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Persisters are a sub-population of genetically sensitive bacteria that survive antibiotic treatment by entering a dormant state. The emergence of persisters from dormancy after antibiotic withdrawal leads to recurrent infection. Indole is an aromatic molecule with diverse signalling roles, including a role in persister formation. Here we demonstrate that indole stimulates the formation of Escherichia coli persisters against quinolone antibiotics which target the GyrA subunit of DNA gyrase. However, indole has no effect on the formation of E. coli persisters against an aminocoumarin, novobiocin, which targets the GyrB subunit of DNA gyrase. Two modes of indole signalling have been described: persistent and pulse. The latter refers to the brief but intense elevation of intracellular indole during stationary phase entry. We show that the stimulation of quinolone persisters is due to indole pulse, rather than persistent, signalling. In silico docking of indole on DNA gyrase predicts that indole docks perfectly to the ATP binding site of the GyrB subunit. We propose that the inhibition of indole production offers a potential route to enhance the activity of quinolones against E. coli persisters. The decreasing effectiveness of antibiotic therapy represents an unprecedented, worldwide threat to human and animal health. The most widely publicised, and best understood, aspect of this problem is antibiotic resistance. This involves genetic change, through mutation or horizontal gene transfer. The target organism is rendered immune to the antibiotic by inactivation of the drug, alteration of its target or export from the cell 1. A less well studied but increasingly important aspect of the anti-bacterial problem is the ability of small sub-populations (< 1%) of genetically sensitive bacteria to survive high concentrations of antibiotic by entering a dormant or partially-dormant state. These cells are known as antibiotic persisters 2-4. When treated with a bactericidal concentration of an antibiotic, persisters display a temporary, non-heritable phenotype where they survive but do not replicate. When the antibiotic therapy is withdrawn, persisters revert to the growing state, giving rise to a population characterised by the same antibiotic sensitivity as the original population. In contrast, resistant cells continue to grow and divide in the presence of the antibiotic and the resistance phenotype is heritable 5-7. The mechanisms by which persister cells enter a dormant state are not well understood. It has been suggested that the activation of chromosome-encoded toxin-antitoxin systems is an important mechanism 8 although this has recently been questioned 9. Links have also been made to the stringent response and carbon source transitions 10. A few reports 11,12 have suggested a role for the signalling molecule indole and this seems plausible because indole has been known for several years to induce reversible E. coli dormancy 13. Indole is an aromatic signalling molecule produced by over 85 species of bacteria encompassing ...

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In silico screening of GMQ-like compounds reveals guanabenz1 and sephin1 as new allosteric modulators of acid-sensing ion 2 channel 3 3 4 Abstract 16Acid-sensing ion channels (ASICs) are voltage-independent cation channels that 17 detect decreases in extracellular pH. Dysregulation of ASICs underpins a number 18 of pathologies. Of particular interest is ASIC3, which is recognised as the key 19 sensor of acid-induced pain and is instrumental in the establishment of pain 20 arising from inflammatory conditions, such as rheumatoid arthritis. Thus, the 21 identification of new ASIC3 modulators and the mechanistic understanding of 22 how these compounds modulate ASIC3 could be important for the development 23of new strategies to counteract the detrimental effects of dysregulated ASIC3 24 activity in inflammation. Here, we report the identification of novel ASIC3 25 modulators based on the ASIC3 specific agonist, 2-guanidine-4-26 methylquinazoline (GMQ). Through a GMQ-guided in silico screening of Food 27and Drug administration (FDA)-approved drugs, 5 compounds were selected and 28 tested for their possible modulation of rat ASIC3 (rASIC3) using whole-cell patch-29 clamp electrophysiology. Of the chosen drugs, guanabenz, an α2-adrenoceptor 30 agonist, produced similar effects to GMQ on rASIC3, activating the channel at 31 neutral pH and potentiating its response to mild acidic stimuli. Sephin1, a 32 guanabenz derivative that lacks α2-adrenoceptor activity, has been proposed to 33 act as a selective inhibitor of a regulatory subunit of the stress-induced protein 34 phosphatase 1 (PPP1R15A) with promising therapeutic potential for the 35 treatment of multiple sclerosis. However, we found that like guanabenz, sephin1 36 activates rASIC3 at neutral pH and potentiates its response to acidic stimulation, 37i.e. sephin1 is a novel modulator of rASIC3. Furthermore, docking experiments 38 showed that, like GMQ, guanabenz and sephin1 likely interact with the nonproton 39 ligand-sensing domain of rASIC3. Overall, these data demonstrate the utility of 40 computational analysis for identifying novel ASIC3 modulators, which can be 41 validated with electrophysiological analysis and may lead to the development of 42 better compounds for targeting ASIC3 in the treatment of inflammatory 43 conditions. 44 48 channel (ASIC) family, these voltage-independent, ligand-gated cation channels 49 are activated by extracellular protons [2][3][4] and belong to the amiloride-sensitive 50 epithelial sodium channel/degenerin (ENaC/DEG) ion channel family [5,6]. In 51 mammals, four genes (accn1-4) encode for at least 6 different ASIC subunits 52 (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4), which can assemble into 53 homo-and heterotrimeric channels displaying different pH sensitivity, current 54 kinetics and pharmacology [7][8][9]. ASICs are widely expressed in the central and 55 peripheral nervous systems [2,10] and are implicated in a range of physiological 56 and pathological processes including nociception, mechanosensation and 57 learning/me...