Flag-TFEB nuclear translocation (EC50 = 2167 nM)
curcumin analog C1 directly binds to TFEB (transcription factor EB) at the N-terminal Gly and Ala-rich domain. The binding affinity (K_D) of C1 to full-length recombinant TFEB is 2.53 ± 0.33 μM (determined by isothermal titration calorimetry). C1 also binds to TFEB truncations Δ(121-330) (K_D = 2.17 μM), ΔC150 (K_D = 1.12 μM), and ΔC461 (K_D = 1.81 μM). No binding was detected for TFEB truncations 221-320, ΔN220, or ΔN44. [1]

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].

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curcumin analog C1 (1 μM, 12 h) significantly increased LC3B-II levels in mouse neuroblastoma N2a cells compared to vehicle control; this effect was further enhanced in the presence of chloroquine (20 μM), indicating enhanced autophagic flux rather than blocked lysosomal degradation. C1 did not inhibit MTOR pathway; instead, it promoted phosphorylation of RPS6KB1 and MTOR. C1 treatment (1 μM, 12 h) induced TFEB nuclear translocation in >80% of N2a cells, and in HeLa cells stably expressing 3xFlag-TFEB, C1 promoted nuclear translocation with an EC₅₀ value of 2167 nM. C1 did not affect total phosphoserine levels on TFEB nor phosphorylation of S142 and S211, and did not inhibit MAPK1/3 activity. C1 directly bound to recombinant TFEB (K_D = 2.53 μM). C1 reduced the interaction between TFEB and YWHA/14-3-3 as shown by co-immunoprecipitation. C1 (0.5-1 μM, 12 h) increased protein levels of LC3B-II, TFEB, LAMP1, and CTSD (both precursor 46 kDa and mature 28 kDa forms) in N2a and HeLa cells. C1 (1 μM, 12 h) upregulated transcription of TFEB target genes (e.g., LC3B, LAMP1, CTSD, SQSTM1) in HeLa and SH-SY5Y cells. C1 enhanced autophagic degradation of SQSTM1 in cycloheximide chase assay and degraded exogenous Flag-SQSTM1, which was blocked by chloroquine. In N2a cells transfected with tandem fluorescent mRFP-GFP-LC3, C1 significantly increased the number of red-only puncta (autolysosomes). C1 promoted formation of DQ-BSA red puncta colocalized with LC3B puncta, indicating increased lysosomal proteolytic activity. Knockdown of Tfeb or Becn1 abolished C1-induced LC3B-II increase, while Atg5 knockdown partially reduced but did not completely block the effect. [1]

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].

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Oral administration of curcumin analog C1 (10 mg/kg and 25 mg/kg for 24 h, with an additional dose 6 h before sacrifice) dose-dependently increased LC3B-II and TFEB protein levels in rat frontal cortex, striatum, and liver. C1 treatment (25 mg/kg) significantly increased phosphorylation of MTOR and RPS6KB1 in the frontal cortex, confirming that C1 promotes MTOR activity. C1 dose-dependently promoted TFEB nuclear translocation in the brain. C1 (25 mg/kg) inhibited the interaction between endogenous TFEB and MTOR in the frontal cortex. Chronic oral administration of C1 (10 mg/kg per day for 21 days, with an additional dose 6 h before sacrifice) increased TFEB and LC3B-II levels, and significantly decreased endogenous SQSTM1 levels in rat frontal cortex and striatum, indicating enhanced autophagy-mediated degradation. No changes in body weight or behavioral abnormalities were observed during chronic treatment. Histological evaluation revealed no morphological abnormalities in major organs (liver, lung, kidneys, pancreas). [1]

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.

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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.

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Solid-phase binding assay: Recombinant His-tagged TFEB was incubated with C1 or curcumin. After centrifugation, a specific His-TFEB band at ~55 kDa was observed only in the pellet of C1, indicating direct binding of C1 to TFEB. [1]
Isothermal titration calorimetry (ITC) binding assay: ITC experiments were carried out in a VP-ITC microcalorimeter or MicroCal-ITC 200. Protein and ligand solutions were diluted with protein storage buffer containing 6% DMSO and degassed under vacuum for 5 min. Typically, ligand solutions at 100-200 μM in a syringe were titrated into 1.4 mL of protein solutions at 10-20 μM in the sample cell. Titrations were performed at 25°C with a reference cell power of 9. Injections of 10-14 μL of ligand were made at intervals of 120-150 s with stirring at 307 rpm. For MicroCal-ITC 200, 200 μL of ligand (200 μM) and 200 μL of protein (10-20 μM) were used, with an initial 0.1 μL injection excluded, followed by 16 injections of 2 μL each at 120 s intervals. Raw data were processed using a one-site binding model in MicroCal ORIGIN software, corrected by subtracting ligand dilution heat. The dissociation constant (K_D) of C1 to full-length TFEB was 2.53 ± 0.33 μM, with n = 0.99 ± 0.03, ΔH = 5.09 ± 0.24 kcal/mol, ΔS = 42.7 cal/mol/K. C1 also bound to TFEB truncations Δ(121-330) (K_D = 2.17 μM), ΔC150 (K_D = 1.12 μM), and ΔC461 (K_D = 1.81 μM), but not to 221-320, ΔN220, or ΔN44. Curcumin, B1, and E4 showed weak or no binding. [1]
Co-immunoprecipitation (Co-IP) for TFEB-YWHA/MTOR interaction: HeLa cells stably expressing 3xFlag-TFEB were treated with C1 (1 μM, 12 h). Anti-Flag antibody was added to whole cell lysates and Dynabeads Protein G was used for immunoprecipitation. Immunoprecipitated proteins were separated by SDS-PAGE and blotted with anti-MTOR or anti-YWHA antibodies. C1 treatment significantly decreased the levels of YWHA and MTOR co-immunoprecipitated with Flag-TFEB compared to control, indicating reduced TFEB-YWHA and TFEB-MTOR interactions. [1]

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).

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Cell culture and drug treatment: N2a and HeLa cells were cultured in DMEM with 10% FBS; SH-SY5Y cells in DMEM/F12 with 10% FBS. HeLa cells stably expressing 3xFlag-TFEB were maintained in DMEM with 10% FBS and G418. For drug treatment, medium was replaced with fresh Opti-MEM I containing compounds (in 0.1% DMSO) for indicated times. [1]
LDH cytotoxicity assay: Cytotoxicity was determined by measurement of LDH release from damaged cells using an LDH kit according to the manufacturer's protocol. The tested compounds were nontoxic at 1 μM. [1]
High-content TFEB nuclear translocation assay: HeLa cells stably overexpressing 3xFlag-TFEB were used. Cells were treated with compounds, fixed, stained, and imaged. Nuclear translocation was quantified to calculate EC₅₀ value (2167 nM for C1). [1]
Western blotting: Cells were lysed in 1x lysis buffer with protease and phosphatase inhibitors. For SQSTM1 detection, 1% SDS was added to lysis buffer. Proteins were separated by 10-15% SDS-PAGE, transferred, and blotted with indicated antibodies. Protein signals were detected by ECL and quantified using ImageJ software. [1]
Immunoprecipitation: Anti-Flag or anti-TFEB antibody was added to whole cell lysates and Dynabeads Protein G was used for immunoprecipitation. Clean-Blot IP Detection Reagent was used for secondary detection. [1]
Quantitative real-time PCR: Total RNA was extracted using RNeasy Plus Mini Kit. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit. Real-time PCR was carried out with Fast SYBR Green Master Mix using a ViiA 7 Real-Time PCR System. Fold changes were calculated using the ΔΔCT method, normalized to GAPDH or ACTB. [1]
Gene knockdown assay: N2a cells were transfected with mouse Atg5, Becn1, or Tfeb siRNA (25 nM or 50 nM) and nontarget siRNA using Lipofectamine RNAi-MAX for 48 h, then treated with C1 for 12 h. For stable TFEB knockdown, HeLa cells were infected with lentivirus expressing nontarget shRNA or TFEB shRNA for 48 h, then selected with puromycin. [1]
Autophagy flux assay (tandem fluorescent mRFP-GFP-LC3): N2a cells were transfected with tflC3 plasmid for 24 h, then treated with C1 (1 μM, 12 h), chloroquine (50 μM, 12 h), or torin1 (0.5 μM, 3 h). Cells were fixed, stained with DAPI, and imaged. The number of red-only puncta per cell was quantified (n=20 randomly selected cells from 3 independent experiments). C1 significantly increased red-only puncta compared to control. [1]
Lysosomal degradation assay (DQ-BSA): N2a cells were preincubated with DQ-BSA-Red (10 μg/mL) for 1 h, washed, then treated with C1 (1 μM, 12 h), chloroquine (50 μM, 12 h), or torin1 (0.5 μM, 3 h). Cells were fixed and stained with LC3B antibody. The number of DQ-BSA red puncta per cell and colocalization with LC3B puncta (Pearson correlation coefficient) were quantified. C1 promoted formation of DQ-BSA red puncta colocalized with LC3B. [1]
Immunocytochemistry: Cells on coverslips were fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% BSA, then stained with anti-TFEB, anti-Flag, or anti-LC3B antibodies overnight at 4°C, followed by Alexa Fluor secondary antibodies for 1 h at room temperature. Nuclei were stained with DAPI. Cells were visualized using fluorescence microscope or deconvolution microscope. [1]

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.

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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.

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Acute toxicity study: Rats received single-dose intravenous tail vein injection of C1 to determine median lethal dose (LD₅₀ = 175 mg/kg). [1]
Short-term oral administration (24 h): Adult male Sprague-Dawley rats (350-400 g, n=6 per group) were orally administered by gavage with C1 (10 mg/kg and 25 mg/kg per day) or vehicle (1% sodium carboxymethylcellulose, CMC-Na) for 24 h. An additional dose of C1 was given 6 h before sacrifice. Livers and brain regions (frontal cortex, striatum) were dissected and snap-frozen in liquid nitrogen. [1]
Chronic oral administration (21 days): Rats (n=6 per group) were orally administered by gavage with C1 (10 mg/kg per day) or vehicle (1% CMC-Na) for 21 days. An additional dose of C1 was given 6 h before sacrifice. Brain tissues were collected for Western blot and histological analysis. [1]
Tissue processing for Western blot and immunohistochemistry: Animal tissues were homogenized in 9 volumes of ice-cold PBS supplemented with protease inhibitors. Cytosolic and nuclear fractions were isolated using standard protocols. For determining SQSTM1 levels, 1% SDS was added to lysis buffer. [1]
Real-time PCR from brain tissues: Total RNA was extracted from brain lysates, reverse transcribed, and analyzed using specific primers for TFEB, LC3B, LAMP1, CTSD, and other autophagy/lysosomal genes. [1]

Blood-brain barrier (BBB) permeability: curcumin analog C1 could pass the BBB. After oral administration of C1 (10 mg/kg) for 6 h, the average concentration in brain tissues was 0.26 ± 0.063 μg/g (equivalent to 0.885 ± 0.213 μM) as determined by HPLC. [1]
Brain concentration after chronic dosing: After oral administration of C1 (10 mg/kg per day) for 21 days, with an additional dose given 6 h before sacrifice, the average concentration in brain tissues was 0.849 ± 0.302 μg/g (equivalent to 2.884 ± 1.028 μM). [1]

Acute toxicity (LD₅₀): Single-dose intravenous tail vein injection of curcumin analog C1 in rats gave a median lethal dose (LD₅₀) of 175 mg/kg. [1]
Sub-chronic toxicity: During chronic oral administration of C1 (10 mg/kg per day for 21 days), no changes in body weight and no behavioral abnormalities were observed. Histological evaluation revealed no morphological abnormalities in major organs such as liver, lung, kidneys, and pancreas. [1]

[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.

Autophagy dysfunction is a common feature of neurodegenerative diseases, characterized by the accumulation of toxic protein aggregates. Increasing evidence suggests that TFEB (transcription factor EB) is a key regulator of autophagy and lysosomal biosynthesis, and its activation can improve neurotoxicity and rescue neurodegeneration in animal models. Currently known TFEB activators are mainly inhibitors of mTOR (target of rapamycin [serine/threonine kinase]), which is a key regulator of cell growth and metabolism and participates in a variety of biological functions. Therefore, it would be more advantageous to find regulators that do not inhibit the mTOR pathway and act on TFEB, and may cause less damage to cells. In this study, a synthetic curcumin derivative C1 was identified as a novel mTOR-independent TFEB activator. Compound C1 specifically binds to the N-terminus of TFEB, promotes TFEB nuclear translocation, and does not inhibit mTOR activity. C1 enhances autophagy and lysosomal biosynthesis in vitro and in vivo by activating TFEB. In summary, compound C1 is an orally effective TFEB activator and a potential therapeutic agent for the treatment of neurodegenerative diseases. [1]

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curcumin analog C1 is also referred to as B63 in a previous study (Xiao et al., 2012). It is a monocarbonyl analog of curcumin lacking the β-diketone moiety, which exhibits enhanced stability and improved pharmacokinetic profiles compared to curcumin. C1 shows better cellular uptake and delayed degradation than curcumin. C1 treatment increases TFEB protein levels, likely through positive autoregulatory transcription after nuclear translocation. C1 binds to TFEB with endothermic, entropy-driven interaction (positive ΔS and ΔH values), indicating hydrophobic interaction and release of bound water. The binding site is localized within the N-terminal Gly and Ala-rich domain (first 44 amino acids). C1 is an orally effective TFEB activator with good brain penetration and low toxicity, making it a potential therapeutic agent for neurodegenerative diseases such as Parkinson disease, Alzheimer disease, and Huntington disease. [1]

C19H18O3

294.35

294.126

C, 77.53; H, 6.16; O, 16.31

39777-61-2

830608

Light yellow to yellow solid

3.999

0

3

6

22

364

0

COC1=CC=CC=C1/C=C/C(=O)/C=C/C2=CC=CC=C2OC

RCZMPCUUTSDNAJ-PHEQNACWSA-N

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+

(1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one

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;

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~424.68 mM)

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.

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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.

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