mTOR; FKBP1A
FKBP1A (FK506-binding protein 1A, 12 kDa). 3BDO binds to the same sites (TYR82A and ILE56A) on FKBP1A as rapamycin. [1]
MTOR (mechanistic target of rapamycin). 3BDO activates MTOR signaling pathway. [1][2]

3BDO inhibits autophagy in human umbilical vein endothelial cells (HUVECs) and neuronal cells. By reducing the levels of the tumor proteins TP53 and NUPR1, TP53 nuclear translocation, and the production of excessive reactive oxygen species, it prevents lipopolysaccharide-induced HUVEC autophagic injury. FKBP1A (FK506-binding protein 1A, 12 kDa), which is the target of 3BDO, stimulates MTOR. While having no effect on the expression of TGFB2, 3BDO significantly reduces the level of FLJ11812, a long noncoding RNA (lncRNA) derived from the 3′ untranslated region (3′UTR) of TGFB2. In addition to FLJ11812 level decline caused by 3BDO, ATG13 protein level also declines. In PC12 neuronal cells, 3BDO inhibits excessive A (25 to 35) peptide-induced autophagy and elevates RPS6KB1 phosphorylation[1]. Human umbilical vein EC (HUVEC) senescence and apoptosis brought on by basic fibroblast growth factor 2 and serum deprivation could be prevented by 3BDO. It prevents the proliferation and migration of vascular smooth muscle cells (VSMCs) while protecting vascular ECs (VECs) specifically[2]. Inhibiting VEC apoptosis and suppressing integrin 4 expression were both possible with 3BDO (20–60 g/ml), but it was unable to reduce the level of ROS brought on by the lack of serum and FGF-2[3].

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In human umbilical vein endothelial cells (HUVECs), 3BDO (60 μM, 24 h) increased the phosphorylation of MTOR substrates RPS6KB1 and EIF4EBP1. This effect was suppressed by FKBP1A overexpression. [1]
In HUVECs, 3BDO (60 μM, 30 min pretreatment) antagonized the effect of rapamycin (10 μM, 6 h), reversing the rapamycin-induced decrease in phosphorylation of MTOR (Ser2448) and RPS6KB1 (Thr389). [1]
3BDO (60 μM, 30 min pretreatment) suppressed the increase in MAP1LC3B puncta, increased MAP1LC3B-II protein level, and decreased SQSTM1 protein level induced by rapamycin (10 μM, 6 h) in HUVECs, indicating inhibition of autophagy. [1]
3BDO (60 μM, 6 h) increased the phosphorylation of Ser residues on TIA1 protein in HUVECs, reversing the decrease induced by rapamycin (10 μM). [1]
In HUVECs, 3BDO (60 μM, 24 h) significantly downregulated the level of lncRNA FLJ11812, as shown by microarray (most downregulated transcript, >2-fold change) and RT-PCR. This effect was dose- and time-dependent (24 h with various concentrations; 120 μM for various times). 3BDO did not affect the mRNA or protein levels of TGFB2. [1]
3BDO (120 μM, 10 h) decreased the interaction between TIA1 and the 3'UTR of TGFB2 in RNA-ChIP assay. [1]
Overexpression of FLJ11812 reversed the inhibition of autophagy induced by 3BDO in HUVECs, suggesting the effect of 3BDO on autophagy suppression is mediated by FLJ11812. [1]
In HUVECs, 3BDO (7.5, 15, 30, 60, 120 μM, 30 min pretreatment) inhibited the rapamycin (10 μM)-induced decrease in ATG13 phosphorylation (p-ATG13, Ser318) and increase in total ATG13 protein level in a dose-dependent manner. [1]
In HUVECs, 3BDO (60 or 120 μM, 12 h) inhibited oxLDL (50 μg/ml, 12 h)-induced autophagy, as shown by: decreasing the protein level of ATG13 and increasing its phosphorylation (p-ATG13/ATG13 ratio); decreasing the MAP1LC3B-II level and increasing the SQSTM1 level (with 60 and 120 μM). [2]
In HUVECs, 3BDO (60 or 120 μM, 12 h) inhibited oxLDL (50 μg/ml)-induced upregulation of ICAM-1 and VCAM-1 protein levels, and inhibited the secretion of IL-6 and IL-8. [2]
In HUVECs, 3BDO (20-60 μg/ml) inhibited apoptosis induced by deprivation of serum and FGF-2, as shown by improved cell morphology, increased cell viability (MTT assay), reduced nuclear DNA condensation/fragmentation (acridine orange staining), and decreased percentage of apoptotic cells (TUNEL assay, reduced from 30.89±9.29% to 9.89±5.66% with 40 μg/ml for 24 h). [3]
In VECs deprived of serum and FGF-2, 3BDO (40 μg/ml, 24 h) significantly depressed the increased expression of integrin β4 but had no effect on the elevated intracellular ROS level. [3]
In RAW246.7 macrophages and vascular smooth muscle cells (VSMCs) from apoE-/- mice, 3BDO did not affect oxLDL-induced changes in the phosphorylation of p70S6K and 4EBP1, nor the levels of LC3-II and ATG13, indicating cell-type-specific activity on VECs. [2]

In vivo tests revealed that 3BDO had a high level of safety. In App and Psen1 transgenic mice, 3BDO treatment could significantly lower the number of autophagosomes and enhance neuronal function[1]. In the plaque endothelium of apoE-/- mice, 3BDO decreased the protein level of ATG13 while activating mTOR in vivo. Although it had no effect on the activity of mTOR or autophagy in the vascular smooth muscle cells of apoE-/- mice or the macrophage cell line RAW246.7, it did suppress plaque endothelial cell death and restrain the development of atherosclerosis in the mice. In apoE-/- mice, 3BDO stabilized atherosclerotic lesions by protecting VECs by activating mTOR[2].

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In App and Psen1 transgenic mice, 3BDO treatment significantly reduced the number of autophagosomes and improved neuronal function. [1]
In apoE-/- mice fed an atherogenic diet, 3BDO (50 or 100 mg/kg/d, injection for 8 weeks) activated endothelial mTOR, as shown by increased p-p70S6K level in plaque endothelium. [2]
In apoE-/- mice, 3BDO (50 or 100 mg/kg/d, 8 weeks) decreased the protein level of ATG13 in the plaque endothelium, confirming inhibition of endothelial autophagy. [2]
In apoE-/- mice, 3BDO (100 mg/kg/d) treatment resulted in decreased endothelium autophagy (fewer LC3 dots by en face staining) and decreased apoptosis (fewer TUNEL-positive cells) in plaque endothelium. [2]
In apoE-/- mice, 3BDO (100 mg/kg/d, 8 weeks) significantly decreased atherosclerotic lesion areas (Oil-red O staining of whole aortas and H&E staining of aortic roots), promoted plaque stability (decreased lipid deposition, increased SMCs, reduced macrophage area and MMP-2/9 activity), and decreased serum levels of IL-6 and IL-8. [2]

Using IP lysis buffer, total protein is extracted from HUVECs that have been exposed to rapamycin (10 μM), 3BDO (60 M), or both for 6 hours. The supernatant is collected after centrifuging at 4°C, and it is then incubated with protein A/G agarose beads, TIA1 antibody, or regular mouse IgG as a control at 4°C overnight. The beads are cleaned with IP lysis buffer three times before being eluted with 4×SDS loading buffer. Using a Ser phosphorylation antibody and a western blot assay, Ser phosphorylation can be found.

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Molecular docking was performed using SYBYL software. The three-dimensional coordinates of the FKBP1A structure (PBD ID: 2PPN; 1FAP) were obtained from the Protein Data Bank. 3BDO was docked to the pocket site of FKBP1A to examine possible binding modes. Docking conformations were ranked according to the CScore, and the best-ranked results (CScore 5) were checked visually. The results predicted that 3BDO could form hydrogen bonds with TYR82A and ILE56A sites in FKBP1A, the two amino acid sites for rapamycin binding. [1]

HUVECs were isolated from umbilical cords and cultured in M199 medium with 20% fetal bovine serum and 10 IU/mL fibroblast growth factor 2. For experiments, cells up to passage 10 were used. Cells were grown to 80% confluency before treatment. [1][2]
For Western blot analysis, HUVECs were lysed in lysis buffer (25 mM Tris-HCl pH 6.8, 2% SDS, 6% glycerol, 1% 2-mercaptoethanol, 2 mM PMSF, 0.2% bromophenol blue, and a protease inhibitor cocktail). Equal amounts of protein (15 μg) were separated by 15% SDS-PAGE, transferred to PVDF membranes, blocked with 5% nonfat dry milk, incubated with primary antibodies (1:1000), then with HRP-conjugated secondary antibodies (1:5000). Bands were detected by enhanced chemiluminescence. [1]
For immunofluorescence assay, HUVECs were fixed with 4% paraformaldehyde, blocked with 3% normal goat serum, incubated with primary antibody (e.g., anti-MAP1LC3B) at 4°C overnight, then with FITC-conjugated secondary antibody (1:200). Cells were evaluated by confocal microscopy. [1][2]
For TIA1 phosphorylation detection, total protein was obtained using IP lysis buffer. After centrifugation, the supernatant was incubated with protein A/G agarose beads and TIA1 antibody at 4°C overnight. Beads were washed and eluted. Ser phosphorylation was detected by western blot with a Ser phosphorylation antibody. [1]
For RNA interference (TIA1 or FLJ11812 siRNA), cells at 80% confluence were transfected with siRNA (40 nM) using a transfection reagent. After 24 h, the medium was substituted with normal medium, and cells were cultured for another 24 h for assays. [1]
For transient overexpression, HUVECs were transfected with pCMV6-FLJ11812, pCMV6-XL5 (control), or FKBP1A-GFP vectors using Lipofectamine 2000 for 4-6 h, then cultured for 24 or 48 h before treatment. [1]
For luciferase reporter assay, HEK293 cells were seeded in 96-well plates. Cells were cotransfected with luciferase reporter constructs (e.g., Luc-FLJ11812-WT or Luc-ATG13-WT) and MIR4459 mimics (10 or 40 nM) using Lipofectamine 2000. After 24 h, firefly and Renilla luciferase activities were measured using a luminometer. [1]
To measure cell viability (MTT assay), VECs were treated, and MTT solution was added. The formazan crystals were dissolved, and absorbance was measured. [3]
Apoptosis was quantified by TUNEL assay. Cells were fixed and stained with a TUNEL kit, and the percentage of apoptotic cells was counted. Nuclear morphology was assessed by acridine orange staining. [3]
For ROS detection, cells were loaded with the fluorescent probe DCHF, which is oxidized to fluorescent DCF by intracellular ROS. Fluorescence intensity was measured. [3]

Male apoE-/- mice (8 weeks old) are used in this study. ApoE-/- mice are fed an atherogenic diet that contains 21% fat and 0.15% cholesterol. A single batch of the diet is set aside and used throughout the experiment to avoid any potential confounding effects from variation among diet batches. Mice that are 20 weeks older are divided into 3 groups for treatment (n=8 mice/group) for 8 weeks: control (DMSO), low-dose (3BDO; 50 mg/kg/d; 3BDO-L), and high-dose (3BDO; 100 mg/kg/d; 3BDO-H). Every week, when the mice are being injected with 3BDO, their body weight is recorded. Animals are killed by exsanguination and blood samples are taken from the inferior vena cava[2].

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For the atherosclerosis model, 8-week-old male apoE-/- mice (C57BL/6J-background) were fed an atherogenic diet (21% fat, 0.15% cholesterol). At 20 weeks of age, mice were divided into groups (n=8/group) for an 8-week treatment period: control (DMSO), low-dose 3BDO (50 mg/kg/d), and high-dose 3BDO (100 mg/kg/d). The agent was administered by injection. [2]
For the Alzheimer's disease model, App and Psen1 transgenic mice were used. 3BDO was administered, and its effect on autophagosomes and neuronal function was assessed (detailed regimen not specified in the provided text). [1]
After treatment, animals were sacrificed by exsanguination. Blood was collected from the inferior vena cava to obtain serum. Hearts and whole aortas were extracted. Aortic roots were embedded in OCT compound for histology. The remaining aorta was opened longitudinally, fixed, and stained with Oil-red O for en face lesion analysis. [2]

3BDO had a good safety profile in vivo (in App and Psen1 transgenic mice). [1]
In apoE-/- mice treated with 3BDO (50 or 100 mg/kg/d for 8 weeks), there was no significant difference in body weight or the weights of various organs (heart, liver, spleen, lungs, kidney, brain) between control and treated groups. [2]
3BDO inhibited the inflammatory response in oxLDL-treated HUVECs and in apoE-/- mice (decreased serum IL-6 and IL-8). [2]

[1]. Autophagy. 2014 Jun;10(6):957-71.

[2]. Sci Rep. 2014 Jul 1;4:5519. doi: 10.1038/srep05519.

[3]. Bioorg Med Chem Lett. 2007 Jan 15;17(2):482-5.

3BDO (3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one) is a butyrolactone derivative. Prior to this study, no chemical activators of MTOR had been found. 3BDO is identified as a novel MTOR activator that acts as an antagonist of rapamycin. [1]
3BDO was found to inhibit chloroquine-induced and lipopolysaccharide-induced autophagic injury in HUVECs. It also inhibited excessive Aβ peptide-induced autophagy in PC12 neuronal cells. [1]
The study discovered that 3BDO decreases the level of a lncRNA (FLJ11812) derived from the TGFB2 3'UTR, which acts as a competing endogenous RNA (ceRNA) for MIR4459, thereby regulating the expression of ATG13 and autophagy. [1]
In the context of atherosclerosis, the study proposes that activating VEC mTOR with 3BDO is a novel strategy for treatment. 3BDO selectively protects endothelial cells by modulating mTOR-dependent autophagy, without affecting macrophages or smooth muscle cells. [2]
3BDO inhibited VEC apoptosis induced by deprivation of serum and FGF-2, an effect linked to the downregulation of integrin β4 expression. Its anti-apoptotic effect was independent of ROS levels. [3]

C18H17NO5

327.3313

327.11

C, 62.60; H, 5.55; N, 4.06; O, 27.80

890405-51-3

890405-51-3

16216349

Off-white to light yellow solid powder

1.3±0.1 g/cm3

540.0±20.0 °C at 760 mmHg

233.2±23.8 °C

0.0±1.4 mmHg at 25°C

1.594

2.56

0

5

5

24

443

0

C1(=C(C=CC=C1)OCC1CC(CC2C=CC=CC=2)C(=O)O1)N(=O)=O

AXPZIVKEZRHGAS-UHFFFAOYSA-N

InChI=1S/C18H17NO5/c20-18-14(10-13-6-2-1-3-7-13)11-15(24-18)12-23-17-9-5-4-8-16(17)19(21)22/h1-9,14-15H,10-12H2

3-benzyl-5-[(2-nitrophenoxy)methyl]oxolan-2-one

3BDO

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)

DMSO: 65~100 mg/mL (198.6~305.5 mM)
Ethanol: ~19.7 mg/mL (~60.2 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.64 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 (7.64 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.64 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.)