Antibiotic That Inhibits the ATPase Activity of an ATP-Binding Cassette Transporter by Binding to a Remote Extracellular Site

Abstract

Antibiotic-resistant strains of Staphylococcus aureus pose a major threat to human health and there is an ongoing need for new antibiotics to treat resistant infections. In a high throughput screen (HTS) of 230 000 small molecules designed to identify bioactive wall teichoic acid (WTA) inhibitors, we identified one hit, which was expanded through chemical synthesis into a small panel of potent compounds. We showed that these compounds target TarG, the transmembrane component of the two-component ATP-binding cassette (ABC) transporter TarGH, which exports WTA precursors to the cell surface for attachment to peptidoglycan. We purified, for the first time, a WTA transporter and have reconstituted ATPase activity in proteoliposomes. We showed that this new compound series inhibits TarH-catalyzed ATP hydrolysis even though the binding site maps to TarG near the opposite side of the membrane. These are the first ABC transporter inhibitors shown to block ATPase activity by binding to the transmembrane domain. The compounds have potential as therapeutic agents to treat S. aureus infections, and purification of the transmembrane transporter will enable further development.

Figure 1

Schematic of cell wall biosynthetic pathways showing the sites of action of inhibitors mentioned in the text. Blue arrows denote the peptidoglycan pathway and red arrows denote the WTA pathway; these pathways use the same undecaprenyl (UndP) carrier. Antibiotic structures and legend abbreviations are explained in Figure S1.

Figure 2

A HTS screening hit led to potent anti-MRSA compounds 2 and 4. (A) Plot of HTS results. Each circle represents the average OD₆₀₀ of the strains in the presence of a library compound tested in duplicate. One compound (compound 1, red circle) inhibited growth of the WT Newman strain but not ΔtarO. (B) Synthesized analogs of 1 with activities against S. aureus Newman. MICs against MRSA strains are identical (Table S1).

Figure 3

TarG is the target of 2. (A) Assay to detect Lipid II abundance after antibiotic treatment, with results for control antibiotics and 2 shown. Extracted Lipid II is labeled with biotin-d-Lys using S. aureus PBP4 to enable detection with HRP-streptavidin. (B) Mutants resistant to 2 (lanes 1–3) were sorted into two groups by plating on amsacrine. Susceptible mutants 1 and 2 had mutations in tarA while amsacrine-resistant mutant 3 had a mutation in tarG (see Table S3, S4 for full list and comparison to other TarG inhibitors). (C) Substitutions in TarG that conferred high level resistance to 2. (D) Disk diffusion assay shows that strain KS002, in which B. subtilis TagGH was replaced with S. aureus TarGH, is sensitive to 2.

Figure 4

Compound 2 inhibits the ATPase activity of TarGH in proteoliposomes but binds in a remote location. (A) Averaged ATPase activity (n=3; error bars=SD) of reconstituted TarGH (200 nM) in the absence (black) and presence (red) of compound 2 (1 µM). Saturating levels of ATP (1 mM) were used. (B) Homology model of TarGH. TarH is cytoplasmic and much of TarG is embedded in the membrane. C) Top view of the TarG dimer. Mutations in residues shown in pink give high level resistance to 2.