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Purity: ≥98%
NSC 185058 (NSC-185058) is an inhibitor of ATG4B which is a major cysteine protease, and inhibition of ATG4B by NSC185058 markedly attenuates autophagic activity. NSC185058 acts by inhibiting autophagy and showing a negative impact on osteosarcoma tumors.
| Targets |
NSC 185058 is an antagonist of ATG4B.[1]
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| ln Vitro |
NSC 185058 decreased ATG4B-induced conversion of LC3-I to lipidated LC3-II and inhibited the degradation of the autophagy substrate p62/SQSTM1 in GSC JKB3 cells treated for 72 hours.[1]
Treatment with NSC 185058 or chloroquine (CQ) decreased GSC JKB3 cell viability and sphere-forming frequency to similar extents after 72 hours of treatment.[1] In GSC JKB3 cells, inhibition of ATG4B by NSC 185058 sensitized cells to ionizing radiation (IR) by suppressing LC3B lipidation and increasing p62 levels. This molecular response was accompanied by decreased cell viability from the combination treatment (NSC + IR) compared to treatment with NSC 185058 or IR alone.[1] In GSC 23 cells, combined treatment of IR and NSC 185058 resulted in decreased cell viability compared to single-agent treatments.[1] Combined IR and NSC 185058 treatments in GSCs resulted in a pronounced reduction in mitotic cells due to G2 arrest and significant increases in apoptosis (measured by annexin V staining) compared to single-agent treatments.[1] |
| ln Vivo |
An antagonist of ATG4B is NSC185058. By reversibly altering ATG8 to encourage autophagosomes, ATG4B promotes autophagy. Radiation therapy (RT) has an enhanced antitumor activity when it contains ATG4B-conjugated NSC185058. NSC185058 was found to decrease the tumorigenicity of astroblastoma (GBM) cells and increase the antitumor activity of RT in an orthotopic GBM xenograft model [1].
In a subcutaneous tumor xenograft model using GSC M83 cells, treatment with NSC 185058 (150 mg/kg, i.p., every other day) significantly decreased tumor growth.[1] NSC 185058 did not enhance the effects of STK26 (MST4) knockout on M83 xenograft growth and autophagic activity in vivo.[1] In an intracranial GSC JKB3 xenograft model, monotherapy with NSC 185058 (150 mg/kg, i.p., Monday, Wednesday, Friday for 3 weeks) showed a growth-suppressive effect.[1] In the same intracranial model, all combination treatments of NSC 185058 with radiotherapy (IR) showed significantly increased anti-tumor activity relative to monotherapy. Co-administration and IR followed by NSC 185058 regimens performed the best, as indicated by bioluminescence monitoring and survival analysis.[1] Intracranial JKB3 and 23 GBM tumors treated with combined NSC 185058 + IR had significantly lower proliferation indices (by Ki-67 staining) compared to NSC 185058 or IR alone. Combination treatment also markedly decreased LC3B levels and increased apoptosis indicators (γH2AX and cleaved caspase-3).[1] Administration of NSC 185058 in the preclinical in vivo experiments, in amounts and at frequencies showing substantial anti-tumor activity, both as a single agent and especially in combination with RT, was well tolerated by animal subjects.[1] |
| Cell Assay |
For cell viability assays, GSCs with indicated treatments were plated in 96-well plates at 2000 cells per well. Cell viabilities were evaluated at indicated time points using a luminescent cell viability assay.[1]
For sphere-forming assays (limiting dilution assays), dissociated cells from glioma spheres were seeded in 96-well plates at various densities (e.g., 1, 5, 10, 20, or 50 cells per well for certain GSC lines). After 7 to 14 days, each well was examined for the formation of tumor spheres. Stem cell frequency was calculated using extreme limiting dilution analysis.[1] For apoptosis assay, cells were incubated with an anti-Annexin V antibody and Propidium Iodide and analyzed by flow cytometry. Cells without staining were used as negative controls.[1] For cell cycle analyses, cultured cells were pulsed with a DNA dye stain for 30 minutes followed by processing for fluorescence-activated cell sorting (FACS).[1] For analysis of autophagy markers by immunoblotting (Western blot), cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein samples were quantified, subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against proteins such as LC3B and p62.[1] For the analysis of ATG4B-mediated cleavage of a FRET-LC3B fusion protein substrate, purified FRET-LC3B protein was mixed with cell lysates from GSCs in an appropriate buffer. After incubation at 37°C, the reaction was stopped, and samples were separated by SDS-PAGE and examined by Coomassie Brilliant Blue staining to assess cleavage.[1] |
| Animal Protocol |
For subcutaneous tumor xenograft studies, 1x10^6 GSC M83 cells in 100 μl PBS were injected subcutaneously into athymic nude mice. When palpable tumors formed, tumor-bearing mice were treated by intraperitoneal (i.p.) injection with NSC 185058 (150 mg/kg) or vehicle control (peanut oil) on alternating days. Mice were euthanized when tumor size reached approximately 1500 mm³ or when pathological symptoms developed. Tumor volume was estimated using the formula V = ab²/2, where a and b (a > b) are the tumor's length and width.[1]
For intracranial xenograft models treated with radiation and NSC 185058, mice stereotactically transplanted with GSC JK83 or 23 cells were randomized into six treatment groups: 1) vehicle control (peanut oil), 2) NSC 185058 alone, 3) radiation alone, 4) concurrent NSC 185058 and radiation, 5) sequential radiation followed by NSC 185058, and 6) sequential NSC 185058 followed by radiation. The NSC 185058 treatment groups received the drug at a dose of 150 mg/kg by i.p. injection on Monday, Wednesday, and Friday for three weeks. The radiation groups received 2 Gy per day for five consecutive days. Treatment started one week after transplantation. Tumor growth was monitored by bioluminescence imaging after injecting D-luciferin. Mice were monitored for neurological symptoms and euthanized when symptoms developed.[1] For bioluminescence imaging, tumor-bearing mice were injected with D-luciferin (300 mg/kg) before isoflurane anesthesia. Radiance was measured 15 minutes after substrate injection using an imaging system.[1] |
| References | |
| Additional Infomation |
NSC 185058 is a small molecule inhibitor targeting ATG4B. [1]
This study identified an autophagy-promoting MST4-ATG4B signaling pathway in glioblastoma (GBM). Pharmacological inhibition of ATG4B using NSC 185058 reduced autophagy activity, decreased the tumorigenicity of GBM cells, and enhanced the antitumor activity of radiotherapy in an orthotopic GBM xenograft model. [1] Combined therapy (simultaneous or sequential) affects the antitumor activity of NSC 185058 in combination with radiotherapy. [1] This study suggests that ATG4B may be a viable target for combination therapy of malignant tumors such as GBM, and provides a basis for developing small molecule drugs targeting ATG4B as clinical drugs. [1] |
| Molecular Formula |
C11H9N3S
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|---|---|
| Molecular Weight |
215.27426
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| Exact Mass |
215.052
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| CAS # |
39122-38-8
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| PubChem CID |
750538
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.323g/cm3
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| Boiling Point |
378.2ºC at 760 mmHg
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| Flash Point |
182.5ºC
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| Index of Refraction |
1.72
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| LogP |
2.337
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
15
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| Complexity |
220
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
UGWOJXZJIZUKDP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C11H9N3S/c15-11(9-5-1-3-7-12-9)14-10-6-2-4-8-13-10/h1-8H,(H,13,14,15)
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| Chemical Name |
N-Pyridin-2-ylpyridine-2-carbothioamide
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| Synonyms |
NSC185058; NSC-185058; NSC 185058;
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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 (~580.67 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (9.66 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 (9.66 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 20.8 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: ≥ 2.08 mg/mL (9.66 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.6453 mL | 23.2266 mL | 46.4533 mL | |
| 5 mM | 0.9291 mL | 4.6453 mL | 9.2907 mL | |
| 10 mM | 0.4645 mL | 2.3227 mL | 4.6453 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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