Aromatase (CYP19) catalyzes the last biosynthetic step of estrogens in mammals and is a primary drug target for hormone-related breast cancer. However, treatment with aromatase inhibitors is often associated with...

Heterologous expression of the G protein–coupled estrogen receptor (GPER) comes with a suite of challenges intrinsic to membrane proteins. This receptor's low expression levels and tendency to form insoluble aggregates in Escherichia coli and yeast make it a difficult receptor‐target to study. In this unit, we detail steps to produce monomeric GPER using a precipitation‐based cell‐free system. We provide information on the DNA construct for expression, the pipetting scheme for the reaction supplements to generate a master mix, and the cell‐free reaction setup. In the last portion of this unit, we outline steps for solubilization and purification, and we provide a viable method for qualitatively observing functionality by liquid chromatography–mass spectrometry detection. © 2019 by John Wiley & Sons, Inc.

Aromatase (Cyp19) catalyzes the last biosynthetic step of estrogens in mammals and is a primary therapeutic target for postmenopausal women with hormone-related breast cancer. However, treatment with aromatase inhibitors is often associated with adverse effects and drug resistance. In this study, we used virtual screening to target a potential cytochrome P450 reductase binding site to discover four novel non-steroidal aromatase inhibitors. The inhibitors have potencies comparable to the noncompetitive tamoxifen metabolite, endoxifen. Our two most potent inhibitors, AR11 and AR13, exhibit mixed-type and competitive-type inhibition. The cytochrome P450 reductase binding site likely acts as a transient binding site. Modeling shows that our inhibitors actually bind better at various sites near the catalytic site. These structures may serve as chemical scaffolds to inhibit aromatase with different adverse effects profiles than clinically used aromatase inhibitors.

No abstract