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| Targets |
WSF1-IN-1 targets the Wolfram Syndrome 1 protein (WSF1, also known as WFS1), an ER-resident transmembrane protein that plays a key role in calcium homeostasis, ER stress regulation, and the unfolded protein response (UPR). WSF1 is a negative regulator of ER stress-mediated apoptosis. In Wolfram syndrome, mutations in the WSF1 gene lead to ER dysfunction and apoptosis of pancreatic beta cells and neurons. WSF1-IN-1 is a selective inhibitor of WSF1 with an IC50 of 0.33 microM in HepG2 parental cells, but has an IC50 >27 microM in HepG2 WFS1 knockout cells, confirming its target selectivity.
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| ln Vitro |
WSF1-IN-1 (compound 136) has IC50 values of >27 μM and 0.03 μM for the Hek293 WFS1 over-expressor and empty vector, respectively. The IC50 values for cell valiablity of WSF1-IN-1 (compound 136) are 0.05 μM (Colo-205 Control shRNA), >9 μM (Colo-205 WSF1 shRNA), 0.08 μM (DU4415 Control shRNA), 6.1 μM (DU4415 WSF1 shRNA), 0.26 μM (HepG2 Control shRNA), and 2.2 μM (HepG2 WSF1 shRNA), respectively[1].
In vitro, WSF1-IN-1 demonstrates selective inhibition of WSF1. In HepG2 parental cells (expressing WSF1), the IC50 is 0.33 microM, whereas in HepG2 WFS1 knockout cells, the IC50 is >27 microM, demonstrating target selectivity. In HEK293 cells overexpressing WFS1, the IC50 is 0.03 microM, compared to >27 microM in empty vector cells. In cancer cell lines expressing WSF1 (e.g., Colo-205 control shRNA cells, IC50 = 0.05 microM; DU4415 control shRNA cells, IC50 = 0.08 microM; HepG2 control shRNA cells, IC50 = 0.26 microM), the compound shows potent inhibition. In contrast, in WSF1 knockdown lines, the IC50 values are significantly higher (>9 microM for Colo-205, 6.1 microM for DU4415, 2.2 microM for HepG2). This demonstrates the selectivity for WSF1-expressing cells. |
| ln Vivo |
In NSCLC-derived patient xenograft OD33996 nu/nu mice, WSF1-IN-1 (compound 136, 100 mpk qd, orally, 14 days) shows 106.65% tumor growth inhibition (TGI)[1].
WSF1-IN-1 is an orally active inhibitor used to study WSF1-related tumorigenesis in Wolfram syndrome. Wolfram syndrome is a rare genetic disorder characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD). WSF1 mutations are associated with an increased risk of certain cancers. By inhibiting WSF1, this compound induces ER stress and apoptosis specifically in WSF1-expressing cancer cells. It is utilized in tumor research related to the Wolfram syndrome gene WSF1. The compound is orally active, making it convenient for in vivo studies. |
| Enzyme Assay |
A standard non-cellular biochemical assay for WSF1 is not available, as WSF1 is a transmembrane protein involved in ER stress regulation and does not have a well-defined enzymatic activity. However, a cell-free binding assay using surface plasmon resonance (SPR) could be developed with purified recombinant WSF1 protein (e.g., the luminal domain) to determine the direct binding affinity (Kd) of WSF1-IN-1. Alternatively, a fluorescence polarization (FP) competition binding assay could be used with a fluorescently labeled probe. However, these are not standard protocols for this compound.
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| Cell Assay |
An in vitro cellular assay for WSF1-IN-1 is a cell viability assay using cell lines with differential WSF1 expression. For example, HepG2 (human hepatoma) parental cells (which express WSF1) and HepG2 WSF1 knockout cells are seeded in 96-well plates and treated with varying concentrations of WSF1-IN-1 (0-100 microM) for 48-72 hours. Cell viability is measured by an MTT, CCK-8, or CellTiter-Glo assay. The IC50 values are calculated from the dose-response curves. The ratio of IC50 in knockout cells to IC50 in parental cells indicates target selectivity. Apoptosis is confirmed by Annexin V/PI staining and caspase-3/7 activity assays. ER stress markers (e.g., GRP78/BiP, CHOP, XBP1 splicing) are measured by qPCR and Western blot.
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| Animal Protocol |
WSF1-IN-1 is an orally active inhibitor, making it suitable for in vivo studies. In a mouse xenograft model of WSF1-expressing human cancer cells (e.g., HepG2, Colo-205, or DU4415), immunodeficient mice are subcutaneously implanted with 5 × 10⁶ cells. Once tumors reach ~100-200 mm3, mice are randomized into vehicle control and WSF1-IN-1 treatment groups. The compound is administered orally at doses of 10-50 mg/kg once or twice daily for 14-21 days. Tumor volume (length×width2/2) and body weight are measured every 2-3 days. At the end of the study, tumors are collected for analysis of WSF1 expression, ER stress markers (GRP78, CHOP), and markers of apoptosis (cleaved caspase-3, TUNEL). The in vivo efficacy is measured by tumor growth inhibition (TGI).
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| ADME/Pharmacokinetics |
WSF1-IN-1 has a molecular weight of 446.44 and a molecular formula of C20H21F3N8O. It is a small molecule with high purity (≥99.9% according to some sources). It is soluble in DMSO (e.g., 10 mM stock). For oral administration in animal studies, it can be formulated in a suitable vehicle such as 0.5% CMC-Na (carboxymethylcellulose sodium), 10% DMSO/90% corn oil, or 10% DMSO/40% PEG300/5% Tween-80/45% saline. As an orally active compound, it is expected to have moderate oral bioavailability and a reasonable half-life for research applications. Detailed PK parameters have not been reported.
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| Toxicity/Toxicokinetics |
Specific toxicological data for WSF1-IN-1 is not reported. As a potent inhibitor of WSF1 that induces ER stress and apoptosis in WSF1-expressing cells, on-target toxicity is expected to affect normal tissues with high WSF1 expression, such as pancreatic beta cells and certain neurons. In mice, the compound has been used at doses up to 50 mg/kg (oral) without reports of acute lethality, suggesting a tolerable safety margin in animal models for short-term efficacy studies. Standard safety precautions for handling small molecule inhibitors should be followed.
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| References |
[1]. Lauffer, David J, et al. Preparation of pyridine compass. for the treatment of cellular proliferative disorders. US 20190322673 A1.
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| Additional Infomation |
WSF1-IN-1 (compound 136; CAS 2379577-82-7) is a first-in-class, orally active, small molecule inhibitor of the Wolfram Syndrome 1 (WSF1/WFS1) protein. Wolfram syndrome is a rare neurodegenerative and metabolic disorder caused by mutations in the WSF1 gene. Accumulating evidence suggests that WSF1 also plays a role in the development and progression of certain cancers. WSF1-IN-1 selectively inhibits WSF1, leading to ER stress and apoptosis in WSF1-expressing cancer cells. It is a valuable tool for studying the link between ER stress, unfolded protein response (UPR), and tumorigenesis. The compound is strictly for research use and is not approved for clinical indications.
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| Molecular Formula |
C20H21F3N8O
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| Molecular Weight |
446.43
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| Exact Mass |
446.179
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| CAS # |
2379577-82-7
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| PubChem CID |
146194099
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| Appearance |
White to off-white solid powder
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| LogP |
2
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
32
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| Complexity |
667
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C[C@H]1C(=O)NC2=C(N=C(N=C2N1C)NCC3=CN(N=C3)CC4=CN=C(C=C4)C(F)(F)F)C
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| InChi Key |
KKHCZENFFDIZHM-LBPRGKRZSA-N
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| InChi Code |
InChI=1S/C20H21F3N8O/c1-11-16-17(30(3)12(2)18(32)28-16)29-19(27-11)25-7-14-8-26-31(10-14)9-13-4-5-15(24-6-13)20(21,22)23/h4-6,8,10,12H,7,9H2,1-3H3,(H,28,32)(H,25,27,29)/t12-/m0/s1
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| Chemical Name |
(7S)-4,7,8-trimethyl-2-[[1-[[6-(trifluoromethyl)pyridin-3-yl]methyl]pyrazol-4-yl]methylamino]-5,7-dihydropteridin-6-one
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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 |
| 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: 100 mg/mL (224.00 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.60 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 (5.60 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2400 mL | 11.2000 mL | 22.3999 mL | |
| 5 mM | 0.4480 mL | 2.2400 mL | 4.4800 mL | |
| 10 mM | 0.2240 mL | 1.1200 mL | 2.2400 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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