Quinoline Compounds Targeting the c-Ring of ATP Synthase Inhibit Drug-Resistant Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa (PA) is a Gram-negative, biofilm-forming bacterium and an opportunistic pathogen. The growing drug resistance of PA is a serious threat that necessitates the discovery of novel antibiotics, ideally with previously underexplored mechanisms of action. Due to their central role in cell metabolism, bacterial bioenergetic processes are of increasing interest as drug targets, especially with the success of the ATP synthase inhibitor bedaquiline to treat drug-resistant tuberculosis. Like Mycobacterium tuberculosis, PA requires F₁F_o ATP synthase for growth, even under anaerobic conditions, making the PA ATP synthase an ideal drug target for the treatment of drug-resistant infection. In previous work, we conducted an initial screen for quinoline compounds that inhibit ATP synthesis activity in PA. In the present study, we report additional quinoline derivatives, including one with increased potency against PA ATP synthase in vitro and antibacterial activity against drug-resistant PA. Moreover, by expressing the PA ATP synthase in Escherichia coli, we show that mutations in the H⁺ binding site on the membrane-embedded rotor ring alter inhibition by the reported quinoline compounds. Identification of a potent inhibitor and its probable binding site on ATP synthase enables further development of promising quinoline derivatives into a viable treatment for drug-resistant PA infection.

Keywords: ATP synthase; Pseudomonas aeruginosa; antibiotic resistance; antibiotics; bioenergetics; quinolines.

Figure 1

(A) Cartoon of ATP synthase showing the arrangement of F₁F_o subunits in the bacterial inner membrane. (B) Model of the H⁺ binding site at the interface between two c subunits. The homology model was generated by SWISS MODEL based on the cryoelectron microscopy structure of ATP synthase from Acinetobacter baumannii, which is 77% identical and 92% similar in this region. (C) Compound 1 was previously reported as an inhibitor of PA ATP synthesis activity.

Scheme 1. Synthesis of Compounds 4 and 5

Figure 2

Inhibition of ATP synthesis activity in PA membrane vesicles. NADH-driven ATP synthesis activity of inverted membrane vesicles from PA was measured in the presence of 0–64 μg/mL compound 1 (A), 4 (B), or 5 (C) using an end point luminescence assay as described in Methods section. Luminescence values in each replicate (dots) were normalized to 0 μg/mL compound, and mean values were used to fit a dose–response curve (black line). Panel (C) inset shows the quality of fit at lower concentrations of compound 5 on a logarithmic (base 2) plot.

Figure 3

Functional characterization of PA ATP synthase expressed in E. coli. (A) E. coli DK8 cells transformed with pFV2, encoding the EC ATP synthase, or pASH20, encoding the PA ATP synthase, were grown in minimal medium containing succinate as the sole carbon source. Bars show mean maximum growth (measured as OD₅₅₀) after 8 h normalized to that of DK8/pFV2. Error bars report standard deviation of n ≥ 3 replicates. (B) NADH-driven ATP synthesis activity of membrane vesicles (10 μg protein) from PA (red), E. coli DK8 (gray), DK8 pFV2 (blue), and DK8/pASH20 (black) was measured in real time using a luminescence assay as described in Methods section. Representative traces show raw luminescence in arbitrary units over time. (C) ATP-driven H⁺ pumping activity of inverted membrane vesicles (coloring as in panel B) was measured by quenching of ACMA fluorescence as described in Methodssection. Representative traces show fluorescence values normalized to t = 0, and the addition of ATP and nigericin (N) is indicated.

Figure 4

Inhibition of ATP synthesis activity in DK8/pASH20 membrane vesicles. NADH-driven ATP synthesis activity of inverted membrane vesicles from E. coli DK8/pASH20 was measured in the presence of 0–64 μg/mL compound 1 (A), 4 (B) or 5 (C) using an end point luminescence assay as described in Methods section. Luminescence values in each replicate (black dots) were normalized to 0 μg/mL compound, and mean values were used to fit a dose–response curve (black line). The dotted red line in each panel is the dose–response fit for PA vesicles from Figure 2 for reference.

Figure 5

Inhibition of ATP synthesis activity in mutant DK8/pASH20 membrane vesicles. NADH-driven ATP synthesis activity of inverted membrane vesicles from WT (black), cI65A (orange), or cI65F (green) was measured in the presence of 0–64 μg/mL compound 1 (A) or 4 (B), or 0–32 μg/mL compound 5 (C) using a luminescence assay as described in Methods section. Luminescence values in each replicate (dots) were normalized to 0 μg/mL compound, and mean values were used to fit a dose–response curve (lines).