Abstract
The alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p)ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.
Highlights
The alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials
DksA binds to the secondary channel via which nucleotides enter the catalytic center of the RNAP46 and counteracts the misincorporation events[48]
The higher fidelity of RNAP:DksA – and, better discrimination against nucleotide compounds that can not serve as substrates – is likely to be responsible for the protective effect of DksA against DR-4250 and DR-M014 in in vitro transcription assays (Fig. 5)
Summary
The alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials. (p)ppGpp is a pleotropic intracellular effector targeting numerous unrelated molecular targets It regulates transcription via direct interaction with two allosteric sites of Escherichia coli RNAP5–7; suppresses translation via binding to the GTP-binding pocket of ribosome-associated GTPases[8,9,10], DNA replication via binding to the active site of DNA-dependent RNA polymerase primase DnaG11,12, and nucleotide biosynthesis via direct competition with nucleotide substrates of several enzymes involved in synthesis of GTP13 and ATP14. Due to the central role of the (p)ppGpp in regulation of bacterial virulence[16] and recently proposed connection to formation of antibiotic-tolerant persister cells[18], (p) ppGpp-mediated signaling constitutes a promising target for development of novel antibacterials. Follow up studies have shown that, promising as an antibacterial, 1018 is not a specific inhibitor of the stringent response[22,23]
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