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Purity: ≥98%
VLX1570 is a competitive inhibitor of the 19S proteasome-specific DUB (deubiquitylating enzymes) activity with IC50 ranging from 4.2 uM to 8.6 uM. VLX1570 preferentially inhibits proteasomal DUB activity while not inhibiting the activities of a panel of non-proteasomal DUBs. VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro ipon administration, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). This blocks the ubiquitin proteasome degradation pathway, prevents the degradation of defective proteins, and leads to an accumulation of poly-ubiquitylated proteins, which induces the unfolded protein response (UPR) and results in both the induction of tumor cell apoptosis and the inhibition of tumor cell growth. Therefore, VLX1570 has apoptosis-inducing ability and anticancer activities.
| Targets |
Deubiquitinase(IC50= appr 10 μM)
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| ln Vitro |
USP14 and UCHL5 activity of 19S regulatory particles are inhibited by VLX1570, with USP14 being more strongly inhibited. In KMS-11 myeloma cells, VLX1570 (1 μM) exhibits inhibitory activity against USP14. VLX1570 on HCT116 cells shows an IC50 of 0.58 μM[1].Using two distinct sources of recombinant protein, VLX1570 binds to recombinant USP14 with a Kd of 1.5–18 μM; in contrast, recombinant UCHL5 has a higher Kd (14–18 μM) than recombinant USP14. With IC50s of 43 ± 2 nM, 74 ± 2 nM, 126 ± 3 nM, and 191 ± 1 nM for KMS-11, RPMI8226, OPM-2, and OPM-2-BZR cells, respectively, VLX1570 exhibits strong antiproliferative activities on multiple myeloma cells[2]. BCWM.1 cell viability is suppressed by VLX1570, with an EC50 of 20.22 nM. In all Waldenstrom macroglobulinemia (WM) cell lines tested, including BCWM.1/IR (IR) and BCWM.1/BR (BR) subclones, VLX1570 (100, 250, and 500 nM) dose-dependently induces significant apoptosis by 12 hours. In WM cells, VLX1570 (100, 250, and 500 nM) also damages the mitochondria and the ER stress machinery. In WM cells, VLX1570 (250 nM) downregulates the expression of CXCR4 and the end effectors of BCR-signalosomes[3].
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| ln Vivo |
VLX1570 (3 mg/kg) dramatically reduces the growth of tumors in mice with multiple myeloma cells (KMS-11)[2]. In Waldenstrom macroglobulinemia (WM)-bearing mice, VLX1570 (4.4 mg/kg, i.p.) significantly suppresses tumor growth without causing apparent weight loss or other signs of systemic toxicity[3].
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| Enzyme Assay |
Before adding ubirhodamine, 26S proteasome preparations (1 nM) are pretreated for 2 min in assay buffer (25 mM Tris, 5 mM MgCl2, 10% glycerol, 0.05 mg/mL BSA, 2 mM ATP, and 1 mM DTT) with DMSO, VLX1570, or b-AP15. Using a TECAN infinite 200 instrument, fluorescence is measured at 37°C using Ex/Em = 490 nm/520 nm. Data is read every 10 seconds for 30 minutes. Cell pellets from control or treated cells are lysed with a buffer (50 mM HEPES pH 7.4, 250 mM sucrose, 10 mM MgCl2, 2 mM ATP, 1 mM DTT) on ice for 30 minutes in order to prepare them for UbVS labeling of KMS 11 cells. The debris is then removed by centrifugation. 1 μM UbVS is applied to 25 μg of protein and left for 30 minutes at 37°C. Samples are separated using SDS-PAGE and then immunoblotted. Purified 19S proteasomes (50 nM) are pretreated for 10 min at room temperature with DMSO, VLX1570, or b-AP15 (50 μM). After that, they are labeled with 1 μM HA-UbVS for 30 min at 37°C and then subjected to immunoblotting[1].
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| Cell Assay |
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is used to measure cell viability. Cells are suspended at 5 × 10^5 cells/mL for the MTT assay. 100 μL aliquots are then put into 96-well microtiter plates and exposed to drugs, with DMSO serving as the control. 10μL of a stock solution containing 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is added to each well after the incubations are complete, and the plates are then incubated for 4 hours at 37°C. Overnight at 37°C, 100μL of a 10% SDS/10 mM HCl solution is used to dissolve the formazan crystals.In certain experiments, cell viability is assessed using the acid phosphatase method49 because VLX1570 affects OXPHOS and mitochondrial activity affects MTT assays. Cells are lysed in 100μL of 0.1 M sodium acetate, 0,1% Triton X-100, and p-nitrophenylphosphate after being washed twice with PBS. They are then incubated for 90 minutes at 37°C. After the incubation period, 10μL of NaOH is added to each well, and the value of A405 is obtained [2].
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| Animal Protocol |
An animal experiment is carried out with 7 participants per group and 80% power at the 5% significance level to identify a 1800 mm^3 mean difference between the 2 groups. With a sample size of 7 per group, 80% power at the 5% significance level is computed to detect a 450% difference in means between the 2 groups for the percentage change in IgM from baseline. Subcutaneous implants of 1× 10^6 luciferase-labeled RPCI-WM1 cells (Luc-RPCI-WM1) are made in 14 female NOD/SCID mice (6-8 weeks old). The cells are left to grow until an IVIS imaging signal (Day 20) is seen. On day 21, mice are randomly assigned to two groups (n=7), one of which receives an intraperitoneal injection of VLX1570 at a dose of 4.4 mg/kg and the other a vehicle (cremaphor+PEG+Tween). The group assignment is not hidden from the investigator. For 22 days, each of the two groups receives treatment with a vehicle or a VLX1570 on alternate days. Every three to four days, the tumors' sizes are measured with calipers, and their volumes are computed using the formula (width)^2 × length/2. The Xenogen imaging system is used for bioluminescent tumor imaging on Days 0, 20, 30, 36, and 43 following tumor implantationOn the same days, mice's blood is drawn by submandibular venous puncture, and the sera are then separated so that human IgM levels can be measured using ELISA. Mice are killed on Day 44, and the ultimate tumor volume in the treatment and control groups is measured. A digital camera, Canon D40, was used to capture all of the images. No particular standards for inclusion or exclusion are applied because all of the mice developed tumors and were thus included in the investigation[3].
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| References |
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| Molecular Formula |
C23H17F2N3O6
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| Molecular Weight |
469.39
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| Exact Mass |
469.11
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| Elemental Analysis |
C, 58.85; H, 3.65; F, 8.09; N, 8.95; O, 20.45
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| CAS # |
1431280-51-1
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| Related CAS # |
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| Appearance |
Solid powder
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| SMILES |
O=C1/C(CN(C(C=C)=O)CC/C1=C/C2=CC=C(F)C([N+]([O-])=O)=C2)=C\C3=CC=C(F)C([N+]([O-])=O)=C3
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| InChi Key |
SCKXBVLYWLLALY-CQRYCMKKSA-N
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| InChi Code |
InChI=1S/C23H17F2N3O6/c1-2-22(29)26-8-7-16(9-14-3-5-18(24)20(11-14)27(31)32)23(30)17(13-26)10-15-4-6-19(25)21(12-15)28(33)34/h2-6,9-12H,1,7-8,13H2/b16-9-,17-10-
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| Chemical Name |
1-acryloyl-3,5-bis((Z)-4-fluoro-3-nitrobenzylidene)azepan-4-one
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| Synonyms |
VLX-1570;VLX1570;VLX 1570
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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 |
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| 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 : 32 ~93 mg/mL ( 68.17~198.12 mM )
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1304 mL | 10.6521 mL | 21.3042 mL | |
| 5 mM | 0.4261 mL | 2.1304 mL | 4.2608 mL | |
| 10 mM | 0.2130 mL | 1.0652 mL | 2.1304 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT02372240 | Terminated | Drug: VLX1570 and dexamethasone | Multiple Myeloma | Vivolux AB | April 8, 2015 | Phase 1 Phase 2 |
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