An old system for new applications: perspectives of the yeast-3-hybrid screening for natural product research

Identifying protein targets of small molecules —commonly referred to as ‘target fishing’— is challenging and typically requires intensive effort [ 1 ]. The process involves investigation of numerous possible interaction partners generated by high-throughput screening techniques, followed by validation of promising candidates. This demands a multidisciplinary approach and a high level of technical expertise. As an alternative to classical affinity-based proteomics, the yeast three-hybrid (Y3H) system provides a valuable strategy for target deconvolution in living cells. First introduced by Licitra and Liu in 1996 [ 3 ], this method offers unique advantages, particularly for detecting interactions under physiological conditions. In our group, we have implemented an optimized version of the Y3H system and successfully used it to reveal a previously unidentified protein target of the widely prescribed contraceptive ethinylestradiol [ 4 ]. Natural products have drawn considerable scientific attention due to their biological properties. However, despite this interest, our understanding of their direct interactions with proteins remains limited. While small molecules can have significant biological effects, their precise protein targets and mechanisms of action often remain unidentified. Yet, this knowledge is essential for evaluating a compound’s potential as a therapeutic agent [ 2 ]. Frequently, the key to explaining a compound’s cellular impact lies in uncovering the molecular pathways it influences. We are currently employing an improved Y3H screening to explore protein targets of natural products, and are working to expand its capabilities to improve its adaptability and effectiveness in the context of natural product research. Publication History Article published online: 05 March 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany