Synthesis and Evaluation of Pseudomonas aeruginosa ATP Synthase Inhibitors

Abstract

New antibiotics with unique biological targets are desperately needed to combat the growing number of resistant bacterial pathogens. ATP synthase, a critical protein found in all life, has recently become a target of interest for antibiotic development due to the success of the anti-tuberculosis drug bedaquiline, and while many groups have worked on developing drugs to target bacterial ATP synthase, few have been successful at inhibiting Pseudomonas aeruginosa (PA) ATP synthase specifically. PA is one of the leading causes of resistant nosocomial infections across the world and is extremely challenging to treat due to its various antibiotic resistance mechanisms for most commonly used antibiotics. Herein, we detail the synthesis and evaluation of a series of C1/C2 quinoline analogues for their ability to inhibit PA ATP synthase and act as antibiotics against wild-type PA. From this survey, we found six compounds capable of inhibiting PA ATP synthase in vitro showing that bulky/hydrophobic C1/C2 substitutions are preferred. The strongest inhibitor showed an IC₅₀ of 10 μg/mL and decreased activity of PA ATP synthase to 24% relative to the control. While none of the compounds were able to inhibit wild-type PA in cell culture, two showed improved inhibition of PA growth when permeability of the outer membrane was increased or efflux was knocked out, thus demonstrating that these compounds could be further developed into efficacious antibiotics.

Figure 1

(A) Cartoon structure of bacterial ATP synthase, where the H⁺ translocating c-ring of F_o is shown in green with red dots, indicating locations of the H⁺ binding sites (Glu65 in MT). (B) Structure of BDQ, 1, highlighting substituents at positions C1 and C2 on the quinoline core. (C) Crystal structure of BDQ bound to the MT c-ring (PDB 4V1F). Residues interacting with BDQ are highlighted and correspond to highlighted residues in panel (D). (D) Amino acid sequence alignment of two regions of subunit c from Mycobacterium tuberculosis (MT) and Pseudomonas aeruginosa (PA); MT numbering.

Scheme 1. Synthesis of C1 Derivatives

Scheme 2. Synthesis of Carbamate, Heterocyclic, and Hydrazine C2 Derivatives 8–13

Scheme 3. Synthesis of Amino Acid C2 Derivatives 14–23 via Reductive Amination

Figure 2

Inhibition of ATP synthesis activity by selected compounds. Relative activity was calculated by normalizing luminescence values to a control with no inhibitor. Activity at each concentration of the inhibitor is shown as mean ± standard error (n ≥ 3). Dose–response curves (red) were fitted using GraphPad Prism 9. The dotted line indicates the mean background activity that can be attributed to ATP sources other than ATP synthase (Figure S1).