Novel TPP-riboswitch activators bypass metabolic enzyme dependency

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.

Quantitative and predictive model of kinetic regulation byE. coliTPP riboswitches

“…Indeed, finding TPP analogs that, on one hand, could turn off the expression of genes involved in the biosynthesis of TPP but that, on the other hand, could not be used as cofactors of crucial enzymes would be a valuable route to fight pathogenic bacteria. 46-49 The success of such a strategy depends on the ability of such analogs to lure the regulation mechanism to the point that gene expression is switched off even though the TPP concentration is becoming too low. Our results on kinetic regulation imply that the in-cell concentration of the TPP analog has to be of the order of, or above or, put differently, .…”

Synthetic biology for pharmaceutical drug discovery

“…Thiamine pyrophosphate riboswitches, for example, are involved in the regulation of thiamine metabolism in numerous bacteria and are examples of promising antibacterial targets. 174 Prommana et al described a glucosamine-induced ribozyme activation system to modulate a drug target dihydrofolate reductase-thymidylate synthase in Plasmodium falciparum . 175 …”

ThiL is a valid antibacterial target that is essential for both thiamine biosynthesis and salvage pathway inPseudomonas aeruginosa

“…Thiamine is a crucial molecule in all living organisms, from microorganisms to mammals. Therefore, thiamine metabolism has attracted growing attention in the development of various drugs, including antibiotics (15)(16)(17)(18)(19). The physiologically active form of thiamine, thiamine pyrophosphate (TPP), plays important roles as a cofactor in various essential cellular processes, including carbohydrate, lipid, and amino acid metabolism (20)(21)(22).…”