A Modular Approach to Triazole-Containing Chemical Inducers of Dimerisation for Yeast Three-Hybrid Screening

Abstract

The yeast three-hybrid (Y3H) approach shows considerable promise for the unbiased identification of novel small molecule-protein interactions. In recent years, it has been successfully used to link a number of bioactive molecules to novel protein binding partners. However despite its potential importance as a protein target identification method, the Y3H technique has not yet been widely adopted, in part due to the challenges associated with the synthesis of the complex chemical inducers of dimerisation (CIDs). The development of a modular approach using potentially “off the shelf” synthetic components was achieved and allowed the synthesis of a family of four triazole-containing CIDs, MTX-Cmpd2.2-2.5. These CIDs were then compared using the Y3H approach with three of them giving a strong positive interaction with a known target of compound 2, TgCDPK1. These results showed that the modular nature of our synthetic strategy may help to overcome the challenges currently encountered with CID synthesis and should contribute to the Y3H approach reaching its full potential as an unbiased target identification strategy.

Yeast Three-Hybrid Screen Identifies TgBRADIN/GRA24 as a Negative Regulator of Toxoplasma gondii Bradyzoite Differentiation

“…As a first step in determining how Compound 2 enhances differentiation, we used yeast three-hybrid (Y3H) analysis [ 32 , 42 – 46 ] to examine the target profile of Compound 2 in T . gondii .…”

“…Of the three kinases, only TgCDPK1 interacted with MTX-Cmpd2.1 in the Y3H system ( Fig. 3A and B ; see also [ 46 ]). Reporter activation with TgCDPK1 was dose dependent for MTX-Cmpd2.1 concentrations in the micromolar range ( Fig.…”

“…Reporter activation with TgCDPK1 was dose dependent for MTX-Cmpd2.1 concentrations in the micromolar range ( Fig. 3C and D ; [ 46 ]). Another methotrexate-containing CID (MTX-118793.1 [ 45 ]) did not activate any reporter genes, demonstrating that the interaction between TgCDPK1 and MTX-Cmpd2.1 is CID-specific ( Fig.…”

Efficient Genome Engineering of Toxoplasma gondii Using CRISPR/Cas9

“…All plates were allowed to incubate for a total of eight days before staining with crystal violet. C2 was synthesized by N. Westwood and F. Tran as previously described [4] , [9] , [10] .…”

Structure-activity relationship studies of lipophilic teicoplanin pseudoaglycon derivatives as new anti-influenza virus agents

“…Propargylamine and alkyl thiols were purchased from Sigma Aldrich Chemical Co., dansyl chloride, benzenesulfonyl chloride, pacetamidobenzenesulfonyl chloride, biphenylsulfonyl chloride were purchased from Tokyo Chemical Industry Co., Ltd., alkyl sulfonyl chlorides were prepared from the corresponding thiols using a literature method [28], triethylene glycol 2-aminoethyl propargyl ether, [29] 1-mercapto-11-hydroxy-3,6,9-trioxaundecane, [30] 1-azido-11-hydroxy-3,6,9-trioxaundecane, [31] 2,3dibromomaleimide 3 [32], teicoplanin pseudoaglycon 11 [9] and azido teicoplanin pseudoaglycon 25 [9] were also prepared according to literature procedures. TLC analysis was performed on Kieselgel 60 F 254 (Merck) silica gel plates with visualization by immersing in ammonium-molibdate solution followed by heating or Pauly-reagent in the case of teicoplanin-derivatives.…”