A quinolinol-based small molecule with anti-MRSA activity that targets bacterial membrane and promotes fermentative metabolism

Article metrics


In a loss-of-viability screen of small molecules against methicillin-resistant Staphylococcus aureus (MRSA) USA300, we found a small molecule, designated DNAC-2, which has an MIC of 8 μg ml−1. DNAC-2 is a quinolinol derivative that is bactericidal at 2X MIC. Macromolecular synthesis assays at 2 × MIC of DNAC-2 revealed inhibition of DNA, cell wall, RNA and protein synthesis within fifteen to thirty minutes of treatment when compared to the untreated control. Transmission electron microscopy of DNAC-2-treated cells revealed a significantly thicker cell wall and impaired daughter cell separation. Exposure of USA300 cells to 1 × MIC of DNAC-2 resulted in mislocalization of PBP2 away from the septum in an FtsZ-independent manner. In addition, membrane localization with FM4–64, as well as depolarization study with DiOC2 and lipophilic cation TPP+ displayed membrane irregularities and rapid membrane depolarization, respectively, in DNAC-2-treated cells vs -untreated control. However, DNAC-2 exhibited almost no toxicity toward eukaryotic membranes. Notably, DNAC-2 drives energy generation toward substrate level phosphorylation and the bacteria become more sensitive to DNAC-2 under anaerobic conditions. We propose that DNAC-2 affects USA300 by targeting the membrane, leading to partial membrane depolarization and subsequently affecting aerobic respiration and energy-dependent functional organization of macromolecular biosynthetic pathways. The multiple effects may have the desirable consequence of limiting the emergence of resistance to DNAC-2.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5


  1. 1

    Spellberg, B., Bartlett, J., Wunderink, R. & Gilbert, D. N. Novel approaches are needed to develop tomorrow's antibacterial therapies. Am. J. Respir. Crit Care Med. 191, 135–140 (2015).

  2. 2

    Haydon, D. J. et al. An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science 321, 1673–1675 (2008).

  3. 3

    Rasko, D. A. et al. Targeting QseC signaling and virulence for antibiotic development. Science 321, 1078–1080 (2008).

  4. 4

    Payne, D. J. Microbiology. Desperately seeking new antibiotics. Science 321, 1644–1645 (2008).

  5. 5

    Brown, D. Antibiotic resistance breakers: can repurposed drugs fill the antibiotic discovery void? Nat. Rev. Drug Discov. 14, 821–832 (2015).

  6. 6

    Richter, S. G. et al. Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria. Proc. Natl Acad. Sci. USA 110, 3531–3536 (2013).

  7. 7

    Nair, D. R. et al. Characterization of a novel small molecule that potentiates beta-lactam activity against gram-positive and gram-negative pathogens. Antimicrob. Agents Chemother. 59, 1876–1885 (2015).

  8. 8

    Silverman, J. A., Perlmutter, N. G. & Shapiro, H. M. Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob. Agents Chemother. 47, 2538–2544 (2003).

  9. 9

    Dutter, B. F. et al. Decoupling Activation of Heme Biosynthesis from Anaerobic Toxicity in a Molecule Active in Staphylococcus aureus. ACS Chem. Biol. 11, 1354–1361 (2016).

  10. 10

    Kahl, B. et al. Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J. Infect. Dis. 177, 1023–1029 (1998).

  11. 11

    Magalon, A. et al. Inhibitor binding within the NarI subunit (cytochrome bnr) of Escherichia coli nitrate reductase A. J. Biol. Chem. 273, 10851–10856 (1998).

  12. 12

    Toyofuku, M. et al. Influence of the Pseudomonas quinolone signal on denitrification in Pseudomonas aeruginosa. J. Bacteriol. 190, 7947–7956 (2008).

  13. 13

    Centers for Disease Control and Prevention Active Bacterial Core Surveillance (ABCs): Emerging Infections Program Network Methicillin-Resistant Staphylococcus aureus, 2011 (2011).

  14. 14

    Walsh, C. T. & Wencewicz, T. A. Prospects for new antibiotics: a molecule-centered perspective. J. Antibiot. (Tokyo) 67, 7–22 (2014).

  15. 15

    Drlica, K. & Zhao, X. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61, 377–392 (1997).

  16. 16

    Cunningham, M. L., Kwan, B. P., Nelson, K. J., Bensen, D. C. & Shaw, K. J. Distinguishing on-target versus off-target activity in early antibacterial drug discovery using a macromolecular synthesis assay. J. Biomol. Screen. 18, 1018–1026 (2013).

  17. 17

    Turner, R. D. et al. Peptidoglycan architecture can specify division planes in Staphylococcus aureus. Nat. Commun. 1, 26 (2010).

  18. 18

    Sieradzki, K. & Tomasz, A. Inhibition of the autolytic system by vancomycin causes mimicry of vancomycin-intermediate Staphylococcus aureus-type resistance, cell concentration dependence of the MIC, and antibiotic tolerance in vancomycin-susceptible S. aureus. Antimicrob. Agents Chemother. 50, 527–533 (2006).

  19. 19

    Pinho, M. G. & Errington, J. Recruitment of penicillin-binding protein PBP2 to the division site of Staphylococcus aureus is dependent on its transpeptidation substrates. Mol. Microbiol. 55, 799–807 (2005).

  20. 20

    Mike, L. A. et al. Activation of heme biosynthesis by a small molecule that is toxic to fermenting Staphylococcus aureus. Proc. Natl Acad. Sci. USA 110, 8206–8211 (2013).

  21. 21

    Thomas, V. C. et al. A central role for carbon-overflow pathways in the modulation of bacterial cell death. PLoS Pathog. 10, e1004205 (2014).

  22. 22

    Cui, L. et al. Cell wall thickening is a common feature of vancomycin resistance in Staphylococcus aureus. J. Clin. Microbiol. 41, 5–14 (2003).

  23. 23

    Fuchs, S., Pane-Farre, J., Kohler, C., Hecker, M. & Engelmann, S. Anaerobic gene expression in Staphylococcus aureus. J. Bacteriol. 189, 4275–4289 (2007).

  24. 24

    Tan, C. M. et al. Restoring methicillin-resistant Staphylococcus aureus susceptibility to beta-lactam antibiotics. Sci. Transl. Med. 4, 126ra35 (2012).

  25. 25

    Memmi, G., Filipe, S. R., Pinho, M. G., Fu, Z. & Cheung, A. L. Staphylococcus aureus PBP4 is essential for beta-lactam resistance in community acquired MRSA. Antimicrob. Agents Chemother. 52, 3955–3966 (2008).

  26. 26

    Clinical and Laboratory Standards Institute Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard 9th ed. CLSI, Wayne, PA USA, (2012).

  27. 27

    Saar, K. et al. Cell-penetrating peptides: a comparative membrane toxicity study. Anal. Biochem. 345, 55–65 (2005).

  28. 28

    Schneider, T. et al. Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II. Science 328, 1168–1172 (2010).

Download references


We thank NERCE-BEID for facilitating this research, SRI Biosciences under the auspices of NIH Product Development Services for the pharmacokinetic and toxicity analyses, Louisa Howard for processing the TEM samples and imaging. HGS received support by the German Centre of Infection Research (DZIF). NERCE-BEID was funded by NIH grant U54AI057159. Work in the AC laboratory was partially funded by COBRE (NIH) # P30GM106394 and the Pfeiffer Foundation. MGP laboratory was funded by ERC-2012-StG-310987 grant from the European Research Council and JMM was supported by fellowship SFRH/BD/71993/2010.

Author information

Correspondence to Ambrose Cheung.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on The Journal of Antibiotics website

Supplementary information

Supplementary Table 1 (DOCX 15 kb)

Supplementary Figure (PPT 113 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading