Abstract
Riboswitches are conserved regions within mRNA molecules that bind specific metabolites and regulate gene expression. TPP-riboswitches, which respond to thiamine pyrophosphate (TPP), are involved in the regulation of thiamine metabolism in numerous bacteria. As these regulatory RNAs are often modulating essential biosynthesis pathways they have become increasingly interesting as promising antibacterial targets. Here, we describe thiamine analogs containing a central 1,2,3-triazole group to induce repression of thiM-riboswitch dependent gene expression in different E. coli strains. Additionally, we show that compound activation is dependent on proteins involved in the metabolic pathways of thiamine uptake and synthesis. The most promising molecule, triazolethiamine (TT), shows concentration dependent reporter gene repression that is dependent on the presence of thiamine kinase ThiK, whereas the effect of pyrithiamine (PT), a known TPP-riboswitch modulator, is ThiK independent. We further show that this dependence can be bypassed by triazolethiamine-derivatives that bear phosphate-mimicking moieties. As triazolethiamine reveals superior activity compared to pyrithiamine, it represents a very promising starting point for developing novel antibacterial compounds that target TPP-riboswitches. Riboswitch-targeting compounds engage diverse endogenous mechanisms to attain in vivo activity. These findings are of importance for the understanding of compounds that require metabolic activation to achieve effective riboswitch modulation and they enable the design of novel compound generations that are independent of endogenous activation mechanisms.
Highlights
Riboswitches are RNA elements mostly found in the 5 UTR of bacterial mRNA that sense the concentration of a small metabolite
In order to investigate the dependence of TT activity on endogenous proteins and enzymes involved in thiamine biosynthesis, several strains of the Keio collection were investigated, that all originate from the strain BW25113, and contain deletions of non-essential genes known to be involved in thiamine metabolism of E. coli (Scheme 1) (Baba et al, 2006)
We show that replacing the thiazole heterocycle with 1,2,3-triazole is a valuable strategy to generate thiamine analogs that interact with thiamine pyrophosphate (TPP) riboswitches and, induce repression of gene expression
Summary
Riboswitches are RNA elements mostly found in the 5 UTR of bacterial mRNA that sense the concentration of a small metabolite. Conformational changes of the riboswitch resulting in repression of gene expression are only achieved if the pyrophosphate group of the ligand is recognized by the pyrophosphate sensor helix (P4, J4-5, P5, Figure 1A) (Edwards and Ferre-D’Amare, 2006; Serganov et al, 2006; Thore et al, 2006) While this riboswitch class has been shown to allow a greater degree of variation at the position of the thiazole ring, exemplified by the thiamine analog pyrithiamine (PT, Figure 2A), the presence of the pyrophosphate moiety is mandatory to interact with and, more importantly, to induce switching of the E. coli thiM-riboswitch (Winkler et al, 2002; Rentmeister et al, 2007). This strategy opens new avenues toward riboswitch activating compounds, in particular those that rely on phosphate groups to recognize and switch cognate RNA structures
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