SIRT6 (IC50 = 89 μM); SIRT2 (IC50 = 751 μM); SIRT1 (IC50 = 1578 μM); HBV
OSS_128167 targets SIRT6 [2]

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].

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1. In HepG2.2.15 and HBV-infected HepG2-NTCP cells, OSS_128167 treatment (gradient concentrations) significantly reduced HBV core DNA levels (detected by real-time PCR and Southern blotting) and 3.5-Kb HBV RNA levels (real-time PCR) in a concentration-dependent manner; secretion of HBsAg and HBeAg (ELISA) and intracellular HBsAg expression (Western blot) were also decreased (P < 0.05) [2]
2. MTS assay showed that OSS_128167 had no significant cytotoxicity in HepG2.2.15 and HepG2-NTCP cells at tested concentrations (3 days treatment) [2]
3. In SIRT6-overexpressing HepG2.2.15 and HBV-infected HepG2-NTCP cells, OSS_128167 blocked the promotion of HBV transcription/replication by SIRT6; the upregulation of PPARα by SIRT6 was reversed by OSS_128167 (real-time PCR and Western blot), and PPARα silencing mimicked the anti-HBV effect of OSS_128167 [2]
4. In multiple myeloma (MM) cell lines (MM.1S, NCI-H929), OSS_128167 (200 μM) enhanced the sensitivity of MM cells to DNA-damaging agents (doxorubicin 10-100 nM, melphalan 0.3-3 μM); survival curves showed that OSS_128167 significantly reduced cell viability when combined with doxorubicin/melphalan (P < 0.001) [3]
5. OSS_128167 pretreatment (200 μM) inhibited SIRT6 recruitment to Alu sequences after DNA damage (doxorubicin 1 μM, melphalan 100 μM) in MM cells (ChIP assay, P < 0.01), and increased H3K56 acetylation at DNA damage sites, impairing NHEJ/HR DNA repair mechanisms [3]
6. OSS_128167 treatment in MM cells led to hyperactivation of MEK/ERK signaling (via H3K9 acetylation and ELK1-mediated activity), blockade of RSK2-mediated Chk1 phosphorylation (Ser280), and impairment of ATR/CHK1 DNA damage checkpoint [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].

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1. In HBV transgenic mice (n=8 per group), OSS_128167 was administered intraperitoneally at 50 mg/kg every 4 days for 12 days; serum ALT levels (colorimetric microplate assay) showed no significant liver toxicity; serum HBsAg, HBeAg (ELISA) and HBV DNA (real-time PCR) were significantly reduced, and intrahepatic HBV DNA, total HBV RNAs, and 3.5-Kb RNA (real-time PCR) were also decreased (P < 0.05); HBcAg expression in liver tissues (immunohistochemistry) was downregulated [2]
2. In NCI-H929 MM xenograft model (CB17-SCID mice), combination treatment of OSS_128167 with doxorubicin significantly reduced tumor volume (P = 0.011 vs vehicle + doxorubicin), prolonged median survival time of mice, and increased γH2AX foci (DNA damage marker) in tumor tissues (immunohistochemistry/immunofluorescence); SIRT6 depletion + doxorubicin showed similar effects, with reduced RAD51 focus formation (DNA repair marker) [3]

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.

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1. In MM cells, ChIP assay was performed to detect SIRT6 binding to Alu sequences and gene promoters after OSS_128167 treatment: cells were cross-linked with formaldehyde, lysed, and chromatin was sheared into fragments; SIRT6-specific antibody was used for immunoprecipitation, and quantitative PCR was performed to detect the enrichment of target sequences (IgG as negative control) [3]
2. H3K56 acetylation assay at DNA damage sites: MM cells treated with OSS_128167 and DNA-damaging agents were subjected to ChIP with anti-acetylated H3K56 antibody; the enrichment of acetylated H3K56 at DNA damage sites was quantified by real-time PCR (IgG as control) [3]

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.

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1. Anti-HBV activity assay in HepG2.2.15/HepG2-NTCP cells: Cells were seeded in 96/6-well plates and treated with gradient concentrations of OSS_128167; after 3-4 days, cell supernatants were collected for HBsAg/HBeAg ELISA, and cells were harvested for HBV DNA (genomic extraction) and RNA (TRIzol extraction) detection by real-time PCR/Southern blotting; intracellular HBsAg was detected by Western blot (GAPDH as loading control) [2]
2. Cytotoxicity assay (MTS): HepG2.2.15 and HepG2-NTCP cells were seeded in 96-well plates, treated with serial concentrations of OSS_128167 for 3 days; MTS reagent was added, and absorbance was measured at 490 nm to calculate cell viability [2]
3. SIRT6 overexpression/knockdown combined with OSS_128167 treatment: HepG2.2.15/HepG2-NTCP cells were transfected with Flag-SIRT6 plasmid or SIRT6 shRNA (shSIRT6-1/2) + scramble control; 1 day post-transfection, OSS_128167 was added; after 3-4 days, cells were lysed for Western blot (SIRT6, PPARα, GAPDH) and real-time PCR (HBV RNA, PPARα mRNA) [2]
4. MM cell survival assay: NCI-H929 cells were seeded in 96-well plates, treated with OSS_128167 (200 μM) plus gradient concentrations of doxorubicin/melphalan for 48 hours; cell viability was measured by colorimetric assay, and survival curves were plotted (triplicate experiments, P < 0.001) [3]
5. ChIP assay for SIRT6/ELK1 binding: MM.1S cells treated with OSS_128167 were cross-linked, lysed, and chromatin was immunoprecipitated with SIRT6/ELK1/AcH3K9 antibody; the binding of these proteins to MAPK pathway gene promoters was quantified by real-time PCR (IgG as control, GAPDH as negative reference) [3]
6. DNA repair assay (NHEJ/HR): MM.1S cells (SIRT6-WT/SIRT6-KD) were cotransfected with NHEJ/HR reporter plasmids and DsRed plasmid; OSS_128167 was added, and GFP+/DsRed+ cells were counted by flow cytometry 72 hours later to evaluate repair efficiency (P < 0.03) [3]

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.

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1. HBV transgenic mouse model: HBV transgenic mice (n=8 per group) were randomly divided into vehicle, OSS_128167 (50 mg/kg), and ETV (0.02 mg/kg) groups; OSS_128167 was administered intraperitoneally every 4 days for 12 days, ETV was given by oral gavage every 4 days; serum was collected via tail vein at indicated time points for HBsAg/HBeAg/ALT/HBV DNA detection; mice were euthanized on day 12, liver tissues were collected for intrahepatic HBV DNA/RNA detection (real-time PCR) and HBcAg immunohistochemistry [2]
2. MM xenograft model: CB17-SCID mice were subcutaneously injected with GFP-expressing NCI-H929 scramble/SIRT6-KD cells; when tumors formed, mice were treated with vehicle + doxorubicin or OSS_128167 (no specific dose/route/frequency reported) + doxorubicin; whole-body imaging was performed to monitor tumor growth, tumor volume was measured by caliper (mean ± SD, P = 0.011); mice were euthanized, tumor tissues were collected for Western blot (SIRT6, γH2AX) and immunohistochemistry (γH2AX, RAD51) [3]

1. In vitro experiments: At the tested concentration, OSS_128167 did not show significant cytotoxicity to HepG2.2.15 and HepG2-NTCP cells (MTS method, treatment for 3 days) [2] 2. In vivo experiments: In HBV transgenic mice, OSS_128167 (50 mg/kg, intraperitoneal injection every 4 days for 12 days) did not increase serum ALT levels (a marker of hepatotoxicity), indicating no significant acute hepatotoxicity [2]

[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.

1. OSS_128167 is a selective SIRT6 inhibitor with anti-HBV activity. It restricts HBV transcription and replication by inhibiting SIRT6-mediated upregulation of PPARα and activation of the HBV core promoter [2]. 2. OSS_128167 makes multiple myeloma cells more sensitive to DNA damage agents (doxorubicin, melphalan) by inhibiting SIRT6-dependent DNA damage response: the loss/inhibition of SIRT6 impairs the MAPK/ERK/RSK2/Chk1 signaling pathway, blocks DNA repair (NHEJ/HR), and enhances genomic stress-induced cell death [3]. 3. SIRT6 plays a key role in maintaining the genomic stability of MM cells by deacetylation of H3K9 at the MAPK pathway gene promoter, downregulation of the MAPK signaling pathway, and promotion of DNA repair. OSS_128167 can inhibit this function, leading to excessive proliferation of MM cells and increased sensitivity to DNA damage [3]. 4. OSS_128167 is a potential antiviral drug for HBV and a sensitizer for chemotherapy in multiple myeloma [2,3].

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.)