SIRT6 (IC50 = 89 μM); SIRT2 (IC50 = 751 μM); SIRT1 (IC50 = 1578 μM); HBV

OSS_128167 (Compound 9; 100 μM; 0–24 hours; BxPC3 cells) treatment raises the acetylation of H3K9. and also caused BxPC-3 cells to express more GLUT-1 [1]. Phenol myristate acetate (PMA)-induced TNF-α secretion in cultured BxPC-3 cells was effectively inhibited by OSS_128167 (Compound 9). Cellular uptake of glucose is enhanced by OSS_128167 [1]. HepG2.2.15 and HepG2-NTCP cells treated with 100 μM of OSS_128167 for 96 hours showed a significant reduction in HBV core DNA and 3.5-Kb RNA levels. Additionally, OSS_128167 treatment reduced the expression of HBsAg in cell lysates as well as the secretion of HBsAg and HBeAg, the hepatitis B surface antigens [2]. MM cell lines that are melphalan-resistant (LR-5) and doxorubicin-resistant (Dox40) as well as primary multiple myeloma (MM) cells (NCI-H929) are chemosensitized by OSS_128167 (200 μM)[3].

HBV transgenic mice's HBV DNA and 3.5-Kb RNA levels were markedly suppressed by treatment with OSS_128167 (50 mg/kg; intraperitoneal injection; every 4 days; for 12 days; male HBV transgenic mice) [2].

In Vitro Assays and IC50 Determination[1]
The identified molecules were tested as sirtuin inhibitors using commercial kits for SIRT6, SIRT1, and SIRT2, following manufacturer’s instructions. IC50 values were determined using the same commercial kit assays. All compounds were solubilized at 50 mM concentration in DMSO. The concentrations of the compounds for IC50 determination were in the range from 8 μM to 5 mM. IC50s were determined from the logarithmic nonlinear regression curves in GraphPad Prism. Three independent IC50 measurements were performed for each compound.

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Flow Cytometric GLUT-1 Detection[1]
For flow cytometric detection of GLUT-1 expression at the cell surface, adherent cells were trypsinized and washed in cold PBS. An amount of 5 ×105 cells was subsequently fixed for 10 min in 100% methanol at −20 °C. Thereafter, cells were washed in cold PBS, resuspended in 100 μL cold culture medium, and incubated with or without 2.5 μL of anti-GLUT-1 antibody for 30 min at 4 °C. Subsequently, cells were washed in cold PBS and incubated in 100 μL of cold culture medium with 10 μL of antimouse IgG2a (a kind gift of Dr. Alessandro Poggi, IRCCS AOU San Martino IST, National Cancer Institute, Genoa, Italy) for 30 min at 4 °C. Finally, cells were washed twice with cold PBS and analyzed using a FACS Calibur (Becton Dickinson) by acquiring 10.000 events/sample.

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TNF-α ELISA[1]
BxPC-3 cells (3 × 105 cells/well) were seeded in six-well plates and allowed to adhere for 24 h. Thereafter, cells were incubated overnight with SIRT6 inhibitors (100 μM) or vehicle. Thereafter, to induce cytokine secretion, cells were stimulated for 24 h with 25 ng/mL PMA. Finally, supernatants were collected and assayed for TNF-α concentration using commercially available DuoSet ELISA kits).

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Glucose Uptake Assay with [14C]-2-Deoxy-d-glucose in BxPC-3 and L6 Cells[1]
BxPC-3, pRETROSUPER (pRS) and SIRT6-silent (SIRT6-sh210) BxPC-3, and L6 cells (6 × 105/well), seeded in 12-well plates, were treated in triplicate for 18 h with or without different compounds (200 μM, final concentration), in complete medium. Cells were then washed twice with 1 mL of PBS buffer, and glucose transport was measured by the addition of 0.5 mM d-glucose/[14C]-2-deoxy-d-glucose (0.5 μCi/well) in 0.4 mL of KRH buffer (129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1 mM CaCl2, 1.2 mM MgSO4, 10 mM HEPES, 0.5% bovine serum albumin) for 5 min at 37 °C. Glucose uptake was stopped by immediately removing the labeling mix and washing the cells three times with ice-cold PBS. Cells were then lysed with 0.1% sodium dodecyl sulfate (SDS), and each lysate was used for scintillation counting in a Beta-Counter LS 6500. Unspecific uptake in the presence of 20 μM cytochalasin B and 200 μM phloretin was subtracted from each experimental value.

Western Blot Analysis[1]
Cell Types: BxPC3 cells
Tested Concentrations: 100 µM
Incubation Duration: 0 hrs (hours), 2 hrs (hours), 6 hrs (hours), 18 hrs (hours), 24 hrs (hours)
Experimental Results: Increased H3K9 acetylation.

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RT-PCR[2]
Cell Types: HepG2.2.15 and HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells
Tested Concentrations: 100 µM
Incubation Duration: 96 hrs (hours)
Experimental Results: Dramatically diminished HBV core DNA and 3.5-Kb RNA levels.

Animal/Disease Models: Male HBV transgenic mice (6-8weeks old)[2]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip)injection; every 4 days; for 12 days
Experimental Results: The level of HBV DNA and 3.5-Kb RNA were markedly suppressed in HBV transgenic mice.

[1]. Discovery of novel and selective SIRT6 inhibitors. J Med Chem. 2014 Jun 12;57(11):4796-804.

[2]. SIRT6 Inhibitor, OSS_128167 Restricts Hepatitis B Virus Transcription and Replication Through Targeting Transcription Factor Peroxisome Proliferator-Activated Receptors α. Front Pharmacol. 2019 Oct 25;10:1270.

[3]. Evidence for a role of the histone deacetylase SIRT6 in DNA damage response of multiple myeloma cells. Blood. 2016 Mar 3;127(9):1138-50.

C19H14N2O6

366.32

366.09

C, 62.30; H, 3.85; N, 7.65; O, 26.20

887686-02-4

6496840

White to light yellow solid

2.7

4

6

5

27

568

0

HTJWLEGCECXGSQ-UHFFFAOYSA-N

InChI=1S/C19H14N2O6/c22-15-7-6-13(10-14(15)19(25)26)20-17(23)11-3-1-4-12(9-11)21-18(24)16-5-2-8-27-16/h1-10,22H,(H,20,23)(H,21,24)(H,25,26)

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: ≥ 2.08 mg/mL (5.68 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 20.8 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: ≥ 2.08 mg/mL (5.68 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 20.8 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.)