SIRT2 (IC50 = 10 μM); SIRT1 (IC50 = 21 μM); SIRT3 (IC50 = 67 μM); HDAC8; MDM-2/p53
Tenovin-6 Hydrochloride is more toxic to yeast than the less water-soluble tenovin-1, with an IC50 of 30 M for inhibiting the growth of S. cerevisiae cultures. In MCF-7 cells, endogenous K382-Ac p53 levels are quickly elevated by tenovin-6 hydrochloride[1].
Tenovin-6 Hydrochloride (0 to 15 μM) dose dependently increases the level of LC3-II in diverse cell types, and the increase is ATG5/7 dependent. In addition, the use of tenovin-6 hydrochloride prevents SQSTM1/p62 degradation caused by torin 1 and boosts the quantity and intensity of autophagic vesicles both with and without torin 1. The fusion between autophagosomes and lysosomes is unaffected by tenovin-6 hydrochloride, but it affects the acidification of autolysosomes and hinders lysosome hydrolytic activity. Tenovin-6 Hydrochloride inhibits autophagy, but this is unrelated to p53 activation, and inhibiting SIRT1/2 either through knockdown or knockout cannot mimic Tenovin-6 Hydrochloride's effect on LC3B accumulation[3].
Tenovin-6 Hydrochloride (0, 1, 2.5, 5 or 10 μM) potently inhibits cell proliferation in a dose- and time-dependent manner in all OCI-Ly1, DHL-10, U2932, RIVA, HBL1 and OCI-Ly10 cell lines. Tenovin-6 Hydrochloride consistently raises the level of LC3B-II in DLBCL cell lines by blocking the traditional autophagy pathway without turning on p53, and the rise is unrelated to SIRT1/2/3 or p53. Through the extrinsic cell-death pathway, tenovin-6 hydrochloride causes apoptosis[4].
Tenovin-6 Hydrochloride inhibits UM cell growth with IC50 values of 12.8 μM, 11.0 μM, 14.58 μM and 9.62 μM for 92.1, Mel 270, Omm 1, and Omm 2.3 cells, respectively[5].

Tenovin-6 Hydrochloride is more toxic to yeast than the less water-soluble tenovin-1, with an IC50 of 30 M for inhibiting the growth of S. cerevisiae cultures. In MCF-7 cells, endogenous K382-Ac p53 levels are quickly elevated by tenovin-6 hydrochloride[1].
Tenovin-6 Hydrochloride (0 to 15 μM) dose dependently increases the level of LC3-II in diverse cell types, and the increase is ATG5/7 dependent. In addition, the use of tenovin-6 hydrochloride prevents SQSTM1/p62 degradation caused by torin 1 and boosts the quantity and intensity of autophagic vesicles both with and without torin 1. The fusion between autophagosomes and lysosomes is unaffected by tenovin-6 hydrochloride, but it affects the acidification of autolysosomes and hinders lysosome hydrolytic activity. Tenovin-6 Hydrochloride inhibits autophagy, but this is unrelated to p53 activation, and inhibiting SIRT1/2 either through knockdown or knockout cannot mimic Tenovin-6 Hydrochloride's effect on LC3B accumulation[3].
Tenovin-6 Hydrochloride (0, 1, 2.5, 5 or 10 μM) potently inhibits cell proliferation in a dose- and time-dependent manner in all OCI-Ly1, DHL-10, U2932, RIVA, HBL1 and OCI-Ly10 cell lines. Tenovin-6 Hydrochloride consistently raises the level of LC3B-II in DLBCL cell lines by blocking the traditional autophagy pathway without turning on p53, and the rise is unrelated to SIRT1/2/3 or p53. Through the extrinsic cell-death pathway, tenovin-6 hydrochloride causes apoptosis[4].
Tenovin-6 Hydrochloride inhibits UM cell growth with IC50 values of 12.8 μM, 11.0 μM, 14.58 μM and 9.62 μM for 92.1, Mel 270, Omm 1, and Omm 2.3 cells, respectively[5].

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Tenovin-6 increases p53 protein levels in MCF-7 cells after 6-hour treatment in a concentration-dependent manner, with slightly higher efficacy than tenovin-1. [1]
It elevates acetylated p53 at lysine 382 in H1299 cells transfected with p53 expression vector, even when total p53 levels remain constant. [1]
It increases acetylated α-tubulin at lysine 40 in H1299 cells, an effect weakened by SirT2 overexpression. [1]
It is more toxic to ARN8 melanoma cells than tenovin-1 in a 72-hour viability assay. [1]
A single 2-hour exposure to Tenovin-6 followed by 4 days in fresh medium decreases ARN8 cell growth. [1]
It inhibits the growth of S. cerevisiae cultures with an IC50 of 30 µM. [1]
It does not significantly affect the activity of a panel of 51 purified kinases, a DNA replication assay in Xenopus extracts, or an RNA polymerase I transcription assay. [1]

Tenovin-6 Hydrochloride (50 mg/kg, i.p.) inhibits the growth of tumor in mice[1].

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Daily intraperitoneal administration of Tenovin-6 (50 mg/kg) significantly delays the growth of ARN8 melanoma cell-derived xenograft tumors in SCID mice over a 15-day period. [1]

The Fluor de Lys Fluorescent Assay Systems perform assays using purified components. Useful FdL substrates are used at a concentration of 7 M, and NAD+ is used at a concentration of 1 mM. Tenovins are soluble in DMSO with a final DSMO concentration in the reaction being less than 0.25%. One unit of the enzyme is used in each reaction for SirT1 and HDAC8, and five units are used in each reaction for SirT2 and SirT3. For one hour, reactions are conducted at 37°C.

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In vitro deacetylation assays were performed using purified human SirT1, SirT2, SirT3, or HDAC8 enzymes. Reactions were carried out in a fluorescent assay system with specific acetylated peptide substrates and NAD+ as co-substrate. Compounds were dissolved in DMSO and added to reaction mixtures. Enzyme activity was measured after 1-hour incubation at 37°C. IC50 values were determined by testing increasing concentrations of Tenovin-6. [1]
Lineweaver-Burke analysis was conducted for SirT1 inhibition by Tenovin-6, varying concentrations of either the acetylated peptide substrate or NAD+, to determine the mode of inhibition. [1]

The MTS assay is used to evaluate cell viability. In 96-well plates, UM cells are seeded into each well (5,000 cells/well), treated the following day with control or Tenovin-6 in increasing concentrations from 0 to 20 M for 68 h, and then MTS is added at 20 L/well to be read at a wave length of 490 nm. The IC50 is calculated by curve fitting the sigmoidal dose-response curve.

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For p53 protein level analysis, MCF-7 cells were treated with Tenovin-6 for 6 hours, lysed, and subjected to Western blotting using an antibody against p53. Loading was controlled with an antibody against PCNA. [1]
For acetylation status analysis, H1299 cells were transfected with p53 expression vectors, treated with Tenovin-6 for 6 hours, and analyzed by Western blot using antibodies against acetylated p53 (K382) and total p53. [1]
For acetylated α-tubulin detection, H1299 cells were treated with Tenovin-6 for 16 hours in the presence of trichostatin A to inhibit non-sirtuin HDACs, then analyzed by Western blot with antibodies against acetylated α-tubulin (K40) and total α-tubulin. [1]
Cell viability was assessed using Giemsa staining after treatment with Tenovin-6 for 72 hours or after a 2-hour pulse followed by 4 days in fresh medium. [1]
Cell cycle distribution and death were analyzed by BrdU labeling and propidium iodide staining followed by flow cytometry. [1]

50 mg/kg
ARN8 melanoma cells were injected into the flank of SCID mice and allowed to develop into tumors.

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Female SCID mice were injected subcutaneously with 1 × 10^6 ARN8 melanoma cells suspended in Matrigel. Tumors were allowed to reach approximately 10 mm² in size. Tenovin-6 was dissolved in a vehicle containing 20% cyclodextrin and 10% DMSO. Treatment consisted of daily intraperitoneal injections of Tenovin-6 at 50 mg/kg. Control animals received the vehicle alone. Tumor dimensions were measured regularly with calipers, and volumes were calculated. Statistical significance was assessed using the Mann-Whitney U-test. [1]

The water solubility of Tenovin-6 is seven times that of Tenovin-1. [1]
The improved water solubility compared to Tenovin-1 allows for better pharmacokinetic assessment, but specific pharmacokinetic parameters (e.g., half-life, bioavailability) are not provided in the main text or supplemental data of this publication. [1]

Tenovin-6 primarily exhibits cytotoxic effects against normal human dermal fibroblasts (NHDFs) with minimal cell death, a stark contrast to its cytotoxic effects on ARN8 tumor cells. [1]

[1]. Discovery, in vivo activity, and mechanism of action of a small-molecule p53 activator. Cancer Cell. 2008 May;13(5):454-63.

[2]. Exploitation of DHODH and p53 activation as therapeutic targets - a case study in polypharmacology [published online ahead of print, 2020 Sep 8]. J Biol Chem. 2020;jbc.RA119.012056.

[3]. Class III-specific HDAC inhibitor Tenovin-6 induces apoptosis, suppresses migration and eliminates cancer stem cells in uveal melanoma. Sci Rep. 2016 Mar 4;6:22622.

[4]. Tenovin-6 impairs autophagy by inhibiting autophagic flux. Cell Death Dis. 2017 Feb 9;8(2):e2608.

[5]. Tenovin-6 inhibits proliferation and survival of diffuse large B-cell lymphoma cells by blocking autophagy. Oncotarget. 2017 Feb 28;8(9):14912-14924.

Tenovin-6 was discovered through forward chemogenetic screening of small molecules in mammalian cells that could activate p53-dependent reporter genes. [1]
Its mechanism of action is to inhibit sirtuin deacetylases (SirT1/SirT2), thereby increasing the acetylation and stability of p53, as well as the acetylation of other substrates such as α-tubulin. [1]
Functional p53 contributes to its cytotoxic effects, but is not essential. [1]
Unlike DNA damage agents, Tenovin-6 does not activate DNA damage responses (it does not increase the levels of phosphorylated Ser15 p53 or γH2AX). [1]
It is a potential lead compound for cancer therapy and an effective tool for studying sirtuin biology. [1]

C₂₅H₃₅CLN₄O₂S

491.09

490.216

C, 61.14; H, 7.18; Cl, 7.22; N, 11.41; O, 6.52; S, 6.53

1011301-29-3

Tenovin-6;1011557-82-6

49871498

Yellow solid powder

4

4

9

33

616

0

Cl[H].S=C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O)N([H])C(C1C([H])=C([H])C(=C([H])C=1[H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O

UBNCTIDXQDCEPI-UHFFFAOYSA-N

InChI=1S/C25H34N4O2S.ClH/c1-25(2,3)19-11-9-18(10-12-19)23(31)28-24(32)27-21-15-13-20(14-16-21)26-22(30)8-6-7-17-29(4)5;/h9-16H,6-8,17H2,1-5H3,(H,26,30)(H2,27,28,31,32);1H

4-tert-butyl-N-[[4-[5-(dimethylamino)pentanoylamino]phenyl]carbamothioyl]benzamide;hydrochloride

Tenovin-6 HCl; Tenovin6; Tnv-6; Tenovin 6; Tenovin-6

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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