Discovery of a Pyrrole-pyridinimidazole Derivative as Novel SIRT6 Inhibitor for Sensitizing Pancreatic Cancer Cells to Gemcitabine

doi:10.21203/rs.3.rs-1982889/v1

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Discovery of a Pyrrole-pyridinimidazole Derivative as Novel SIRT6 Inhibitor for Sensitizing Pancreatic Cancer Cells to Gemcitabine

https://doi.org/10.21203/rs.3.rs-1982889/v1

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published 04 Aug, 2023

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Pancreatic cancer is a highly aggressive cancer, and is primarily treated with gemcitabine, with increasing resistance. SIRT6 as a member of sirtuin family plays important roles in lifespan and diverse diseases, such as cancer, diabetes, inflammation and neurodegenerative diseases. Considering the role of SIRT6 in the cytoprotective effect, it might be a potential anticancer drug target, and is associated with resistance to anticancer therapy. However, very few SIRT6 inhibitors have been reported. Here, we reported the discovery of a pyrrole-pyridinimidazole derivative, 8a, as a new non-competitive SIRT6 inhibitor, and studied its roles and mechanisms in the antitumor activity and sensitization of pancreatic cancer to gemcitabine. Firstly, we found a potent SIRT6 inhibitor compound 8a by virtual screening and identified by molecular and cellular SIRT6 activity assays. Then, we found that 8a can inhibit the proliferation, induce cell apoptosis and sensitize pancreatic cancer cells to gemcitabine treatment both in pancreatic cancer cells and xenograft models. Mechanistically, 8a inhibited the cell proliferation and survival signaling pathways, such as PI3K/AKT/mTOR and ERK signal pathway, and markedly exacerbated DNA damage induced by gemcitabine. Overall, we demonstrate that 8a, a novel SIRT6 inhibitor, could be a promising potential drug candidate for pancreatic cancer treatment.

SIRT6

pancreatic cancer

gemcitabine

DNA damage

apoptosis

Pancreatic cancer is a highly aggressive cancer with a 5-year survival rate of 11% [1]. Gemcitabine is still the first-line chemotherapy for pancreatic cancer [2]. However, high rate of chemoresistance severely impedes its efficacy as a cornerstone of pancreatic cancer chemotherapy[3]. Therefore, there is an urgent need for developing innovative targets and novel therapeutic strategies for therapeutic intervention of pancreatic cancer.

SIRT6 as a member of sirtuin family plays important roles in gene transcription, DNA repair and glucose homeostasis [4, 5]. SIRT6-deficient mice have displayed genomic instability, premature senility and finally dying at approximately four weeks [6]. SIRT6 knockout cells fail to repair multiple types of DNA damage, including DNA double-strand break (DSB) repair and base excision repair (BER) responding to genotoxic and oxidative stress [6–8]. There are many factors leading to resistance to chemotherapeutic agents of cancer, including activation of DNA damage repair pathway, the stemness of cancer cells and immune evasion [9]. In particular, the DNA damage repair pathway inhibits tumorigenesis in normal cells, but it leads to resistance of tumors to genotoxic anticancer therapies [10, 11]. Based on the role of DNA damage repair in chemotherapeutic drug resistance, several inhibitors targeting DNA repair mechanisms, such as the single strand break (SSB) repair protein PARP inhibitors, are being studied in clinical trials for the treatment of cancer [12, 13]. Due to the role of SIRT6 in the regulation of DNA repair, mounting evidence points towards SIRT6 may be a target of tumor chemotherapy drug resistance [14]. Therefore, SIRT6 inhibitors might be helpful to improve the efficacy of currently available therapeutics. However, very few SIRT6 inhibitors have been reported and have no ideal inhibitory efficacy [15, 16], and the antitumor efficacy of existing SIRT6 inhibitors is still weak and the underlying mechanism remains largely unknown[17, 18]. Hence, developing highly effective SIRT6 inhibitors is important in improving the therapeutic effect of pancreatic cancer.

Marine environment has demonstrated to be a prolific source of compounds with unique chemical features and structural activity due to the outstanding biological diversity[19, 20]. Marine natural products are ideal candidates as hit or lead compounds in new drug discovery and further development[21–23]. For the development and application of marine drugs, our group has established a fine three-dimensional structure database (http://mc3d.qnlm.ac/) containing 30,117 marine natural products by Ocean University of China that can be directly used for virtual drug screening and intelligent drug design. On this basis, we can quickly and efficiently discover targeted drugs for various diseases.

In this study, to discover novel SIRT6 inhibitors, we initially performed a virtual screening study against the marine natural products and derivatives library using the protein crystal of SIRT6. Verified by biological activity test, we found that compound 8a showed more potent inhibitory effect on the SIRT6 at cellular level. We further investigated the roles and mechanisms of compound 8a in antitumor activity and chemosensitivity in pancreatic cancer cells and xenograft models. This study showed that compound 8a is an effective SIRT6 inhibitor, and a potential drug candidate for pancreatic cancer treatment.

Chemistry.

All commercially available starting materials and solvents were purchased from commercial vendors and used without further purification. Reactions were monitored using analytical thin-layer chromatography (TLC) on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, China) and visualized under ultraviolet light (254 nm and 365 nm). Column chromatography was performed on silica gel (200–300 mesh). ¹H and ¹³C NMR spectra were recorded on the Broker AVANCE NEO and Agilent DD2 500 with 400 or 500 MHz for proton (¹H NMR) and 100 or 125 MHz for carbon (¹³C NMR) with tetramethylsilane (Me₄Si) as the internal standard, respectively. The chemical shifts (δ) were expressed in parts per million (ppm). Abbreviations used: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet. The coupling constant (J) values were described as hertz. High-resolution (ESI) MS spectra were recorded using a QTOF-2 Micromass spectrometer. The purity of the final compounds for biological evaluation was higher than 95% by analytical HPLC analysis with the Primaide 1210 system. General procedure to synthesis compounds (Supplementary information)

Cloning, expression, and purification of recombinant human SIRT6.

The human full-length SIRT6 gene was inserted into the prokaryotic expression vector (pET-28a (+)). The verified recombinant plasmid was transformed into Escherichia coli BL21 (DE3) expression strain and cultured overnight at 37℃ on LB plates supplemented with 50 µg/mL kanamycin. Single colonies were inoculated in fluid kanamycin-containing LB mediums. It was shaken at 160 rpm for 16 hours at 37°C and transferred to kanamycin-containing LB medium at a ratio of 1:100. It was shaken at 160 rpm at 37°C until the OD₆₀₀ reached 0.6. The Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to make the final concentration of solution 0.5 mmol/L, cultured for 15°C and 160 rpm for 24 hours.

Bacterial liquid was harvested at 12,000 rpm at 4°C for 20 min, and then re-suspended in Lysis buffer (20 mmol/L Hepes pH 7.5, 20 mmol/L imidazole, 5% Glycerol, 500 mmol/L NaCl). Cell pellets were disrupted by high-pressure crusher (JNBIO, Guangzhou China) and centrifuged (4°C, 12000 rpm, for 20 min). Supernatant was collected and purified by FPLC with a 1 mL Ni–NTA Sepharose column (GE Healthcare, Stamford, USA). The SIRT6 protein was eluted at containing 50 mM, 100 mM, 150 mM, 200 mM, and 500 mM imidazole in elution buffer. The purified SIRT6 protein was dialyzed in dialysis buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂). The purified SIRT6 protein was analyzed by SDS-PAGE.

In vitro SIRT6 deacetylation assays and IC ₅₀ determination.

Fluor-De-Lys (FDL) assays were used for the examination of the deacetylation activities of sirtuins as described previously [24, 25]. For the detection of SIRT6 activity, the 50-µL reaction mixture contained 5 µM SIRT6, 2.5 mM NAD⁺ (β-Nicotinamide adenine dinucleotide hydrate, Sigma, N1636), 75 µM RHKK-Ac-AMC (GL Biochem, Shanghai), compounds/DMSO, and assay buffer. The reactions were incubated at 37°C for 2 h, added 50 µL stop solution (40 mM nicotinamide, 6 mg/mL trypsin) at 25°C for 30 min. Fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. IC₅₀ values were calculated in GraphPad Prism version 8.00.

Biolayer interferometry (BLI)

Binding assays were performed using Ni-NTA biosensors on the the Octet® Red96 system (ForteBio, Menlo Park, CA) in 96-well microplates and measuring changes wavelength shift (in nm) over time (sec). All steps were carried out at 25℃ with the plate shaking speed of 1000 rpm. The total working volume was 200 µL in 96-well microplate (Greiner Bio-one, Germany). The SIRT6 protein and compounds were diluted with phosphate buffered saline (PBS, pH 7.4). Ni-NTA biosensors were prewetted for at least 10 min in PBS prior to the assay. Afterwards, the Ni-NTA biosensors were immobilized for 180 sec with 200 µL culture containing 20 µg/mL SIRT6. After loading, sensors were washed in PBS buffer for another 120 sec and transferred to wells containing different concentrations of compound 8a (0, 12.5, 25, 50 ,100 µM) for binding. Finally, the dissociation step was carried out in kinetic buffer for 180 sec. The assay data were processed and calculated using the ForteBio Octet RED analysis software.

The cellular thermal shift assay (CETSA).

The CETSA [26] approach builds on the thermal shift assay concept where ligand binding to its target protein affects protein stability at high temperatures. Hela cells were plated in 100 mm plates. After 12 h attachment, Hela cells were incubated with 8a (25 µM) for 6 h, and then the cells were digested with trypsin and collected. The collected cells were subsequently aliquoted into six parts, and incubated each part at different temperatures (40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50°C) for 3 minutes. The heated cells were then snap frozen in liquid nitrogen three times to lyse. After that, the lysed cells were then centrifuged at 12,000 rpm for 20 minutes. Then the level of SIRT6 was detected by western blot.

Cell culture.

The BXPC-3 (RRID:CVCL_0186), PANC-1 (RRID:CVCL_0480), and Hela (RRID:CVCL_0030) cell lines were obtained from the Cell Resource Center of the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences. BXPC-3 were cultured in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum, sodium pyruvate (1 mmol/L), penicillin (50 units/mL), and streptomycin (50 µg/mL). PANC-1 and Hela were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FBS, streptomycin (50 µg/mL) and penicillin (50 units/mL). The cells were maintained in 37°C incubator with 5% CO₂.

Proliferation Assay.

BXPC-3 and PANC-1 cells were plated in 96-well plates at 5,000 cells per well. After 12 h attachment, varying concentrations of 8a were added into the culture medium for 72 h. Then 10 µL MTT (5 mg/ml) solution was added into per well for 4 h at 37℃. After removing medium, formazan crystals were dissolved in 150 µL DMSO. The cells viability was quantified by measuring the absorbance at 570 nm.

Colony Formation Assay.

The BXPC-3 cells were plated in six-well plates at 1000 cells per well. After 12 h, different concentrations of 8a, Gemcitabine or their combination were added for 7–10 days. The cells were fixed for 15 min with paraformaldehyde (4%) and then stained with crystal violet (0.1%) for 15min at room temperature. Surviving colonies consisting of 50 or more cells were counted under microscope.

Cell cycle analysis.

The BXPC-3 cells were seeded onto six-well plates at 1x10⁵ cells per well and different concentrations of 8a, Gemcitabine or their combination were added for 48 h. The cells were washed with PBS, collected after trypsinization and fixed in cool 70% ethanol at -20℃. After fixation, the cells were incubated in the dark with PBS containing 50 µg/mL propidium iodide (PI) and 100 µg/mL RNase A for 30 min at room temperature. Finally, the samples were analyzed on a flow cytometer (Beckman Coulter).

Cell apoptosis analysis.

To quantify cell apoptosis, BXPC-3 cells were treated with different concentrations of compound 8a, Gemcitabine or their combination for a further 72 h. Harvested cells were incubated with Muse™ Annexin V &Dead Cell assay kit (Muse TM Cell Analyzer, Millipore (catalog no. MCH100105)). Cells were then analyzed by flow cytometry (Muse TM Cell Analyzer, Millipore).

Hoechst 33342 stain.

BXPC-3 cells were seeded on the chamber-slides at 12-well plates and treated with the indicated concentration of compound 8a for 48 h. After treatment, cells were fixed with 4% paraformaldehyde for 15 min. Then, cells were stained with 10 µg/mL Hoechst 33342 for 30 min. After washing with PBS, slides were observed with a fluorescent microscopy.

Western blot analysis.

Cells were harvested and lysed in a lysis buffer (solarbio, R0010) supplementing 1 mM PMSF and protease inhibitor (TargetMol, USA). Protein was separated on 7.5%-15% SDS-PAGE gels and transferred to the 0.45 µm or 0.22 µm PVDF membrane (Merck Millipore), and subsequently blocked with 5% bovine serum albumin (BSA) in TBST. The blocked membrane was incubated with primary antibodies:SIRT6 (1:1000, Cell Signaling Technology, Cat#12486S, RRID:AB_2636969), Histone H3 Antibody (1:1000, Cell Signaling Technology, Cat#9715S, RRID:AB_331563), β-Actin (1:2000, Cell Signaling Technology, Cat#4970S, RRID:AB_2223172), Cleaved caspases-3 (1:1000, Cell Signaling Technology, Cat#9661S, RRID:AB_2341188), Cleaved Caspase-9 (Asp315) (1:1000, Cell Signaling Technology, Cat#20750S, RRID:AB_2798848), Cleaved PARP (1:1000, Cell Signaling Technology, Cat#5625S, RRID:AB_10699459), AKT (1:1000, Cell Signaling Technology, Cat#9272S, RRID:AB_329827), Phospho-AKT (Ser473) (D9E) (1:1000, Cell Signaling Technology, Cat#4060S, RRID:AB_2315049), ERK1/2 (1:1000, Cell Signaling Technology, Cat#4695S, RRID:AB_390779), Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, Cell Signaling Technology, Cat#4370S, RRID:AB_2315112), Cyclin D1 (E3P5S) (1:1000, Cell Signaling Technology, Cat#55506S, RRID:AB_2827374), Phospho-mTOR(Ser2448)(1:1000,Cell Signaling Technology, Cat#5536S, RRID:AB_10691552), Phospho-p70S6Kinase (Thr421/Ser424) (1:1000, Cell Signaling Technology, Cat#9204S, RRID:AB_2265913), Phospho-Histone H2A.X (Ser139) (1:1000, Cell Signaling Technology, Cat#9718S, RRID:AB_2118009) at 4℃ overnight. After being washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for one hour. Then, the membranes were detected by Tanon 5200 (Tanon, Beijing, China).

Immunofluorescence assay.

Cells were plated in the chamber-slides in the same conditions, and treated with compound 8a, gemcitabine or their combination for a further 48 h. Cells were washed twice with PBS and fixed with paraformaldehyde (4%) at room temperature for 15 min. After washing twice with PBS, cells were blocked in PBS containing 0.2% Triton X-100, 10% goat serum and 5% BSA for 2 h, and then incubated with primary antibodies (1:500, γ-H2AX, Ser139, Cell Signaling Technology, Cat#9718, RRID:AB_2118009) overnight at 4°C. Cells were stained with secondary antibodies (1:1000, Alexa Fluor 488-conjugated anti-rabbit IgG, Abcam, Cat#ab150077, RRID:AB_2630356) for 2 h at 4℃ in dark. After washing with PBS, the cells were stained with hoechst and observed by confocal microscopy (Leica TCS SP8 STED 3X).

Tumor nude mice model.

All animal experiments were performed in accordance with the principles and procedures approved by the Animal Ethics Committee of the Ocean University of China. Female BALB/c nude mice (14 ± 2 g, 4 weeks old) were purchase from Beijing Vital River Laboratories (Beijing, China). All animals were allowed to adapt to the environment for one week upon arrival. These animals were kept in a standard environment with a 12-hour light/dark cycle and allowing free drinking and eating. About 2 × 10⁶ of BXPC-3 cells were inoculated into nude mouse flanks. When the tumor volume reached approximately 100 mm³, mice were randomly divided into four groups with 6 mice per group before treatment and then treated with vehicle, 8a (20 mg/kg), gemcitabine (10 mg/kg) and their combination every 2 days. Tumor volumes and body weight were measured every 3 days and use the formula to calculate the tumor volume: V = (L × W× W) /2 (V, tumor volume; L, length; and W, width). Mice were sacrificed and tumor tissues were removed. Tumor tissues were fixed with paraformaldehyde (4%) and embedded with paraffin.

Statistical analysis

All the results were expressed as the mean ± SD. Each experiment was repeated at least three times at least triplicate samples. Statistical analyses were performed with unpaired Student’s t-test or one-way ANOVA using GraphPad Prism version 8.0, and p < 0.05 was considered statistically significant.

Discovery of Hit Compounds with the Aid of Molecular Docking-Based Virtual Screening and Biological Activity Test.

To obtain new inhibitors of SIRT6, we implemented virtual screening for marine natural compounds and their derivatives library on SIRT6 (PDB ID:5MF6) protein crystals. According to screening results (Table 1), 16 compounds of Ageladine A derivatives were selected and chemically synthesized for biochemical assays. We first tested the inhibitory activity on each of these compounds at a fixed concentration of 25 µM by the FLUOR DE LYS (FDL) assay using a fluorogenic acetyl peptide substrate (Ac-RHKK(Ac)-AMC). The chemical structures and bioactivities are given in Table 1. We found that compounds 5c, 6b and 8a, containing a different isothiouronium-modified group, showed excellent inhibitory toward SIRT6. Compound 5c and 6b, both of them with isothiourea displayed potent inhibition activity against SIRT6 at 25 µM with inhibition rate of 77.8% and 79.6%, respectively. Compound 6a with the length of four alkly chains, did not show obvious activity against SIRT6 with inhibition rate of 26.7%. Compounds 8a, which contains tetramethyl-isothiourea, displayed modest inhibitory effect, with inhibition rate of 75.6%. It indicated that by introducing isothiourea and tetramethyl-isothiourea to the pyrrole-pyridinimidazole through six alkly chain, the compounds can greatly inhibit SIRT6. It indicated that the different heteroring group hardly contributed to the inhibition toward SIRT6. According to the inhibitory effects toward SIRT6, we choose the three active compounds (5c, 6b and 8a) for follow-up biological evaluation.

Compound 8a Inhibits SIRT6 Deacetylation Activity at Cellular Level

To investigate whether they can inhibit the activity of cellular SIRT6, we examined the acetylation status of H3, which is known to be substrate of SIRT6 [27]. As shown in Fig. 1a, 8a significantly increased the acetylation levels of H3K9 in Hela cells in a dose-dependent manner. 5c and 6b only slightly elevated the acetylation levels of H3, which were inconsistent with the in vitro assay. As these compounds can show violet fluorescence, intracellular localizations of these compounds were evaluated by confocal microscopy and the nuclei were stained by PI with red fluorescence, we found that 8a was localized in the cytoplasm and nuclear, but 5c only accumulated in cell cytoplasm and 6b could not be absorbed into the cells (Fig. 1b). Since SIRT6 is predominantly located in the nucleus [28], we speculated that the most potent SIRT6 inhibitory activity of 8a at cellular levels can be attributed to its good cell penetration.

Compound 8a is a Non-competitive Inhibitor of SIRT6

We subsequently examined the IC₅₀ values of the most potent compounds. The results showed that 8a could effectively inhibit SIRT6 with IC₅₀ values of 7.46 ± 0.79 µM (Fig. 2a). To demonstrate that 8a physically interacts with SIRT6, we used the Octet RED96 system based on the biolayer interferometry technology. The result showed that 8a could directly interact with SIRT6 (KD = 16 ± 0.313 µM) (Fig. 2b). Furthermore, to confirm that 8a reaches and directly binds to SIRT6 in cells, we also carried out the cellular thermal shift assay (CETSA). The CETSA approach builds on the thermal shift assay concept where ligand binding to its target protein affects protein stability at high temperatures. We observed that the thermal stability of SIRT6 increased after 8a treatment compared to the negative group (Fig. 2c and 2d), indicating that 8a directly binds to SIRT6 and inhibits SIRT6 deacetylation activity.

Next, to investigate whether compound 8a is a competitive inhibitor, we performed competition assays between the substrates and compound 8a. As shown in Figure. 2e and 2f, increasing concentrations of the substrate peptide Ac-RHKK-AMC or NAD⁺ had no effect on the inhibitory activity of 8a. Then, we carried out an enzymatic kinetic assay to further clarify the inhibition mode of 8a (Fig. 2g, h). Lineweaver–Burk plot analysis revealed that 8a did not alter the Km but obviously decreased Vmax for both the Ac-RHKK-AMC and NAD⁺ substrates. These findings indicated that 8a is a non-competitive inhibitor.

Additionally, molecular docking was performed for understanding the interaction mechanisms between compound 8a and SIRT6 (PDB ID:6QCJ). The molecular docking of 8a with SIRT6 showed that the hydrogen in the tetramethly-isothiourea group of 8a engages hydrogen bond formation with Glu22 and Gly66. Ser216 forms H-π interactions with the pyrrole ring of 8a, Arg65 forms a hydrogen bond with the pyridine ring and Asp63 forms a hydrogen bond with the H atom on the alkly chains (Fig. 2i, j). Next, we predicted binding mode of the compound 8a, NAD⁺, substrate (Ac-RHKK-AMC) and SIRT6. We found that substrate peptides, NAD⁺, and 8a bind to different sites of SIRT6 (Figure S1), which was consistent with the above findings that 8a is a non-competitive inhibitor. Therefore, our data demonstrated that 8a directly binds to SIRT6 and is a non-competitive inhibitor.

Compound 8a Inhibits Cell Proliferation, Induces Cell-cycle Arrest and Apoptosis in PDAC Cells

PDAC is a highly aggressive malignancy with limited effective therapeutic options [29]. To assess the anti-proliferation effect of compound 8a in PDAC cells, two human PDAC cell lines, BXPC-3 and PANC-1, were used for MTT assay. 8a significantly reduced the proliferation of BXPC-3 and PANC-1 with IC₅₀ values at 72 h of 7.56 µM and 9.28 µM, respectively (Fig. 3a). But, 8a displayed significantly lower potency against normal HUVEC cells (IC₅₀ = 41.01 µM). Colony formation assays reflect the cell growth and survival abilities of a single cell. As shown in Fig. 3b, PDAC cells exhibited much smaller and fewer colonies after being treated with 8a in a dose-dependent manner. The PI3K/AKT/mTOR and MAPK/ERK pathways are important regulators for the cell proliferation and survival [30, 31]. Knockdown of SIRT6 inhibited ERK and PI3K/AKT/mTOR signaling pathway[32, 33]. The effects of 8a on AKT/mTOR/P70S6K and ERK phosphorylation were determined using western blot assays. As expected, p-mTOR, p-70s6k, p-AKT, and p-ERK were significantly down-regulated after treatment with different doses of 8a (Fig. 3c). These results suggested that the suppression of the PI3K/AKT/mTOR and MAPK/ERK signaling pathway might be the reason for inhibiting BXPC-3 cell proliferation by 8a.

To further confirm the effect of 8a on cell survival, cell-cycle and apoptosis were detected by flow cytometry. After incubated with various concentrations of 8a for 48 h, the 8a induced dose-dependent increases in the percentages of the G0-G1 phase (Fig. 3d). Cyclin D1 is well-known genes that regulate the cell cycle[34, 35]. 8a significantly decreased cyclin D1 expression in a dose-dependent manner (Figure S2 a). Hoechst staining showed a markedly shrunk of the chromatin with an increasing concentration of 8a (Figure S2 b and 2c). Annexin V-PE/7-AAD staining was applied to investigate cells apoptosis. As shown in Fig. 6F, BXPC-3 cells undergoing apoptosis after treatment with 0, 6.25, 12.5, 25, 50 µM of 8a were 5.46%, 15.44%, 39.42%, 53.50%, and 85.98%, respectively (Fig. 3e). Subsequently, some apoptosis-associated factors, namely c-PARP, c-caspase3, and c-caspase9, were determined by western blot. The results indicated that 8a significantly up-regulated the expression of c-PARP, c-caspase3, and c-caspase9 (Fig. 3f). Altogether, these results suggested that 8a could inhibits BXPC-3 cell proliferation, induces cell-cycle arrest.

Compound 8a Sensitizes PDAC Cells to Gemcitabine by Inhibiting Cell Survival Signaling

Gemcitabine is a classic drug used for pancreatic cancer therapy, but chemoresistance reduces its therapeutic effect [2]. SIRT6-deficient cells demonstrate enhanced sensitivity to chemotherapeutic agents [14]. Thus, we evaluated the possibility that 8a can sensitize pancreatic cancer cells to gemcitabine. MTT and colony formation assay showed that the 8a significantly enhanced the effects of gemcitabine on the cell viability and clonogenic ability (Fig. 4a and 4c). We found synergistic effects of 8a and gemcitabine by combined effect analysis, with CI index less than 1 (Fig. 4b).

To confirm the synergistic anticancer activity, we performed the cell cycle and apoptosis. 8a significantly enhanced the G0/G1 arrest and S phase inhibition induced by gemcitabine (Figure S3 a and b). Consistently, the expression of cyclin D1 was also decreased following combination treatment (Figure S3 c). Hoechst staining was used for apoptosis detection, the results showed that 8a increased the apoptotic vesicles in BXPC-3 cells compared to gemcitabine alone (Figure S3 d). Flow cytometry assay indicated that cell apoptosis was significantly enhanced when treatment with 8a and gemcitabine together (Fig. 4e). Western blot analysis of apoptosis-related proteins, including cleaved-PARP, cleaved-caspase 3, and cleaved-caspase 9, showed that combination treatment induced much higher level than gemcitabine alone in BXPC-3 cells (Fig. 4f).

Previous studies also showed that gemcitabine activates AKT and ERK signaling pathways in pancreatic cancer cells [36, 37]. AKT and ERK activation may therefore impedes the therapeutic efficacy of gemcitabine [36, 38].Western blot analysis revealed that gemcitabine alone increased phosphorylations of AKT and ERK. 8a could reverse the activation of AKT and ERK signaling pathway induced by gemcitabine (Fig. 4d). These results suggested that the combination of 8a with gemcitabine significantly decreased cell growth and chemoresistance, which may have been due to the 8a-mediated suppression of the AKT and ERK cell survival pathway. Thus, that also explains why there are possible mechanisms for synergies in drug combinations.

Compound 8a Enhances Gemcitabine-induced DNA Damage

Gemcitabine, a deoxycytidine nucleoside analog that inhibits DNA synthesis and induces cell death, shows its killing power mainly through the induction of DNA damage [2]. The presence of DNA repair pathways reduces tumor sensitivity to gemcitabine [39–41]. SIRT6 has been reported to be involved in regulating DNA repair [42]. Based on these studies, we hypothesized that SIRT6 inhibitor can lead to cancer cells hypersensitivity to DNA damaging agents in cancer chemotherapy by blocking DNA repair. γ-H2AX, phosphorylation of the Ser-139 residue of the histone variant γ-H2AX, which is a highly specific and sensitive molecular marker for monitoring DNA damage [43]. We found that gemcitabine can cause γ-H2AX foci, and its combination with 8a treatment can induce much greater expression of γ-H2AX foci (Fig. 5a, 5b and 5c). Consistent with the results of immunofluorescence, western blot analysis also showed that the protein expression of γ-H2AX was significantly enhanced when treatment with gemcitabine and 8a together (Fig. 5d and 5e). Collectively, these results suggested that inhibition of SIRT6 by 8a disturbed DNA repair activity and allowed accumulation of DNA damage resulted from gemcitabine and subsequently induced cell apoptosis.

Compound 8a Augmented the Antitumor Effects of Gemcitabine in BXPC-3 Tumor xenografts

To further confirm whether 8a can enhance the antitumor effects of gemcitabine in vivo, the BXPC-3 PDAC xenografts were used. The mice with tumors received intraperitoneal injection of compound 8a (20 mg/kg), gemcitabine (10 mg/kg), or their combination once every two days for 4 weeks. Tumor volumes and body weight of mice were monitored every three days. As shown in Figure. 6a–c, the compound 8a only had 27% tumor inhibition compared with the control mice, and the mice treated with gemcitabine could inhibit tumor mass by 30.1%. However, the tumor mass was inhibited by up to 71.3% in mice treated with the combinations of 8a and gemcitabine. Notably, mice in different treatment groups showed no significant loss of body weight compared with the control group (Fig. 6d). In addition, immunohistochemistry analysis showed that the combinations of 8a and gemcitabine greatly increased the expression of apoptosis maker, activated caspase 3, and reduced the expression of proliferation marker, Ki 67 (Fig. 6e). Western blotting of the tumor tissues showed that ac-H3 was higher in the 8a-treated groups than the other groups, and the changes in these proteins including cleaved-caspase 3, Ki67 and γ-H2AX were consistent with previous experiments at BXPC-3 cells (Figure. S4). These results showed that 8a significantly enhanced gemcitabine antitumor effects in vivo by inhibition tumor cell proliferation and induction of apoptosis.

SIRT6 has been confirmed to be involved in a variety of biological processes, such as gene transcription, genome stability and DNA repair [6]. Through these functions, SIRT6 plays an important role in multiple diseases, including metabolic diseases, aging and cancer [6, 44]. There are increasing reports suggesting that overexpression of SIRT6 induced resistance to anticancer therapy by mediating the DNA repair [33, 45]. Although SIRT6 has emerging potential as a therapeutic target in cancer, few small molecule inhibitors of SIRT6 have been identified to date, and most of these inhibitors show weak inhibitory activity against SIRT6 and limited antitumor activity [44, 46]. In this study, we focused on the discovery of new small-molecule SIRT6 inhibitors, and we identified the most potent SIRT6 inhibitor compound 8a (IC₅₀ = 7.46 ± 0.79 µM). OSS_128167, the only commercially available SIRT6 inhibitor, also shows a similar weak inhibitory effect with an IC₅₀ of 89 µM [16]. Furthermore, SIRT6 inhibitors (compounds 1, 3 and 8) with a quinazolinedione structure were found with an IC₅₀ of 106 ± 16, 37 ± 2 and 49 ± 4 µM, respectively [17]. Compared with these inhibitors, 8a demonstrated a better inhibitory effect on SIRT6. We identified that 8a directly bound to SIRT6 protein, which was confirmed by biolayer interferometry (Octet) assays and CETSA assay. Moreover, we found 8a was a non-competitive inhibitor by enzymatic kinetic assay.

Previous studies of SIRT6 inhibitors have the ability to modulate proliferation, apoptosis, migration, invasion and cycle of cancer cells [33, 47]. We further confirmed that compound 8a induced pancreatic cancer cell growth inhibition. The PI3K/AKT/mTOR and MAPK/ERK pathways are important regulators for the cell proliferation and survival [30, 31]. RNA-seq data indicated that SIRT6 was functionally enriched in the PI3K/AKT signaling pathway, and knockdown or inhibition of SIRT6 could inhibit the activation of PI3K/AKT and ERK signaling pathway [32, 33, 48]. Our data suggested that 8a could inhibited pancreatic cancer cell proliferation by inhibiting PI3K/AKT/mTOR and ERK signal pathway. The cell cycle plays a pivotal role in the modulation of the growth and proliferation of cells, regulation of DNA damage repair during the development of organisms, and occurrence of diseases [49, 50]. We found that 8a significantly induces G0/G1 cell cycle arrest and apoptosis in pancreatic cancer cell. Taken together, these data offer evidence the potential of SIRT6 as an anti-pancreatic cancer drug target. 8a may be a potential anti-pancreatic cancer drug or lead drug, providing a new direction for drug design against pancreatic cancer.

Gemcitabine is the first-line chemotherapy for pancreatic cancer [2]. However, high rate of chemoresistance severely impedes its efficacy as a cornerstone of pancreatic cancer chemotherapy [2, 3, 51]. We found that the combination of 8a and gemcitabine synergistically inhibited the proliferation of pancreatic cancer cells. Some studies also showed that gemcitabine activates AKT and ERK signaling pathways to cause resistance in pancreatic cancer [36, 37]. Several agents including PI3K inhibitor (LY294002) or AKT inhibitor (MK2206) have been demonstrated alone or in combinations with DNA-targeted drugs (gemcitabine and 5-fluorouracil) in pancreatic cancer [52, 53]. Our data suggested that compound 8a reversed the activation of AKT and ERK signaling pathways induced by gemcitabine and augment the efficacy of gemcitabine. Therefore, the ability of compound 8a to inhibit AKT and ERK signaling pathways may be an important reason for its synergistic effect with gemcitabine.

The nucleoside analogue gemcitabine inhibits DNA synthesis and ribonucleotide reductase, therefore inducing significant DNA damage which is needed for subsequent cancer cell apoptosis [41]. However, the existence of DNA repair pathways reduces cancer cell sensitivity to gemcitabine [39–41]. SIRT6 has been described to be involved in regulating DNA repair, thereby influencing the resistance of cancer cells to DNA damaging agents [42]. Considering the essential role of SIRT6 in the DNA damage repair pathway, we evaluated the possibility that SIRT6 inhibitors may be involved in chemoresistance. Our results suggested that 8a impaired DNA repair activity and allowed accumulation of DNA damage resulted from gemcitabine and subsequently induced cell apoptosis. These results were consistent with other reports that SIRT6 inhibitors sensitize cancer cells to gemcitabine via inducing DNA damage and apoptosis [15, 17]. More importantly, in solid tumors, 8a significantly inhibited the tumor proliferation, induced apoptosis and improved pancreatic cancer sensitivity to gemcitabine treatment. These experiments demonstrate for the first time that small molecule SIRT6 inhibitors promote antitumor drug sensitivity in vivo tumor models. Our result also suggests that SIRT6 inhibitor might be helpful for improving sensitivity of DNA-target chemotherapeutic agents in cancer and we might conduct more in-depth research on this aspect in the future.

In conclusion, in this investigation, we identified a potent SIRT6 inhibitor compound 8a with promising inhibition efficacy. More importantly, 8a exhibited considerable anti-PDAC activities and enhances sensitivity of PDAC to gemcitabine in vitro and in vivo by inhibiting cell proliferation and survival signaling pathways, including AKT/mTOR/P70S6K and ERK signaling pathways, and DNA repair. Overall, 8a could be a promising therapeutic candidate for sensitizing pancreatic cancer to gemcitabine, and it is worthy of further intensive study.

AUTHOR CONTRIBUTIONS

TJ, YG and WY designed this study. NS conducted the main experiments and manuscript writing with the assistance of XW and ZL. XG synthesized compounds with the assistance of SZ and RY. DC and JY-W conducted histological analyses. All authors have read and approved the final manuscript.

ACKNOWLEDGEMENTS

The authors are grateful for financial support granted by the Shandong Province Special Fund “Frontier Technology and Free Exploration” Project of Pilot National Laboratory for Marine Science and Technology (Qingdao) (No. 8-01), the Natural Science Foundation of China (Grant Nos. 82073759, 31870795, and 81871868), the Fund of Greater Bay Area Institute of Precision Medicine (Guangzhou) (No. IPM2021C009), National Science and Technology Major Project for Significant New Drugs Development (2018ZX09735004), the Fundamental Research Funds for the Central Universities (No. 202042011).

COMPETING INTERESTS

The authors declare no other conflicts of interest.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All aspects of this study were approved by Institutional Research Ethics Committee of Ocean University of China.

ASSOCIATED CONTENT

The online version contains supplementary material available at www.nature.com/cddis/index.html

Supplementary Information

Copies of 1H and 13C NMR spectra of the final compounds; copies of HRMS (ESI) spectra of compounds: 5c, 6b, 8a; other experimental data of compound 8a in vitro and in vivo.

Correspondence and requests for materials should be addressed to Wengong Yu, Yuchao Gu, and Tao Jiang.

Reprints and permission information is available at http://www.nature.com/

reprints

DATA AVAILABILITY

All data are available from the corresponding author upon reasonable request.

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Discovery of a Pyrrole-pyridinimidazole Derivative as Novel SIRT6 Inhibitor for Sensitizing Pancreatic Cancer Cells to Gemcitabine

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Abstract

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Introduction

Materials And Methods

Results

Discovery of Hit Compounds with the Aid of Molecular Docking-Based Virtual Screening and Biological Activity Test.

Compound 8a Inhibits SIRT6 Deacetylation Activity at Cellular Level

Compound 8a is a Non-competitive Inhibitor of SIRT6

Compound 8a Inhibits Cell Proliferation, Induces Cell-cycle Arrest and Apoptosis in PDAC Cells

Compound 8a Sensitizes PDAC Cells to Gemcitabine by Inhibiting Cell Survival Signaling

Compound 8a Enhances Gemcitabine-induced DNA Damage

Compound 8a Augmented the Antitumor Effects of Gemcitabine in BXPC-3 Tumor xenografts

Discussion

Declarations

References

table

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Supplementary Files

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