Description: TFEB activator 1 is a potent, orally bioactive, and mTOR-independent activator of TFEB with neuroprotective effects. TFEB activator 1 significantly promotes the nuclear translocation of Flag-TFEB with an EC50 of 2167 nM. TFEB activator 1 enhances autophagy without inhibiting the mTOR pathway and has the potential for neurodegenerative diseases treatment. Autophagy dysfunction is a common feature in neurodegenerative disorders characterized by accumulation of toxic protein aggregates. Increasing evidence has demonstrated that activation of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal biogenesis, can ameliorate neurotoxicity and rescue neurodegeneration in animal models. Currently known TFEB activators are mainly inhibitors of MTOR (mechanistic target of rapamycin [serine/threonine kinase]), which, as a master regulator of cell growth and metabolism, is involved in a wide range of biological functions. Thus, the identification of TFEB modulators acting without inhibiting the MTOR pathway would be preferred and probably less deleterious to cells. In this study, a synthesized curcumin derivative termed C1 is identified as a novel MTOR-independent activator of TFEB. Compound C1 specifically binds to TFEB at the N terminus and promotes TFEB nuclear translocation without inhibiting MTOR activity. By activating TFEB, C1 enhances autophagy and lysosome biogenesis in vitro and in vivo. Collectively, compound C1 is an orally effective activator of TFEB and is a potential therapeutic agent for the treatment of neurodegenerative diseases.
Physicochemical Properties
| Molecular Formula | C19H18O3 |
| Molecular Weight | 294.35 |
| Exact Mass | 294.126 |
| Elemental Analysis | C, 77.53; H, 6.16; O, 16.31 |
| CAS # | 39777-61-2 |
| PubChem CID | 830608 |
| Appearance | Light yellow to yellow solid |
| LogP | 3.999 |
| Hydrogen Bond Donor Count | 0 |
| Hydrogen Bond Acceptor Count | 3 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 22 |
| Complexity | 364 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | COC1=CC=CC=C1/C=C/C(=O)/C=C/C2=CC=CC=C2OC |
| InChi Key | RCZMPCUUTSDNAJ-PHEQNACWSA-N |
| InChi Code | InChI=1S/C19H18O3/c1-21-18-9-5-3-7-15(18)11-13-17(20)14-12-16-8-4-6-10-19(16)22-2/h3-14H,1-2H3/b13-11+,14-12+ |
| Chemical Name | (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one |
| Synonyms | TFEB activator 1; (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one; 39777-61-2; TFEB activator 1; 41973-42-6; Go-Y019; CHEMBL477053; RPN77612; 1,5-bis(2-methoxyphenyl)-1,4-pentadien-3-one; |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product is not stable in solution, please use freshly prepared working solution for optimal results. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Flag-TFEB nuclear translocation (EC50 = 2167 nM) |
| ln Vitro | TFEB activator 1 (compound C1) binds directly to regulatory factor EB (TFEB) to facilitate its cellular entrance into the nucleus without influencing TFEB phosphorylation or blocking MTOR and MAPK1/ERK2-MAPK3/ERK1 activity [1]. 1 (1 μM; 12 hours) markedly raises the levels of LC3B-II, a lipidated and autophagosome-associated or cell-killing form of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta). N2a Cellular TFEB activator 1 (0.2-1 μM) dose-dependently raises the levels of SQSTM1/p62 (sequestosome 1) and LC3-II in N2a cells [1]. |
| ln Vivo | The intermediate lethal dosage (LD50) of TFEB activator 1 (Compound C1) in an acute toxicity investigation (single dose, tail vein injection; dose) was 175 mg/kg [1]. Short-term wall TFEB activator 1 (low dosage 10 mg/kg and high dose 25 mg/kg; 24 hours) promotes elevated expression of LC3B-II and TFEB in heart, frontal myocardium and cerebral striatum [1]. Long-term administration of TFEB activator 1 (10 mg/kg per day; delivered via gavage) activates TFEB and increases autophagy in the brain center [1]. |
| Enzyme Assay |
High-content TFEB nuclear translocation assay[1] To quantify TFEB subcellular localization, a high-content assay was performed using stable HeLa cells overexpressing 3xFlag-TFEB according to our previous protocols. Isothermal titration calorimetry (ITC) binding assay[1] ITC experiments including C1 to FL-TFEB, C1 to ΔC150, C1 to ΔC461, C1 to Δ(121 to 330), curcumin to the FL-TFEB, B1 to FL-TFEB and E4 to FL-TFEB were carried out in a VP-ITC microcalorimeter. Protein and ligand solutions were prepared by dilution with the corresponding protein storage buffer containing 6% DMSO, to the required concentration. All solutions were thoroughly degased under vacuum for 5 min with gentle stirring before use. Typically, ligand solutions at 100 to 200 μM in a 250-μl syringe were titrated into 1.4 ml solution of the proteins at 10 to 20 μM in the sample cell, respectively. Titration trials were performed at 25°C with a reference cell power of 9. Injections of 10 to 14 μl of ligand solutions were performed at an interval of 120 to 150 s in to the protein solution with stirring at 307 rpm. Titration experiments of C1 to other truncations were performed using MicroCal-ITC 200. The 40-μl syringe and 200-μl cell were filled with 200 μM ligand solution and 10 to 20 μM protein solution, respectively. The experiments were carried out at 25°C. An initial injection of 0.1 μl was excluded for data analysis, followed by 16 injections of 2 μl each, separated by 120 s. The raw data were processed using the one binding site model in the MicroCal ORIGIN software. Prior to analysis, data were corrected by subtracting the dilution heat of the ligands. |
| Cell Assay |
Western Blot Analysis[1] Cell Types: N2a. Cell Tested Concentrations: 0, 0.2, 0.4, 0.6, 0.8 and 1 μM Incubation Duration: 12 hrs (hours) Experimental Results: Treatment dose-dependently increased the levels of LC3-II and SQSTM1/p62 (dotosome 1). |
| Animal Protocol |
Animal/Disease Models: Adult male SD (SD (Sprague-Dawley)) rat, body weight 350 to 400 g[1] Doses: 10 mg/kg Route of Administration: Chronic oral administration; daily; for 21 days Experimental Results: TFEB in rat brain is activated and autophagy is enhanced. All animal care and experimental procedures were approved by the Hong Kong Baptist University Committee on the Use of Human and Animal Subjects in Teaching and Research. Adult male Sprague-Dawley (SD) rats weighing 350 to 400 g were maintained on ad libitum food and water with a 12-h light/dark cycle in a controlled environment. Rats (n = 6 per group) were orally administered by gavage with C1 (10 mg/kg and 25 mg/kg per day) or vehicle (1% sodium carbonyl methylcellulose [CMC-Na]) for 24 h. At the end of the treatment, an additional dosage of C1 was given for 6 h before the rats were killed. Livers and major brain regions dissected according to the previous protocol were snap-frozen in liquid nitrogen. |
| References |
[1]. A novel curcumin analog binds to and activates TFEB in vitro and in vivo independent of MTOR inhibition. Autophagy. 2016 Aug 2;12(8):1372-89. |
| Additional Infomation | Autophagy dysfunction is a common feature in neurodegenerative disorders characterized by accumulation of toxic protein aggregates. Increasing evidence has demonstrated that activation of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal biogenesis, can ameliorate neurotoxicity and rescue neurodegeneration in animal models. Currently known TFEB activators are mainly inhibitors of MTOR (mechanistic target of rapamycin [serine/threonine kinase]), which, as a master regulator of cell growth and metabolism, is involved in a wide range of biological functions. Thus, the identification of TFEB modulators acting without inhibiting the MTOR pathway would be preferred and probably less deleterious to cells. In this study, a synthesized curcumin derivative termed C1 is identified as a novel MTOR-independent activator of TFEB. Compound C1 specifically binds to TFEB at the N terminus and promotes TFEB nuclear translocation without inhibiting MTOR activity. By activating TFEB, C1 enhances autophagy and lysosome biogenesis in vitro and in vivo. Collectively, compound C1 is an orally effective activator of TFEB and is a potential therapeutic agent for the treatment of neurodegenerative diseases.[1] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~125 mg/mL (~424.68 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 6.25 mg/mL (21.23 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly. Solubility in Formulation 2: 4 mg/mL (13.59 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3973 mL | 16.9866 mL | 33.9732 mL | |
| 5 mM | 0.6795 mL | 3.3973 mL | 6.7946 mL | |
| 10 mM | 0.3397 mL | 1.6987 mL | 3.3973 mL |