1. Volume 22, Issue 10, Pages 1362-1373 (October 2015)
Elucidation of DnaE as the Antibacterial Target of the Natural Product, Nargenicin 
Ronald E. Painter, Gregory C. Adam, Marta Arocho, Edward DiNunzio, Robert G.K. Donald, Karen Dorso, Olga Genilloud, Charles Gill, Michael Goetz, Nichelle N. Hairston, Nicholas Murgolo, Bakela Nare, David B. Olsen, Maryann Powles, Fred Racine, Jing Su, Francisca Vicente, Douglas Wisniewski, Li Xiao, Milton Hammond, Katherine Young 
Chemistry & Biology 
Volume 22, Issue 10, Pages (October 2015)
DOI: /j.chembiol
Copyright © 2015 Elsevier Ltd Terms and Conditions

2. Figure 1 Antibacterial Activity of Nargenicin
(A) Structures of nargenicin A1 and (B) nargenicin B1.
(C) Minimum inhibitory activity (MIC) of nargenicin in a panel of wild-type bacteria, pathogenic isolates, and defined strains with mutations in permeability and efflux (Phillips et al., 2011; Kodali et al., 2005; Robertson et al., 2007; Singh et al., 2015; Huber et al., 2009; Gefter et al., 1971).
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2015 Elsevier Ltd Terms and Conditions

3. Figure 2
Characterization of Nargenicin as an Inhibitor of DNA Synthesis
(A and B) Selective inhibition of DNA synthesis by nargenicin in S. aureus (A) and E. coli (B) across the indicated drug concentrations was determined by measuring incorporation of radiolabeled precursors of either DNA (14C-thymidine), RNA (3H-uridine), phospholipid (2-3H-glycerol), protein (3H-leucine), or cell wall (14C-glycine) synthesis.
(C) Nargenicin turned on the SOS response, eliciting a blue ring due to induction of the sulA promoter driving β-galactosidase. Nargenicin had equal activity against a UvrA mutant, indicating inhibition of DNA replication was not due to the addition of bulky adducts. Nargenicin had equal activity in the presence and absence of DNA, indicating lack of DNA binding or intercalation. Positive controls ciprofloxacin, mitomycin C, griseolutein, and actinomycin D behaved as expected.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2015 Elsevier Ltd Terms and Conditions

4. Figure 3
Further Characterization of the Biological Activity of Nargenicin
Upon addition of nargenicin to logarithmically growing cells of Col MRSA, immediate killing was observed. A 3-log10 reduction in viable colony-forming units was observed by 2 hr.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2015 Elsevier Ltd Terms and Conditions

5. Figure 4
Biochemical and Genetic Evidence that DnaE is the Target of Nargenicin
(A and B) S. aureus (A) or E. coli (B) DnaE single-enzyme polymerization assays were incubated with activated calf thymus DNA and four dNTPs, including 3H-dTTP, in the presence of several inhibitors of DNA synthesis. Compounds such as ciprofloxacin and novobiocin, known to target gyrase, show no effect against DnaE. Dose-response curves were plotted using GraphPad Prism software; where samples were tested in triplicate, error bars represent the SEM.
(C) Episomal expression of mutant DnaE conferred resistance to nargenicin when S. aureus COL was grown for 20 hr in cation-adjusted Mueller-Hinton broth supplemented with chloramphenicol (25 μg/ml) and anhydrotetracycline inducer (20 ng/ml).
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2015 Elsevier Ltd Terms and Conditions

6. Figure 5
Binding of Nargenicin to DnaE in the Absence and Presence of DNA
(A) The amount of nargenicin bound to 3.9 μM S. aureus DnaE is increased in the presence of 0.5 mg/ml activated DNA. Binding to DnaE was selective for nargenicin, and nargenicin did not bind to activated DNA alone like the DNA intercalator daunorubicin. Data are reported as the integrated mass spectral response for the extracted ion chromatogram (XIC) of the ligand.
(B) However, for E. coli DnaE, binding of nargenicin to the protein was not increased by the presence of activated DNA.
(C) The increase in binding in the presence of activated DNA is selective for the S. aureus nargenicin-DnaE interaction. The binding of warfarin and SB to human serum albumin and P-38α MAPK, respectively, was the same in the presence and absence of 0.5 mg/ml activated DNA. Binding of nargenicin was selective for DnaE with only minimal non-specific binding detected to HSA. Data are reported as the normalized integrated mass spectral response for the XIC of the ligand where the largest signal for each ligand is normalized to 1.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2015 Elsevier Ltd Terms and Conditions

7. Figure 6
Nargenicin Is Active in Systemic In Vivo Model of MSSA by Both Oral and Subcutaneous Routes of Administration
Balb/c mice (five per group) were challenged intraperitoneally with S. aureus MB 2865 in 5% hog gastric mucin at 2.2 × 103 cfu/mouse. Mice were treated orally or subcutaneously with compounds receiving two total doses (0.5 ml, twice daily for 1 day). Kidneys were collected aseptically at 24 hr after challenge.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2015 Elsevier Ltd Terms and Conditions