Chemical screening of an inhibitor for gibberellin receptors based on a yeast two-hybrid system Jung-Min Yoon a , Masatoshi Nakajima a,b,⇑ , Kiyoshi Mashiguchi a , Seung-Hyun Park a , Masato Otani a,b , Tadao Asami a,b a Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Japan article info Article history: Received 13 October 2012 Revised 14 November 2012 Accepted 5 December 2012 Available online 14 December 2012 Keywords: Gibberellin Inhibitor Receptor Screening Y2H abstract We applied a yeast two-hybrid (Y2H) system to the high-throughput monitoring of two proteins’ inter- action, a receptor for phytohormone gibberellin (GA) and its direct signal transducer DELLA. With this system, we screened inhibitors to the interaction. As a result, we discovered a chemical, 3-(2- thienylsulfonyl)pyrazine-2-carbonitrile (TSPC), and we confirmed that TSPC is an inhibitor for GA perception by in vitro and in planta evaluations. Ó 2012 Elsevier Ltd. All rights reserved. As one of the biochemical approaches for analyzing an inter- action of two macromolecules, we often utilize Y2H systems supplied by some manufacturers. To quantify the interaction, we generally measure the activity of any reporter enzyme, whose induction is designed in response to the interaction of two molecules. b-galactosidase is the most popular reporter, although yeasts need to be cracked-up to be able to measure their activity because this enzyme is not secreted from yeast cells. In this Letter, we utilized an another reporter a-galactosi- dase, whose use is often avoided because of its lower quantita- tive nature than b-galactosidase, even though the secretory characteristic of a-galactosidase could be advantageous to detect enzyme activity without any pretreatments. Based on reports of a-galactosidase, we established a high throughput Y2H system performed in 96-well plates, and then applied it to chemical screenings. The signal transduction of GA in plants is triggered by the perception of GA by its receptor GID1. The GID1-GA complex then shows an affinity to DELLAs, negative regulators of its sig- naling. The interaction between a DELLA and a GID1 represses DELLA’s negative function, which leads to transduction of the GA signal. 1,2 In our previous report, 3 we prepared yeast lines to evaluate the interaction between a GID1 and a DELLA from Arabidopsis, and all yeasts examined grew and showed the b- galactosidase activity in a GA-dependent manner under a nutri- tionally limited condition (SD-AHLWU) in which yeasts grow only when there is an interaction between the target macromol- ecules within them. Then we picked up one line which was used for the detection of an interaction between AtGID1b and RGL2, tried to cultivate it in liquid medium with a 96-deep-well plate, and we detected blue color that originated in the secretion of a- galactosidase only in the presence of GA (Fig. S1A). Hereafter we call this system SS1 (screening system 1). As expectedly, we con- firmed that an increase in GA in SS1 strengthened the blue color (Fig. S1B). Should an effective inhibitor exist in the system, the interaction of two molecules could be interrupted and then both the growth of yeast and the expression of a-galactosidase gene could be blocked (Fig. S2). We expected that inhibitors should decrease the amount of color in SS1 and that candidates could be easily visually selected. We thus began to screen chemicals that might inhibit the interaction of GID1 and DELLA by adding a commercially- supplied chemical library to SS1. After screening SS1 twice, we selected 268 chemicals that caused color to disappear with a good reproducibility (Fig. S1C). The disappearance of color in SS1 allows us judge a chemical as a positive candidate, even when that the chemical inhibits the growth of yeast, regardless 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.12.007 Abbreviations: GA, gibberellin; Y2H, yeast two-hybrid; qRT, quantitative reverse transcription. ⇑ Corresponding author. Tel.: +81 3 5841 5192; fax: +81 3 5841 8025. E-mail address: nkjm@pgr1.ch.a.u-tokyo.ac.jp (M. Nakajima). Bioorganic & Medicinal Chemistry Letters 23 (2013) 1096–1098 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl