Imidazole Derivative As a Novel Translation Inhibitor

Abstract

Searching for novel compounds with antibiotic activity and understanding their mechanism of action is extremely important. The ribosome is one of the main targets for antibiotics in bacterial cells. Even if the molecule does not suit the clinical application for whatever reasons, an investigation of its mechanism of action can deepen our understanding of the ribosome function. Such data can inform us on how the already used translational inhibitors can be modified. In this study, we demonstrate that 1-(2-oxo-2-((4-phenoxyphenyl).

Keywords: bacteria; compounds with antibiotic activity; imidazole derivatives; translation; translation inhibitor.

Fig. 1

Transferring 96 individual molecules onto the cell lawn

Fig. 2

(A) – the composition of the pDualrep2 reporter plasmid. (B) – induction of a two-color dual-reporter system sensitive to inhibitors of ribosome progression or DNA replication. Drops of erythromycin (right-hand side, 2 μg) and levofloxacin (left-hand side, 0.05 μg) were placed on the surface of an agar plate containing E. coli JW5503 cells transformed with the pDualrep2 plasmid. Expression of Katushka2S (red) is induced by translation inhibitors, whereas RFP expression (green) is induced upon DNA damage. (C) – induction of a two-color dual-reporter system induced by 1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3-(p-tolyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazol-1-ium chloride (30 μg)

Fig. 3

(A) – structural formula of the active compound 1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3-(p-tolyl)- 6,7-dihydro-5H-pyrrolo[1,2-a]imidazol-1-ium chloride (STOCK4S-33513). (B) – structural formula of the active compound analog 1-(2-((2,5-dimethoxyphenyl)amino)-2- oxoethyl)-3-(p-tolyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazol- 1-ium chloride (STOCK4S-37310). (C) – structural formula of the active compound analog 1-(2-((2-methoxyphenyl) amino)-2-oxoethyl)-3-(p-tolyl)-6,7-dihydro-5Hpyrrolo[ 1,2-a]imidazol-1-ium chloride (STOCK4S-72264)

Fig. 4

Protein synthesis inhibition with 200 μg/mL of 1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3-(p-tolyl)- 6,7-dihydro-5H-pyrrolo[1,2-a]imidazol-1-ium chloride using an in vitro cell-free translation system. The activity of luciferase synthesised using an in vitro cell-free translation system without translation inhibitors is taken as 100%

Fig. 5

The scheme of toeprinting assay on the RST1 template: lane 1 (STOCK4S-33513) corresponds to the in vitro cell-free translation system supplemented with 200 μg/mL 1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3- (p-tolyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazol-1-ium chloride; lane 2 (DMSO) corresponds to the negative control (1% DMSO); lane 3 (Ths) corresponds to 50 μM thiostrepton (Ths inhibits translation at the start codon [18]); T, G, A, C are the lanes corresponding to sequencing reactions with serial stops at the corresponding nucleotides