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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

HTRF assay verification. (A) HTRF signal in the presence of known aromatase inhibitors, letrozole. Lines shown are fits giving IC50 of 26.0±6.6 nmol/L. (B) BCA is isoflavone natural product. Lines shown are fits giving IC50 of 30.0±3.3 μmol/L. Values are represented as mean±SEM of three independent experiments (n=3).
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fig3: HTRF assay verification. (A) HTRF signal in the presence of known aromatase inhibitors, letrozole. Lines shown are fits giving IC50 of 26.0±6.6 nmol/L. (B) BCA is isoflavone natural product. Lines shown are fits giving IC50 of 30.0±3.3 μmol/L. Values are represented as mean±SEM of three independent experiments (n=3).

Mentions: To further test the utility of the HTRF assay for measuring inhibitor potency, the IC50 value of letrozole, a known inhibitor of aromatase, was determined. Letrozole was dissolved in DMSO to make a stock solution of 10 mmol/L, and the solution was serially diluted using assay buffer. A representative concentration-response curve of letrozole was plotted using PRISM version 5.0 software (GraphPad Software Inc, CA, USA) (Figure 3A), and IC50 value was 26.0±6.6 nmol/L, which coincides with previously reported data36,37,38. Our results indicate that the aromatase HTRF assay is a potentially robust assay that can be used to determine the potency of aromatase inhibitors.


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)

HTRF assay verification. (A) HTRF signal in the presence of known aromatase inhibitors, letrozole. Lines shown are fits giving IC50 of 26.0±6.6 nmol/L. (B) BCA is isoflavone natural product. Lines shown are fits giving IC50 of 30.0±3.3 μmol/L. Values are represented as mean±SEM of three independent experiments (n=3).
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Related In: Results  -  Collection

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

fig3: HTRF assay verification. (A) HTRF signal in the presence of known aromatase inhibitors, letrozole. Lines shown are fits giving IC50 of 26.0±6.6 nmol/L. (B) BCA is isoflavone natural product. Lines shown are fits giving IC50 of 30.0±3.3 μmol/L. Values are represented as mean±SEM of three independent experiments (n=3).
Mentions: To further test the utility of the HTRF assay for measuring inhibitor potency, the IC50 value of letrozole, a known inhibitor of aromatase, was determined. Letrozole was dissolved in DMSO to make a stock solution of 10 mmol/L, and the solution was serially diluted using assay buffer. A representative concentration-response curve of letrozole was plotted using PRISM version 5.0 software (GraphPad Software Inc, CA, USA) (Figure 3A), and IC50 value was 26.0±6.6 nmol/L, which coincides with previously reported data36,37,38. Our results indicate that the aromatase HTRF assay is a potentially robust assay that can be used to determine the potency of aromatase inhibitors.

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