Abstract
Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t(1/2) value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The Km values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 +/- 0.3 and 10 +/- 2.7 microM in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn2+ > Ca2+ > Mn2+ > Mg2+; no inhibition was found with beta-mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC50 values of 11.4 and 35.3 microg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 microg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.
Highlights
Infectious disease is a global problem, and the development of drug resistance is a major issue for all classes of antibiotics
Our FASII assay measures the cumulative activities of enzymes involved in the fatty acid elongation cycle only, avoiding the complexity of the chain initiation reaction steps catalyzed by acetyl-CoA carboxylase (AccABCD) and FabH
Acetyl-CoA and malonyl-CoA have been used as substrates for the in vitro fatty acid synthesis assay for decades
Summary
Other radiolabeled chemicals used in this study were from PerkinElmer Life Sciences. ACP (Sigma, A7303) was pretreated with 3 mM DTT on ice for 20 min, aliquoted, and stored at Ϫ80 °C. 3 g of the partially purified protein containing fatty acid synthesis enzymes were preincubated with a serial dilution of natural products or synthetic compounds at room temperature for 20 min in 50 l of buffer containing 100 mM sodium phosphate (pH 7.0), 5 mM EDTA, 1 mM NADPH, 1 mM NADH, 150 M DTT, 5 mM -mercaptoethanol, 20 M n-octanoyl-CoA (or lauroyl-CoA), 4% Me2SO, and 5 M of the pretreated ACP. Mid-log (A600 ϭ 0.5– 0.6) growth bacteria (E. coli and S. aureus) were incubated with 1 Ci/ml 2-[3H]glycerol, 1 Ci/ml 6-[3H]thymidine, 1 Ci/ml 5,6-[3H]uracil, 5 Ci/ml 4,5-[3H]leucine, and 5 Ci/ml 2,3-[3H]alanine (or 2-[3H]glycine) for phospholipid, DNA, RNA, protein, and cell wall, respectively, at an increasing concentration of each inhibitor at 37 °C for 20 min. Concentrations of quinaldic acid and chlortetracycline were adjusted due to the increased sensitivity of the lpxC mutant
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