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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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Related in: MedlinePlus

The application of HTRF estradiol detection method in determining aromatase activity. (A) The standard curve of estradiol was generated from increasing the concentration of the standard. Estradiol was detected by the binding of homogenous time-resolved fluorescence (HTRF) donor anti-estradiol antibody conjugated with cryptate to d 2 acceptor conjugated estradiol, producing a time-resolved fluorescence resonance energy transfer signal. Enzyme-produced estradiol competes with this interaction and reduces overall time-resolved fluorescence resonance energy transfer signals. (B) The concentration-responsive curve of aromatase was generated by adding 30 nmol/L testosterone, 1 μmol/L NADPH and increasing amount of aromatase from 0.04 nmol/L to 60 nmol/L in the reaction system. The detection buffer was added after incubation for 1 h at 37 °C. Values are represented as mean±SEM of three independent experiments (n=3).
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fig1: The application of HTRF estradiol detection method in determining aromatase activity. (A) The standard curve of estradiol was generated from increasing the concentration of the standard. Estradiol was detected by the binding of homogenous time-resolved fluorescence (HTRF) donor anti-estradiol antibody conjugated with cryptate to d 2 acceptor conjugated estradiol, producing a time-resolved fluorescence resonance energy transfer signal. Enzyme-produced estradiol competes with this interaction and reduces overall time-resolved fluorescence resonance energy transfer signals. (B) The concentration-responsive curve of aromatase was generated by adding 30 nmol/L testosterone, 1 μmol/L NADPH and increasing amount of aromatase from 0.04 nmol/L to 60 nmol/L in the reaction system. The detection buffer was added after incubation for 1 h at 37 °C. Values are represented as mean±SEM of three independent experiments (n=3).

Mentions: A 384-well aromatase HTRF assay was established by quantifying estradiol production during the aromatase reaction. This detection method relies on competitive binding of exogenous, free estradiol disrupting the donor-acceptor complex. Therefore, the observed HTRF signal decreased with increasing amounts of product estradiol (Figure 1A and 1B). As it is typical for HTRF, the long lifetime of the donor fluorophore employed minimizes fluorescence interference due to buffers or test compounds.


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)

The application of HTRF estradiol detection method in determining aromatase activity. (A) The standard curve of estradiol was generated from increasing the concentration of the standard. Estradiol was detected by the binding of homogenous time-resolved fluorescence (HTRF) donor anti-estradiol antibody conjugated with cryptate to d 2 acceptor conjugated estradiol, producing a time-resolved fluorescence resonance energy transfer signal. Enzyme-produced estradiol competes with this interaction and reduces overall time-resolved fluorescence resonance energy transfer signals. (B) The concentration-responsive curve of aromatase was generated by adding 30 nmol/L testosterone, 1 μmol/L NADPH and increasing amount of aromatase from 0.04 nmol/L to 60 nmol/L in the reaction system. The detection buffer was added after incubation for 1 h at 37 °C. Values are represented as mean±SEM of three independent experiments (n=3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125720&req=5

fig1: The application of HTRF estradiol detection method in determining aromatase activity. (A) The standard curve of estradiol was generated from increasing the concentration of the standard. Estradiol was detected by the binding of homogenous time-resolved fluorescence (HTRF) donor anti-estradiol antibody conjugated with cryptate to d 2 acceptor conjugated estradiol, producing a time-resolved fluorescence resonance energy transfer signal. Enzyme-produced estradiol competes with this interaction and reduces overall time-resolved fluorescence resonance energy transfer signals. (B) The concentration-responsive curve of aromatase was generated by adding 30 nmol/L testosterone, 1 μmol/L NADPH and increasing amount of aromatase from 0.04 nmol/L to 60 nmol/L in the reaction system. The detection buffer was added after incubation for 1 h at 37 °C. Values are represented as mean±SEM of three independent experiments (n=3).
Mentions: A 384-well aromatase HTRF assay was established by quantifying estradiol production during the aromatase reaction. This detection method relies on competitive binding of exogenous, free estradiol disrupting the donor-acceptor complex. Therefore, the observed HTRF signal decreased with increasing amounts of product estradiol (Figure 1A and 1B). As it is typical for HTRF, the long lifetime of the donor fluorophore employed minimizes fluorescence interference due to buffers or test compounds.

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