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Discovery of novel aromatase inhibitors using a homogeneous time-resolved fluorescence assay.

Ji JZ, Lao KJ, Hu J, Pang T, Jiang ZZ, Yuan HL, Miao JS, Chen X, Ning SS, Xiang H, Guo YM, Yan M, Zhang LY - Acta Pharmacol. Sin. (2014)

Bottom Line: Among the 7000 compounds, 4 hits (XHN22, XHN26, XHN27 and triptoquinone A) were found to inhibit aromatase with IC50 values of 1.60±0.07, 2.76±0.24, 0.81±0.08 and 45.8±11.3 μmol /L, respectively.Moreover, the most potent hit XHN27 at 10 and 50 μmol/L inhibited the proliferation of T47D cells by 45.3% and 35.2%, respectively.The docking study revealed that XHN27 docked within the active site of aromatase and might form a hydrogen bond and had a π-cation interaction with amino acid residues of the protein.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China.

ABSTRACT

Aim: Aromatase is an important target for drugs to treat hormone-dependent diseases, including breast cancer. The aim of this study was to develop a homogeneous time-resolved fluorescence (HTRF) aromatase assay suitable for high-throughput screening (HTS).

Methods: A 384-well aromatase HTRF assay was established, and used to screen about 7000 compounds from a compound library. Anti-proliferation activity of the hit was evaluated using alamarBlue(R) assay in a hormone-dependent breast cancer cell line T47D. Molecular docking was conducted to elucidate the binding mode of the hit using the Discovery Studio program.

Results: The Z' value and signal to background (S/B) ratio were 0.74 and 5.4, respectively. Among the 7000 compounds, 4 hits (XHN22, XHN26, XHN27 and triptoquinone A) were found to inhibit aromatase with IC50 values of 1.60±0.07, 2.76±0.24, 0.81±0.08 and 45.8±11.3 μmol /L, respectively. The hits XHN22, XHN26 and XHN27 shared the same chemical scaffold of 4-imidazolyl quinoline. Moreover, the most potent hit XHN27 at 10 and 50 μmol/L inhibited the proliferation of T47D cells by 45.3% and 35.2%, respectively. The docking study revealed that XHN27 docked within the active site of aromatase and might form a hydrogen bond and had a π-cation interaction with amino acid residues of the protein.

Conclusion: XHN27, an imidazolyl quinoline derivative of flavonoid, is a potent aromatase inhibitor with anti-proliferation activity against breast cancer in vitro. The established assay can be used in HTS for discovering novel aromatase inhibitor.

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Effects of letrozole, BCA, and XHN27 on the proliferation of T47D cell cultured with 10 nmol/L Δ4A (A, B) or 1 nmol/L E2 (C, D). Cells were cultured with different concentrations of each compound (0.2–50 μmol/L) for 3 d (A, C) or 6 d (B, D). Values are represented as mean±SEM of three independent experiments (n=3). Significant differences between the compounds-treated groups versus negative control are denoted by bP<0.05, cP<0.01. One-way ANOVA followed by Dunnett multiple comparison post-test. Significant differences between the E2-treated cells (dashed lines) versus Δ4A-treated cells (solid lines) after administration with XHN27 are denoted by bP<0.05, cP<0.01. Significant differences between the E2-treated cells versus Δ4A-treated cells after administration with letrozole are denoted by eP<0.05, fP<0.01.
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fig6: Effects of letrozole, BCA, and XHN27 on the proliferation of T47D cell cultured with 10 nmol/L Δ4A (A, B) or 1 nmol/L E2 (C, D). Cells were cultured with different concentrations of each compound (0.2–50 μmol/L) for 3 d (A, C) or 6 d (B, D). Values are represented as mean±SEM of three independent experiments (n=3). Significant differences between the compounds-treated groups versus negative control are denoted by bP<0.05, cP<0.01. One-way ANOVA followed by Dunnett multiple comparison post-test. Significant differences between the E2-treated cells (dashed lines) versus Δ4A-treated cells (solid lines) after administration with XHN27 are denoted by bP<0.05, cP<0.01. Significant differences between the E2-treated cells versus Δ4A-treated cells after administration with letrozole are denoted by eP<0.05, fP<0.01.

Mentions: To examine the pharmacological properties of the hit compounds on aromatase, cell proliferation assays were performed. T47D cells were treated with Δ4A and increasing concentrations of XHN27, BCA or letrozole for 3 d and 6 d, and the inhibition of cell proliferation was evaluated using the alamarBlue® assay. Letrozole was a more potent inhibitor of cell proliferation than XHN27 and BCA (Figure 6A, 6B). The inhibition rate of letrozole (3 μmol/L) was 54.6% after 6 d (Figure 6B), while XHN27 at 50 μmol/L was 45.3%, and 35.2% at 10 μmol/L. Moreover, XHN27 exhibited increased cytotoxicity against T47D cells compared with BCA, which inhibited T47D cell proliferation by 30.6% at 50 μmol/L and 18.7% at 10 μmol/L after 6 d.


Discovery of novel aromatase inhibitors using a homogeneous time-resolved fluorescence assay.

Ji JZ, Lao KJ, Hu J, Pang T, Jiang ZZ, Yuan HL, Miao JS, Chen X, Ning SS, Xiang H, Guo YM, Yan M, Zhang LY - Acta Pharmacol. Sin. (2014)

Effects of letrozole, BCA, and XHN27 on the proliferation of T47D cell cultured with 10 nmol/L Δ4A (A, B) or 1 nmol/L E2 (C, D). Cells were cultured with different concentrations of each compound (0.2–50 μmol/L) for 3 d (A, C) or 6 d (B, D). Values are represented as mean±SEM of three independent experiments (n=3). Significant differences between the compounds-treated groups versus negative control are denoted by bP<0.05, cP<0.01. One-way ANOVA followed by Dunnett multiple comparison post-test. Significant differences between the E2-treated cells (dashed lines) versus Δ4A-treated cells (solid lines) after administration with XHN27 are denoted by bP<0.05, cP<0.01. Significant differences between the E2-treated cells versus Δ4A-treated cells after administration with letrozole are denoted by eP<0.05, fP<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125720&req=5

fig6: Effects of letrozole, BCA, and XHN27 on the proliferation of T47D cell cultured with 10 nmol/L Δ4A (A, B) or 1 nmol/L E2 (C, D). Cells were cultured with different concentrations of each compound (0.2–50 μmol/L) for 3 d (A, C) or 6 d (B, D). Values are represented as mean±SEM of three independent experiments (n=3). Significant differences between the compounds-treated groups versus negative control are denoted by bP<0.05, cP<0.01. One-way ANOVA followed by Dunnett multiple comparison post-test. Significant differences between the E2-treated cells (dashed lines) versus Δ4A-treated cells (solid lines) after administration with XHN27 are denoted by bP<0.05, cP<0.01. Significant differences between the E2-treated cells versus Δ4A-treated cells after administration with letrozole are denoted by eP<0.05, fP<0.01.
Mentions: To examine the pharmacological properties of the hit compounds on aromatase, cell proliferation assays were performed. T47D cells were treated with Δ4A and increasing concentrations of XHN27, BCA or letrozole for 3 d and 6 d, and the inhibition of cell proliferation was evaluated using the alamarBlue® assay. Letrozole was a more potent inhibitor of cell proliferation than XHN27 and BCA (Figure 6A, 6B). The inhibition rate of letrozole (3 μmol/L) was 54.6% after 6 d (Figure 6B), while XHN27 at 50 μmol/L was 45.3%, and 35.2% at 10 μmol/L. Moreover, XHN27 exhibited increased cytotoxicity against T47D cells compared with BCA, which inhibited T47D cell proliferation by 30.6% at 50 μmol/L and 18.7% at 10 μmol/L after 6 d.

Bottom Line: Among the 7000 compounds, 4 hits (XHN22, XHN26, XHN27 and triptoquinone A) were found to inhibit aromatase with IC50 values of 1.60±0.07, 2.76±0.24, 0.81±0.08 and 45.8±11.3 μmol /L, respectively.Moreover, the most potent hit XHN27 at 10 and 50 μmol/L inhibited the proliferation of T47D cells by 45.3% and 35.2%, respectively.The docking study revealed that XHN27 docked within the active site of aromatase and might form a hydrogen bond and had a π-cation interaction with amino acid residues of the protein.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China.

ABSTRACT

Aim: Aromatase is an important target for drugs to treat hormone-dependent diseases, including breast cancer. The aim of this study was to develop a homogeneous time-resolved fluorescence (HTRF) aromatase assay suitable for high-throughput screening (HTS).

Methods: A 384-well aromatase HTRF assay was established, and used to screen about 7000 compounds from a compound library. Anti-proliferation activity of the hit was evaluated using alamarBlue(R) assay in a hormone-dependent breast cancer cell line T47D. Molecular docking was conducted to elucidate the binding mode of the hit using the Discovery Studio program.

Results: The Z' value and signal to background (S/B) ratio were 0.74 and 5.4, respectively. Among the 7000 compounds, 4 hits (XHN22, XHN26, XHN27 and triptoquinone A) were found to inhibit aromatase with IC50 values of 1.60±0.07, 2.76±0.24, 0.81±0.08 and 45.8±11.3 μmol /L, respectively. The hits XHN22, XHN26 and XHN27 shared the same chemical scaffold of 4-imidazolyl quinoline. Moreover, the most potent hit XHN27 at 10 and 50 μmol/L inhibited the proliferation of T47D cells by 45.3% and 35.2%, respectively. The docking study revealed that XHN27 docked within the active site of aromatase and might form a hydrogen bond and had a π-cation interaction with amino acid residues of the protein.

Conclusion: XHN27, an imidazolyl quinoline derivative of flavonoid, is a potent aromatase inhibitor with anti-proliferation activity against breast cancer in vitro. The established assay can be used in HTS for discovering novel aromatase inhibitor.

Show MeSH
Related in: MedlinePlus