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

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 IC 50 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.Keywords: aromatase; breast cancer; imidazolyl quinoline; triptoquinone A; homogeneous time-resolved fluorescence assay; highthroughput screening; molecular docking; drug discovery Acta Pharmacologica Sinica (2014Sinica ( ) 35: 1082Sinica ( -1092 doi: 10.1038/aps.2014 [12,13] . Previous studies have demonstrated that AIs provide an increased survival benefit compared with other therapies and have acceptable toxicity profiles with decreased virginal bleeding and thromboembolism and increased rash, diarrhea and vomiting [14,15] . As AIs sometimes have more severe bone, brain and heart side effects, research for alternative compounds is necessary [15][16][17] .Natural products, extracted from traditional medicines and foods, may be helpful for discovering novel AIs that may selectively target aromatase in the breast and reduce systemic toxicity [18] . Among these compounds, flavonoids [19] are the most commonly investigated agents due to their prominent aromatase inhibitory activity and high breast selectivity [18] . Moreover, flavonoids may modulate the multi-step process of carcinogenesis through cellular and molecular mechanisms [19] . Biochanin A (BCA), isolated from red clover (Trifolium pretense), is a common isoflavone product that can inhibit aromatase activity and cell growth in MCF-7 cells [20] . Furthermore, cyp19a1 mRNA abundance was significantly reduced by BCA through promoter regulation in SK-BR-3 cells [20] .The classical tritiated water release assay [21,22] is ...

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