Using the yeast three-hybrid system for the identification of small molecule-protein interactions with the example of ethinylestradiol.

Pengyu Wang, Thomas Klassmüller, Cornelia A Karg, Maibritt Kretschmer, Stefan Zahler, Simone Braig, Franz Bracher, Angelika M Vollmar, Simone Moser

Biological chemistry 2022 Mar 28

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Since the first report on a yeast three-hybrid system, several approaches have successfully utilized different setups for discovering targets of small molecule drugs. Compared to broadly applied MS based target identification approaches, the yeast three-hybrid system represents a complementary method that allows for the straightforward identification of direct protein binders of selected small molecules. One major drawback of this system, however, is that the drug has to be taken up by the yeast cells in sufficient concentrations. Here, we report the establishment of a yeast three-hybrid screen in the deletion strain ABC9Δ, which is characterized by being highly permeable to small molecules. We used this system to screen for protein binding partners of ethinylestradiol, a widely used drug mainly for contraception and hormone replacement therapy. We identified procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2 or lysyl hydroxylase, LH2) as a novel direct target and were able to confirm the interaction identified with the yeast three-hybrid system by a complementary method, affinity chromatography, to prove the validity of the hit. Furthermore, we provide evidence for an interaction between the drug and PLOD2 in vitro and in cellulo. © 2022 Pengyu Wang et al., published by De Gruyter, Berlin/Boston.

Citation

Pengyu Wang, Thomas Klassmüller, Cornelia A Karg, Maibritt Kretschmer, Stefan Zahler, Simone Braig, Franz Bracher, Angelika M Vollmar, Simone Moser. Using the yeast three-hybrid system for the identification of small molecule-protein interactions with the example of ethinylestradiol. Biological chemistry. 2022 Mar 28;403(4):421-431