An orthogonal dexamethasone–trimethoprim yeast three-hybrid system

Abstract

The yeast three-hybrid assay is an important tool for the detection of protein-ligand interactions in vivo and has recently been used successfully for the discovery of novel drug targets and the directed evolution of enzymes[1–6]. Schreiber and co-workers developed the first chemical inducer of dimerization (CID)3, a dimer of the immunosuppressant FK506[7]. Building from this work, a number of yeast three-hybrid systems based on different CIDs have been reported[2, 8]. We previously developed and optimized a three-hybrid system built around the small molecule CID dexamethasone-methotrexate (Dex-Mtx)[9–11]. The Dex and Mtx ligands were chosen because of their high affinities for their respective protein receptors, the rat glucocorticoid receptor and Escherichia coli dihydrofolate reductase, respectively[12, 13]. However, we hypothesized that the cross-reactivity of Mtx with endogenous DHFR in the yeast cells could impair transcription activation by Dex-Mtx in the yeast three-hybrid assay. In order to overcome this partial limitation we set out to design a CID that would selectively bind to E. coli DHFR and not to endogenous yeast DHFR. As an alternative to Mtx, we chose the DHFR inhibitor trimethoprim (TMP), which is known for its selectivity for bacterial forms of DHFR[14]. Studies have confirmed that while Mtx inhibits growth of wild type Saccrharomyces cerevisiae, TMP does not[15], suggesting that TMP could be a superior CID in yeast. Here we report the design, synthesis, and in vivo activity of Dex-TMP in the yeast three-hybrid assay. By analogy to our dexamethasone-methotrexate system, we chose to build a heterodimeric CID using the ligand-receptor pairs dexamethasone (Dex)-rat glucocorticoid receptor (GR) and trimethoprim (TMP)-E. coli dihydrofolate reductase (DHFR). Both Dex and TMP can be modified without disrupting receptor binding, making them suitable CIDs[16–18]. Both ligands are cell-permeable and commercially available. The ligand-receptor pair Dex-GR has a KD of 5 nM and has been used successfully in yeast three-hybrid systems[9]. E. coli dihydrofolate reductase (DHFR) has a KI of 1.3 nM for inhibition by TMP[14]. We anticipated that the two interactions would be sufficiently strong to induce protein dimerization and transcription activation in the yeast three-hybrid assay.