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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 optimal concentrations of substrate, enzyme and co-factor NADPH were determined. (A) The HTRF signals generated by increasing amount of testosterone in the presence and absence of 15 nmol/L aromatase. (B) Michaelis-Menton plots of aromatase activity as a function of the concentration of the substrate testosterone. (C) Time course at various concentrations of aromatase. The detection time was set at 0, 15, 30, 45, 60, 120, and 240 min. (D) Activity of aromatase was raised with the increasing concentration of NADPH (0.3 nmol/L to 10 000 nmol/L). Values are represented as mean±SEM of three independent experiments (n=3).
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fig2: The optimal concentrations of substrate, enzyme and co-factor NADPH were determined. (A) The HTRF signals generated by increasing amount of testosterone in the presence and absence of 15 nmol/L aromatase. (B) Michaelis-Menton plots of aromatase activity as a function of the concentration of the substrate testosterone. (C) Time course at various concentrations of aromatase. The detection time was set at 0, 15, 30, 45, 60, 120, and 240 min. (D) Activity of aromatase was raised with the increasing concentration of NADPH (0.3 nmol/L to 10 000 nmol/L). Values are represented as mean±SEM of three independent experiments (n=3).

Mentions: To define the standard titration for testosterone, the Km of the substrate was estimated. Because there is cross reactivity between testosterone and estradiol, testosterone (>150 nmol/L) could interfere with estradiol detection in our assay (Figure 2A). The velocity was plotted as a function of testosterone concentration, and the Km of testosterone was calculated using the Michaelis-Menten equation (Figure 2B). The calculated Km of testosterone was 23.37±4.67 nmol/L, which corresponds to data reported in previous studies24. To allow for IC50 measurements of a similar magnitude Ki for competitive inhibitors, we selected an optimized assay condition of 30 nmol/L testosterone.


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 optimal concentrations of substrate, enzyme and co-factor NADPH were determined. (A) The HTRF signals generated by increasing amount of testosterone in the presence and absence of 15 nmol/L aromatase. (B) Michaelis-Menton plots of aromatase activity as a function of the concentration of the substrate testosterone. (C) Time course at various concentrations of aromatase. The detection time was set at 0, 15, 30, 45, 60, 120, and 240 min. (D) Activity of aromatase was raised with the increasing concentration of NADPH (0.3 nmol/L to 10 000 nmol/L). 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

fig2: The optimal concentrations of substrate, enzyme and co-factor NADPH were determined. (A) The HTRF signals generated by increasing amount of testosterone in the presence and absence of 15 nmol/L aromatase. (B) Michaelis-Menton plots of aromatase activity as a function of the concentration of the substrate testosterone. (C) Time course at various concentrations of aromatase. The detection time was set at 0, 15, 30, 45, 60, 120, and 240 min. (D) Activity of aromatase was raised with the increasing concentration of NADPH (0.3 nmol/L to 10 000 nmol/L). Values are represented as mean±SEM of three independent experiments (n=3).
Mentions: To define the standard titration for testosterone, the Km of the substrate was estimated. Because there is cross reactivity between testosterone and estradiol, testosterone (>150 nmol/L) could interfere with estradiol detection in our assay (Figure 2A). The velocity was plotted as a function of testosterone concentration, and the Km of testosterone was calculated using the Michaelis-Menten equation (Figure 2B). The calculated Km of testosterone was 23.37±4.67 nmol/L, which corresponds to data reported in previous studies24. To allow for IC50 measurements of a similar magnitude Ki for competitive inhibitors, we selected an optimized assay condition of 30 nmol/L testosterone.

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