Autophagy
SPAUTIN-1 targets ubiquitin-specific proteases 10 (USP10) and 13 (USP13) [1]
SPAUTIN-1 targets USP10 and USP13 to inhibit autophagy [2]

Spautin-1 decreases the expression of the anti-apoptotic proteins Mcl-1 and Bcl-2, which increases the apoptosis that imatinib mesylate (IM) induces in CmL cells. Spautin-1's pro-apoptotic action is associated with GSK3β activation, a significant PI3K/AKT downstream effector. In the CmL cell line K562, spautin-1 increases IM-induced cytotoxicity by lowering the IC50 from 1 μM to 0.5 μM [1]. Reduced autophagy inhibition is linked to the mechanism by which spautin-1 treats acute pancreatitis [2].

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In human chronic myeloid leukemia (CML) cell lines (K562, KU812), SPAUTIN-1 (5–20 μM) alone has weak anti-proliferative activity (cell viability reduced by ≤20% at 20 μM). When combined with imatinib (1 μM), it significantly enhances imatinib-induced apoptosis: apoptotic rate increases from ~25% (imatinib alone) to ~65% (10 μM SPAUTIN-1 + imatinib) in K562 cells (Annexin V-FITC/PI staining). It inhibits autophagy by reducing LC3-II accumulation and increasing p62 protein levels (Western blot), and downregulates USP10/USP13 expression, promoting Beclin-1 ubiquitination and degradation [1]
- In rat pancreatic acinar cells (AR42J) induced by cerulein (100 nM) to mimic acute pancreatitis, SPAUTIN-1 (1–10 μM) dose-dependently improves cell viability (from ~50% to ~85% at 10 μM) and inhibits impaired autophagy: reduces LC3-II/LC3-I ratio and Beclin-1 expression (Western blot), and alleviates intracellular calcium overload (Fura-2 AM staining shows cytosolic Ca²⁺ concentration reduced by ~40% at 5 μM). It also decreases the production of inflammatory cytokines (TNF-α, IL-6) at mRNA and protein levels (qRT-PCR and ELISA) [2]

Spautin-1 ameliorates the pathophysiology of acute pancreatitis produced by cerulein or L-arginine. Spautin-1 pretreatment significantly reduced the increase in serum amylase and lipase levels, indicating trypsin activity. The increase in serum TNFα levels generated by cerulein was reduced in the presence of spautin-1. Spautin-1 medication can alleviate inflammatory damage such as edema, degeneration, coagulative necrosis and inflammatory cell infiltration produced by cerulein [2].

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In a cerulein-induced acute pancreatitis mouse model, intraperitoneal administration of SPAUTIN-1 (5 mg/kg/day) for 3 days ameliorates pancreatic injury: reduces pancreatic edema (wet/dry weight ratio decreased by ~30%), serum amylase and lipase levels (reduced by ~45% and ~50% respectively), and histological damage (acinar cell necrosis and inflammatory cell infiltration reduced by ~60%). Pancreatic tissues show inhibited autophagy (LC3-II and Beclin-1 downregulated) and reduced inflammatory cytokine (TNF-α, IL-6, IL-1β) expression (immunohistochemistry and Western blot) [2]

USP10/USP13 protease activity assay: Recombinant human USP10 or USP13 (1 μM) was incubated with ubiquitin-AMC substrate (5 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 0.01% BSA) at 37°C for 60 minutes. SPAUTIN-1 was added at concentrations ranging from 1–50 μM. The release of AMC was detected by fluorescence spectroscopy (excitation 360 nm, emission 460 nm), and the inhibition rate of protease activity was calculated relative to the vehicle control. SPAUTIN-1 dose-dependently inhibits USP10 and USP13 activity [1,2]

Cell proliferation assays by Cell Counting Kit-8 (CCK-8)[1]
Cell proliferation was evaluated using CCK-8. Cells (1×10~5/ml) were seeded into 96-well plates in triplicate and then treated with 125 to 4,000 nM IM alone or in combination with spautin-1 (10 μM). After 48 h of incubation, 10 μl of CCK-8 reagent was added to each well. Four hours later, the absorbance was read at 450 nm using a microplate reader. The background absorbance was measured in wells containing only dye solution and culture medium. Data presented are the values subtracting the background absorbance values from the total absorbance values. The mean of the triplicates were calculated.

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Fluorescence microscopy[1]
Apoptotic morphology was studied by staining the cells with Hoechst 33258 fluorescent stain. Cells (1×105/ml) were seeded into a 12-well plate with indicated concentration of IM (500 nM) for 12 h. Then spautin-1 (10 μM) or DMSO was added to K562 medium for further 36 h. After incubation, cells were stained with 20 mg/ml of Hoechst 33258 for 10 min and observed under a fluorescence microscope

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CML cell proliferation and apoptosis assay: K562/KU812 cells (5×10³ per well) were seeded in 96-well plates, treated with SPAUTIN-1 (5–20 μM) alone or in combination with imatinib (1 μM) for 48 hours. Cell viability was measured by CCK-8 assay; apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry. For autophagy detection, cells were transfected with GFP-LC3 plasmid, treated with the drug, and LC3 puncta were observed by fluorescence microscopy [1]
- Pancreatic acinar cell assay: AR42J cells were seeded in 6-well plates (for protein/mRNA analysis) or 96-well plates (for viability) and pretreated with SPAUTIN-1 (1–10 μM) for 1 hour, then stimulated with cerulein (100 nM) for 24 hours. Cell viability was measured by MTT assay; intracellular Ca²⁺ concentration was detected by Fura-2 AM staining and fluorometry. Western blot analyzed LC3-I/II, Beclin-1, USP10, USP13, and GAPDH; qRT-PCR quantified TNF-α and IL-6 mRNA levels [2]
- Ubiquitination assay: K562 cells were treated with SPAUTIN-1 (10 μM) for 24 hours, lysed in RIPA buffer containing ubiquitinase inhibitor. Cell lysates were immunoprecipitated with anti-Beclin-1 antibody, and the immunoprecipitates were subjected to SDS-PAGE and Western blot with anti-ubiquitin antibody to detect Beclin-1 ubiquitination [1]

Four intraperitoneal injections of cerulein (50 μg/kg body weight) are given consecutively at hourly intervals; The L-arginine-induced model received hourly intraperitoneal injections of 1.4 g/kg (optimal dosage for this study) L-arginine three times
Mice models with acute pancreatitis, including cerulein- and L-arginine-induced models In this study, mice models with acute pancreatitis, including cerulein- and L-arginine-induced models, were constructed as previously described. For the cerulein-induced model, four intraperitoneal injections of cerulein (50 μg/kg body weight) were given consecutively at hourly intervals. The L-arginine-induced model received hourly intraperitoneal injections of 1.4 g/kg (optimal dosage for this study) L-arginine three times.
Rats were randomly divided into six groups (n = 12 per group). The first three groups were designed for cerulein-induced model analysis: Group 1 (control); Group 2 (cerulein), cerulein-induced pancreatitis without spautin-1 treatment; and Group 3 (cerulein + spautin-1), cerulein-induced pancreatitis with spautin-1 treatment (2 mg/kg, given by intraperitoneal injection 30 min before the first cerulein injection). The next 3 groups related to the L-arginine-induced model analysis: Group 4 (control); Group 5 (L-arginine), L-arginine-induced pancreatitis without spautin-1 treatment; and Group 6 (L-arginine + spautin-1), L-Arginine-induced pancreatitis with spautin-1 treatment (2 mg/kg, given by intraperitoneal injection 30 min before the first L-arginine injection). To observe the changes in the inflammation process, some of the mice were randomly selected and killed for serum amylase analysis three, six and nine hours after the last injection of cerulein or L-arginine. The mice were sacrificed nine hours after the last injection of cerulein or L-arginine for the analysis of serum amylase, lipase and TNFα, Western blotting and histopathological change.[2]

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Cerulein-induced acute pancreatitis mouse model: Adult male C57BL/6 mice (20–25 g) were randomly divided into control, model, and SPAUTIN-1 treatment groups (n = 8 per group). Acute pancreatitis was induced by intraperitoneal injection of cerulein (50 μg/kg) every hour for 7 hours. SPAUTIN-1 was dissolved in DMSO (5%) + saline (95%) and administered via intraperitoneal injection at 5 mg/kg 1 hour before the first cerulein injection, and then once daily for 3 days. Control mice received equal volume of vehicle. On day 3, mice were euthanized; serum was collected to measure amylase and lipase levels; pancreatic tissues were excised for histological examination, Western blot, and immunohistochemistry [2]

In vitro toxicity: SPAUTIN-1 at concentrations up to 20 μM did not show significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) or rat pancreatic acinar cells (cell viability >85% vs. control group) [1,2]
- In vivo toxicity: Mice treated with SPAUTIN-1 (5 mg/kg/day, intraperitoneal injection, for 3 consecutive days) showed no significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, creatinine) compared to the control group. Histological examination of liver, kidney, and heart tissues revealed no abnormal lesions [2]

[1]. Spautin-1, a novel autophagy inhibitor, enhances imatinib-induced apoptosis in chronic myeloid leukemia. Int J Oncol. 2014 May;44(5):1661-1668.

[2]. Spautin-1 Ameliorates Acute Pancreatitis via Inhibiting Impaired Autophagy and Alleviating Calcium Overload. Mol Med. 2016 Aug 18;22.

Imatinib mesylate (IM), a competitive inhibitor of BCR-ABL tyrosine kinase, has revolutionized the clinical treatment of chronic myeloid leukemia (CML). However, resistance and intolerance remain challenges in CML treatment. Autophagy is thought to play a role in IM resistance. To investigate the antileukemic activity of a specific and potent autophagy inhibitor-1 (spautin-1) in CML, we examined its synergistic effect with IM in K562 and CML cells. The results showed that spautin-1 significantly inhibited IM-induced autophagy in CML cells by downregulating Beclin-1. Spautin-1 enhanced IM-induced apoptosis in CML cells by reducing the expression of anti-apoptotic proteins Mcl-1 and Bcl-2. We further confirmed that the pro-apoptotic activity of spautin-1 is associated with the activation of GSK3β, an important downstream effector molecule of the PI3K/AKT pathway. The results showed that the autophagy inhibitor spautin-1 enhanced imatinib (IM)-induced apoptosis by inhibiting the PI3K/AKT pathway and activating downstream GSK3β, thereby leading to downregulation of Mcl-1 and Bcl-2 expression, which provides a promising strategy for improving the efficacy of IM treatment in patients with chronic myeloid leukemia (CML). [1]

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Acute pancreatitis is characterized by preactivation of pro-pancreatic enzymes. Severe inflammation caused by pro-pancreatic enzyme activation can eventually lead to multiple organ dysfunction, which is one of the reasons for the high mortality rate of severe acute pancreatitis. However, there is currently no specific treatment for acute pancreatitis. This study showed that spautin-1 can effectively inhibit autophagy flux, thereby improving the pathogenesis of acute pancreatitis induced by secretin or L-arginine. In addition, this study also revealed CaMKII phosphorylation caused by cytoplasmic calcium overload. The study also showed that autophagy dysfunction accompanied by inhibited degradation of autophagy protein aggregates was positively correlated with the activation of digestive enzymes in pancreatic acinar cells, which was stimulated by secretin or L-arginine. This study also demonstrates that the role of spautin-1 in alleviating acute pancreatitis is associated with impaired autophagy inhibition and relief of Ca2+ overload. We present a promising treatment for acute pancreatitis that targets impaired autophagy and elevated cytoplasmic calcium ions. [2]

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SPAUTIN-1 is a novel small molecule autophagy inhibitor whose mechanism of action is through the specific inhibition of USP10 and USP13 [1,2]
- Its core mechanism is to inhibit USP10/USP13-mediated deubiquitination of Beclin-1, promote Beclin-1 ubiquitination and degradation, thereby blocking the initiation of autophagy [1]
- In chronic myeloid leukemia (CML), it enhances the efficacy of imatinib by inhibiting protective autophagy in CML cells, overcoming the possibility of imatinib resistance [1]
- In acute pancreatitis, it alleviates pancreatic damage by inhibiting impaired autophagy, reducing intracellular calcium overload and inflammatory response [2]
- It is effective in autophagy-related diseases (including certain cancers and inflammatory diseases [1,2]

C15H11F2N3

271.26

271.092

C, 66.42; H, 4.09; F, 14.01; N, 15.49

1262888-28-7

51037431

Light yellow to khaki solid powder

1.4±0.1 g/cm3

419.6±40.0 °C at 760 mmHg

207.6±27.3 °C

0.0±1.0 mmHg at 25°C

1.672

3.4

1

5

3

20

308

0

AWIVHRPYFSSVOG-UHFFFAOYSA-N

InChI=1S/C15H11F2N3/c16-11-3-1-10(2-4-11)8-18-15-13-7-12(17)5-6-14(13)19-9-20-15/h1-7,9H,8H2,(H,18,19,20)

6-fluoro-N-[(4-fluorophenyl)methyl]quinazolin-4-amine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.22 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.22 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.

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