Rational evolution for altering the ligand preference of estrogen receptor alpha

• DOI: 10.1002/pro.4940
• Publication Date: 2024-03-21T09:55:16Z
• Authors: Roy Eerlings; Purvi Gupta; Xiao Yin Lee; Tien Nguyen; Sarah El Kharraz; Florian Handle; Elien Smeets; Lisa Moris; Wout Devlies; Bram Vandewinkel; Irina Thiry; Duy Tien Ta; Anton Gorkovskiy; Karin Voordeckers; Els Henckaerts; Vitor B. Pinheiro; Frank Claessens; Kevin J. Verstrepen; Arnout Voet; Christine Helsen
• Keywords: Synthetic Biology; Estrogen receptor beta; Estrogen receptor alpha; Estrogen-related receptor gamma; GPER

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting applicability this receptor. Altering its ligand preference chemicals choice solves hurdle but requires adaptation unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design multi-site-directed mutagenesis and directed evolution stably integrated variants Saccharomyces cerevisiae. This method yielded an estrogen variant, named TERRA, that lost responsiveness became activated tamoxifen, anti-estrogenic drug for breast cancer treatment. tamoxifen TERRA was maintained mammalian cells mice, even when fused Cre recombinase, expanding toolbox. Not only our platform transferable engineer any steroid receptor, it can also profile drug-resistance landscapes receptor-targeted therapies.