Discovery, characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases

Abstract

Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC(50)25 microM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC approximately 5 microg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC(50)/MIC>16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (K(i)=8 microM) and is rapidly bactericidal at 4 x MIC.

Figure 1

Kinetic analysis of coumarin-type inhibitor compound 2 vs. B. anthracis helicase in the FRET quenching assay. Data are displayed in the following linear transformations: (A) Dixon plot with 0.625 mM (◊), 1.25 mM (□), 2.5 mM (△), and 5 mM (○) ATP substrate present; (B) Lineweaver-Burk plot with 12.5 μM (□), 6.25 μM (◊), 3.13 μM (△), and 0 μM (○) inhibitor present; (C) Dixon plot with 5 nM (◊), 10 nM (□), 30 nM (△), and 100 nM (○) annealed oligonucleotide substrate present. Lines are drawn based on a linear or polynomial regression analysis of the data.

Figure 2

Viability of B. anthracis Sterne cells incubated with compound 2 in broth culture at multiple concentrations. Compound 2 was added to LB cultures of B. anthracis Sterne cells at 0.5× MIC (○), 1× MIC (△), 4× MIC (□), or omitted from the culture ◊, and aliquots were spread on LB agar plates at various times indicated on the abscissa to determine the number of viable colony-forming units. Lines are drawn based on an exponential regression analysis of the data.

Figure 3

Summary of preliminary SAR results for coumarin type inhibitor, compound 2. (A) The substructure shared by all five coumarin-type helicase inhibitors is outlined in a box, and approximate effects of specific structural alterations are noted. (B) A pharmacophore representation of the coumarin type helicase inhibitors.