A High-Throughput Screen to Identify Inhibitors of Aromatase (CYP19)

Stresser, David M.; Turner, Stephanie D.; McNamara, J. Judson; Stocker, Penny; Miller, Vaughn P.; Crespi, Charles L.; Patten, Christopher

doi:10.1006/abio.2000.4729

Cited by 127 publications

(86 citation statements)

References 17 publications

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“…The velocity was plotted as a function of testosterone concentration, and the K m of testosterone was calculated using the Michaelis-Menten equation ( Figure 2B). The calculated K m of testosterone was 23.37±4.67 nmol/L, which corresponds to data reported in previous studies [24] . To allow for IC 50 measurements of a similar magnitude K i for competitive inhibitors, we selected an optimized assay condition of 30 nmol/L testosterone.…”

Section: Resultssupporting

confidence: 90%

“…The substrate dose-response curve indicated that when testosterone was used at concentrations above 100 nmol/L, the fluorescent signal may have experienced interference due to excessive substrate for the analogical structures of estradiol and testosterone. However, the Michaelis-Menten fit of the reaction velocity as a function of the substrate resulted in an approximate K m value less than 30 nmol/L for testosterone, which was in agreement with an earlier reported value [24] . Thus, the optimized assay condition of 30 nmol/L testosterone was selected in our protocol.…”

supporting

confidence: 91%

“…However, these methods require strict experimental environments and are not amenable for screening large compound libraries [23,44] . Moreover, the purified aromatase used in our assay may avoid many of the variables that affect compound performance on target binding and signal reading such as cell membrane permeability, serum stability, plasma protein binding, and the interference of other microsome enzymes in catalyzing the substrate.Here, we reported a rational approach for discovering novel aromatase inhibitors with a robust HTRF detection method that avoids the limitations of currently available methods [23,24,45] . The detection signal is inversely proportional to the estradiol concentration because the HTRF detection method is a competition assay.…”

mentioning

confidence: 99%

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

Lao

et al. 2014

Acta Pharmacol Sin

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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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Section: Resultssupporting

confidence: 90%

supporting

confidence: 91%

mentioning

confidence: 99%

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

Lao

et al. 2014

Acta Pharmacol Sin

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“…However, these assays require the use of radioactive materials and the detection procedures are time-and labor-consuming, which means they are not suitable for high-throughput screening. An alternative fluorescent cell-free method has been developed using recombinant human aromatase protein and dibenzylfluorescein, but it cannot detect aromatase induction because it utilizes a cell-free system (Stresser et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

“…An in vitro recombinant expressed aromatase activity assay was conducted as described previously with minor modifications (Stresser et al, 2000;Maiti et al, 2007). In brief, the test compounds (5 ml) were preincubated with an NADPH regenerating system (45 ml of 2.6 mM NADP þ , 7.6 mM glucose-6-phosphate, 0.8 U/ml glucose-6-phosphate dehydrogenase, and 1 mg/ml albumin, in 50 mM potassium phosphate, pH 7.4) for 10 min at 37 1C before 50 ml of the enzyme and substrate mixture (40 pM recombinant aromatase and 0.4 mM dibenzylfluorescein in 50 mM potassium phosphate, pH 7.4) were added.…”

Section: Recombinant Expressed Aromatase Activity Assaymentioning

confidence: 99%

Egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae promote the biosynthesis of estrogen by aromatase

Yang

et al. 2012

European Journal of Pharmacology

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Synthesis of Resveratrol Derivatives and In Vitro Screening for Potential Cancer Chemopreventive Activities

Orsini

Verotta

Klimo

et al. 2016

Archiv der Pharmazie

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New resveratrol (trans-3,4',5-trihydroxystilbene) analogs were synthesized and screened for their in vitro cancer chemopreventive potential using various bioassays relevant for the prevention of carcinogenesis in humans: two assays to detect modulators of carcinogen metabolism (Cyp1A inhibition; determination of NAD(P)H/quinone reductase (QR) activity), three assays to identify radical scavenging and antioxidant properties (DPPH, ORAC, superoxide anion radicals in differentiated HL-60 cells), four assays to determine anti-inflammatory and anti-hormonal effects (iNOS, Cox-1 and aromatase inhibition, anti-estrogenic potential). 3,4',5-Tri-O-methyl resveratrol 1a was about sevenfold more active than resveratrol in inhibiting Cyp1A activity, it was a potent inducer of QR activity, and it showed pure anti-estrogenic activity (whereas resveratrol is a known mixed estrogen (ant)agonist with both estrogenic and anti-estrogenic properties). Dual estrogen ant-/agonist activity was restored in the mono-O-benzyl-substituted derivatives 4b (4'-O-benzyl resveratrol) and 5b (3-O-benzyl resveratrol). With respect to aromatase inhibition (Cyp19), which provided the highest number of actives, the benzyl-substituted series was more potent than the methyl-substituted derivatives of resveratrol, and 3-O-benzyl resveratrol 5b was about eightfold more active than resveratrol. Overall, 3,4',5-tri-O-pivaloyl resveratrol oxide 7c was identified as a potent inducer of phase 2 enzymes concomitant with inhibition of LPS-mediated iNOS induction.

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A High-Throughput Screen to Identify Inhibitors of Aromatase (CYP19)

Cited by 127 publications

References 17 publications

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

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

Egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae promote the biosynthesis of estrogen by aromatase

Synthesis of Resveratrol Derivatives and In Vitro Screening for Potential Cancer Chemopreventive Activities

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