-ZP7-4 (5, 10 nM) for 4 hours, then fluorescence intensity is detected by flow cytometry or confocal microscopy [1] - Relative zinc concentration is calculated by comparing fluorescence intensity of drug-treated groups to the control [1] 4. Western blot and RT-qPCR assays: - NVS-ZP7-4-treated Jurkat cells are lysed with RIPA buffer containing protease and phosphatase inhibitors [1] - For Western blot: Equal amounts of protein are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against NICD, HES1, HEY1, cleaved caspase-3/9, Bax, Bcl-2, and GAPDH [1] - For RT-qPCR: Total RNA is extracted, reverse-transcribed into cDNA, and qPCR is performed with specific primers for HES1, HEY1, and GAPDH (internal control) [1] |
| Animal Protocol |
1. Jurkat T-ALL subcutaneous xenograft model:
- Male BALB/c nude mice (6-8 weeks old) are subcutaneously injected with 5×10^6 Jurkat cells suspended in Matrigel (1:1 with PBS) [1] - When tumors reach a volume of ~100 mm³, mice are randomized into vehicle control and NVS-ZP7-4 treatment groups (n=8 per group) [1] - NVS-ZP7-4 is dissolved in 0.5% carboxymethyl cellulose (CMC) + 0.1% Tween 80, administered orally at 10 or 30 mg/kg twice daily for 21 days [1] - Tumor volume is measured every 3 days using calipers, and body weight is monitored weekly [1] - At the end of treatment, mice are euthanized, tumors are excised, weighed, and stored for protein, mRNA, and immunohistochemical analysis [1] |
| ADME/Pharmacokinetics |
NVS-ZP7-4 has oral bioavailability of 41% and 38% in mice (oral 30 mg/kg) and rats (oral 10 mg/kg), respectively[1]
- In mice, the plasma half-life (t1/2) after intravenous injection (5 mg/kg) was 3.8 h, the volume of distribution (Vd) was 1.1 L/kg, and the total clearance (CL) was 190 ml/kg/h[1] - Mice reached peak plasma concentration (Cmax) of 1.9 μg/ml 1 h after oral administration of 30 mg/kg, and the 24-hour AUC₀-24h was 15.7 μg·h/ml[1] - Plasma protein binding was 94% (human plasma) and 92% (mouse plasma), respectively[1] - It showed good stability in human liver microsomes (t1/2 > 3) (several hours) mainly through CYP2C9 and CYP3A4 Metabolism [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No deaths or adverse reactions were observed in mice after a single oral dose of up to 200 mg/kg [1]
- Subacute toxicity: No significant changes were observed in body weight, food intake, or hematological parameters (white blood cells, red blood cells, platelets) after mice were given NVS-ZP7-4 twice daily (30 mg/kg) for 28 consecutive days [1] - Serum ALT, AST, creatinine, and blood urea nitrogen levels in treated mice were within the normal range [1] - No histopathological abnormalities were detected in the liver, kidney, heart, lung, or spleen tissues of treated mice [1] |
| References | |
| Additional Infomation |
NVS-ZP7-4 is a highly effective, selective, and orally bioavailable ZIP7 (SLC39A7) inhibitor, discovered through screening for the Notch pathway [1]
- Its binding mode is to occupy the zinc-binding pocket of ZIP7, blocking the transport of zinc ions from the endoplasmic reticulum to the cytoplasm, which is crucial for the maturation and activation of the Notch receptor [1] - It exerts its anti-tumor effect by inhibiting the Notch signaling pathway, inhibiting cancer cell proliferation, and inducing apoptosis in Notch-dependent tumors (such as T-cell acute lymphoblastic leukemia, T-ALL) [1] - ZIP7 is overexpressed in a variety of Notch-driven cancers, therefore NVS-ZP7-4 is expected to be a potential drug for the treatment of Notch-dependent malignancies to meet unmet medical needs [1] |
| Molecular Formula |
C28H28FN5OS
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|---|---|
| Molecular Weight |
501.618227958679
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| Exact Mass |
501.2
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| Elemental Analysis |
C, 67.04; H, 5.63; F, 3.79; N, 13.96; O, 3.19; S, 6.39
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| CAS # |
2349367-89-9
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| Related CAS # |
(R)-NVS-ZP7-4;2517682-14-1
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| PubChem CID |
134823928
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
5.5
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
36
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| Complexity |
758
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1CN(CCC12C3=CC=CC=C3NC(=O)N2)C[C@H](CC4=CC=CC=C4)NC5=NC6=C(S5)C=C(C=C6)F
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| InChi Key |
FZOFDZMKSAUTHT-NRFANRHFSA-N
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| InChi Code |
InChI=1S/C28H28FN5OS/c29-20-10-11-24-25(17-20)36-27(32-24)30-21(16-19-6-2-1-3-7-19)18-34-14-12-28(13-15-34)22-8-4-5-9-23(22)31-26(35)33-28/h1-11,17,21H,12-16,18H2,(H,30,32)(H2,31,33,35)/t21-/m0/s1
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| Chemical Name |
1'-[(2S)-2-[(6-fluoro-1,3-benzothiazol-2-yl)amino]-3-phenylpropyl]spiro[1,3-dihydroquinazoline-4,4'-piperidine]-2-one
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| Synonyms |
NVS-ZP7-4; NVS-ZP7 4; NVS-ZP74; NVSZP7-4; NVSZP7 4; NVSZP74;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 125 mg/mL (~249.19 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.15 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 20.8 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: ≥ 2.08 mg/mL (4.15 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 20.8 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9935 mL | 9.9677 mL | 19.9354 mL | |
| 5 mM | 0.3987 mL | 1.9935 mL | 3.9871 mL | |
| 10 mM | 0.1994 mL | 0.9968 mL | 1.9935 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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