An orthogonal dexamethasone-trimethoprim yeast three-hybrid system.

摘要: The yeast three-hybrid assay is an important tool for the detection of protein-ligand interactions in vivo and has recently been used successfully discovery novel drug targets directed evolution enzymes[1–6]. Schreiber co-workers developed first chemical inducer dimerization (CID)3, a dimer immunosuppressant FK506[7]. Building from this work, number systems based on different CIDs have reported[2, 8]. We previously optimized system built around small molecule CID dexamethasone-methotrexate (Dex-Mtx)[9–11]. Dex Mtx ligands were chosen because their high affinities respective protein receptors, rat glucocorticoid receptor Escherichia coli dihydrofolate reductase, respectively[12, 13]. However, we hypothesized that cross-reactivity with endogenous DHFR cells could impair transcription activation by Dex-Mtx assay. In order to overcome partial limitation set out design would selectively bind E. not DHFR. As alternative Mtx, chose inhibitor trimethoprim (TMP), which known its selectivity bacterial forms DHFR[14]. Studies confirmed while inhibits growth wild type Saccrharomyces cerevisiae, TMP does not[15], suggesting be superior yeast. Here report design, synthesis, activity Dex-TMP