Synthesis and Evaluation of Pyridine-Based Antibacterial Agents that Inhibit ATP Synthase in Acinetobacter baumannii

Multidrug-resistant Acinetobacter baumannii (MDR AB) poses a critical global health challenge due to limited treatment options and robust resistance mechanisms. Targeting bacterial bioenergetics, particularly ATP synthase, represents a promising alternative approach inspired by the success of bedaquiline against Mycobacterium tuberculosis.

In this study, a library of trisubstituted pyridines was synthesized and evaluated for ATP synthase inhibition in A. baumannii. Four lead compounds demonstrated potent and selective inhibition, exhibiting strong antibacterial activity against both susceptible and MDR clinical isolates. Furthermore, these compounds acted synergistically with colistin, underscoring their potential as novel antibiotics to combat resistant A. baumannii infections.

The original article

Synthesis and Evaluation of Pyridine-Based Antibacterial Agents that Inhibit ATP Synthase in Acinetobacter baumannii

Angelina L. Dennison, Armaan Singh, Toni A. Marchlewski, Sierra N. Ghee, Kevin C. Gence, lAva G. Hammock, Sabrina Liu, Shaylla Wilson, Alexander P. L. Williams, Katie T. Ward, Thomas E. Meigs, P. Ryan Steed*, and Amanda L. Wolfe*

ACS Omega 2025, 10, 39, 45823–45839

https://doi.org/10.1021/acsomega.5c06380

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

Antimicrobial resistance─a leading cause of death globally─was responsible for >4 million premature deaths in 2019, with projections suggesting this number could rise to over 10 million by 2050. (1,2) The nosocomial ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) contribute significantly to these deaths. Multidrug resistant A. baumannii (MDR AB), an opportunistic Gram-negative pathogen, poses a serious challenge due to a multitude of resistance pathways, including a robust outer membrane (OM), active efflux pumps, and exiguous aperture porins. Moreover, AB is considered one of the most difficult-to-treat ESKAPE pathogens due to its resistance to safety net antibiotics (including colistin, tigecycline, and carbapenems), which demonstrates the dire need for development of novel antimicrobials. (3)

Recently, efforts in antibiotic discovery and development have shifted toward identifying new bacterial targets to overcome current resistance mechanisms in pathogens. One emerging target is ATP synthase, an essential enzyme in bacterial bioenergetics and all life (Figure 1A). (4−6) F₁F_o ATP synthase is a protein complex comprised of two rotary motors that catalyzes the final step in oxidative phosphorylation. (7) In bacteria, the membrane-embedded F_o motor is composed of stator ab₂ subunits adjacent to a homooligomeric ring of c subunits (c₁₀ in AB). (8) Rotation of the c-ring, driven by an electrochemical gradient of H⁺, is coupled to rotation of a central stalk within the cytoplasmic F₁ motor (composed of α₃β₃γδε), which catalyzes phosphorylation of ADP to ATP. ATP synthase has proven to be a druggable antibiotic target through the success of the FDA-approved antitubercular drug bedaquiline (BDQ). (9) BDQ inhibits ATP production in Mycobacterium tuberculosis (MT) by binding to the H⁺ binding site at the subunit ac interface in F_o, halting ATP synthesis in the F₁ complex and causing cell death. (10−12) BDQ was the first antibiotic to target bacterial energy metabolism and the first drug approved for MDR MT in over four decades. (13)

Due to the success of BDQ against MT, our group (14−17) and others (18−21) have begun to explore whether functionalized quinolines can inhibit ATP synthase in other bacterial pathogens via a similar binding interaction with the F_o c subunit and act as antibiotics against resistant strains. One challenge of targeting ATP synthase in Gram-negative pathogens, like AB specifically, is that the F_o complex of ATP synthase is embedded in the inner membrane. Therefore, the drugs must first penetrate the negatively charged OM and then embed in the hydrophobic inner membrane to inhibit the enzyme. Recently, we have developed and interrogated a series of functionalized quinolines that are able to inhibit ATP synthase in AB and penetrate the OM to act as antibiotics against MDR AB strains. (17) Specifically, the quinoline core was substituted with a benzyl sulfide at the C1 position and a flexible and basic nitrogen-containing side chain at C2. Functionalization on the western portion of the quinoline increased enzyme inhibition and overall antibacterial activity against both susceptible and MDR AB clinical isolates. However, increasing steric bulk beyond a certain threshold decreased both enzymatic and bacterial activity significantly, which was attributed to overall larger steric bulk limiting the interactions with the c subunit and reducing membrane permeability. Quinoline WSA 261 (Figure 1B) demonstrated the most potent antibacterial activity against both susceptible and MDR AB of the series. (17) To explore the necessity of the quinoline core and to interrogate the limitations on molecular flexibility and size on AB ATP synthase inhibition more broadly, we have synthesized a library of 34 novel trisubstituted pyridine analogs (Figure 1C) and evaluated this library in enzymatic, antibacterial, and cytotoxicity assays to assess the viability of this series as potential antibacterial agents for treating AB infections.

Methods

Reagents and solvents were purchased reagent-grade and used without further purification. All reactions were performed in flame-dried glassware under an Ar or N₂ atmosphere. Evaporation and concentration in vacuo was performed at 40–45 °C. TLC was conducted using precoated SiO₂ 60 F254 glass plates from EMD with visualization by UV light (254 or 366 nm). NMR (¹H or ¹³C) were recorded on an Varian INOVA-400 MHz spectrometer or a Bruker AVANCE-400 MHz spectrometer at 298 K. Residual solvent peaks were used as an internal reference (CDCl₃ with 0.1% TMS). Coupling constants (J) (H,H) are given in Hz. Coupling patterns are designated as singlet (s), doublet (d), triplet (t), multiplet (m) or quintet (qu). IR spectra were recorded on a Shimadzu IRSpirit FT-IR spectrophotometer and measured neat. Low-resolution mass spectral data were acquired on a Shimadzu single quadrupole LCMS-2020. High-resolution mass spectral Samples were analyzed with a Q Exactive HF-X (ThermoFisher, Bremen, Germany) mass spectrometer. Samples were introduced via a heated electrospray source (HESI) at a flow rate of 10 μL/min. HESI source conditions were set as: nebulizer temperature 400 °C, sheath gas (nitrogen) 20 arb, auxiliary gas (nitrogen) 0 arb, sweep gas (nitrogen) 0 arb, capillary temperature 320 °C, RF voltage 45 V. The mass range was set to 100–1000 m/z. All measurements were recorded at a resolution setting of 120,000. Solutions were analyzed at 0.1 mg/mL or less based on responsiveness to the ESI mechanism. Xcalibur (ThermoFisher, Breman, Germany) was used to analyze the data. Molecular formula assignments were determined with Molecular Formula Calculator (v 1.3.0). All observed species were singly charged, as verified by unit m/z separation between mass spectral peaks corresponding to the ¹²C and ¹³C¹²C_c-1 isotope for each elemental composition.

Results and Discussion

ATP Synthase Inhibition

Once synthesized, the pyridine compounds were tested for their ability to inhibit ATP synthase activity using our previously reported luciferin/luciferase assay. (17) Briefly, endogenous electron transport chains of inverted inner membrane vesicles were energized with NADH to generate a proton gradient and to drive ATP synthesis. ATP was measured via luminescence from the luciferase-catalyzed oxidation of d-luciferin. Luminescence activities across increasing inhibitor concentrations were corrected for gradient independent background sources of ATP by comparison with a control containing the protonophore CCCP (carbonyl cyanide 3-chlorophenylhydrazone). The data were then fit to a four-parameter logistic dose–response curve, from which IC₅₀ values and Hill coefficients were determined using a nonlinear least-squares regression.

Assessing the SAR of ATP synthase inhibition, pyridines with H or Br at C4 exhibited the lowest inhibitory activity across all amine series (Table S1, Figure 2 and S1). Among compounds having C4 phenyl groups, the 4-methoxyphenyl, 3-fluoro-4-methoxyphenyl, and 4-methylphenyl analogs showed potent IC₅₀ values across all amine types, while the 2,4-difluorophenyl, phenyl, and 4-fluorophenyl substituents generally demonstrated weaker inhibitory activity. Compounds WSA 290, 289, 275, 288, and 305 showed the strongest inhibitory activity among the pyridines, with IC₅₀ values between 190 and 270 ng/mL. These in vitro IC₅₀ values are significantly lower than that of WSA 261 (IC₅₀ = 770 ng/mL), suggesting that the pyridine scaffold improves AB ATP synthase inhibitory activity. WSA 290, 289, and 288 all belonged to the C2 cyclopentyl piperidine substituted series, while WSA 275 belonged to the methyl piperazine series and WSA 305 belonged to the ethyl piperidine series. WSA 290 and WSA 275, the strongest performing compounds in their amine series, contained 3-fluoro-4-methoxyphenyl at C4; similarly, WSA 297 (IC₅₀ = 270 ng/mL) with the same 3-fluoro-methoxyphenyl C4 substituent was the strongest performing compound within the benzyl pyrrolidine series. WSA 288 and WSA 305 had methoxyphenyl C4 substituents, and WSA 289 contained a methylphenyl group off C4. The presence of a methoxy group, in both ortho disubstituted and monosubstituted forms, on the C4 phenyl seems to confer stronger inhibition.

ACS Omega 2025, 10, 39, 45823–45839: Figure 2. Potent inhibition of ATP synthesis activity. ATP synthesis activities of inverted membrane vesicles in the presence of 0–16 μg/mL inhibitor (log2 scale) are plotted relative to the DMSO control containing no inhibitor. Replicate measurements are shown as dots. The fitted dose response curves are shown as solid lines, and the dashed lines indicate the 95% confidence bounds of the fit.

Computational Docking

Computational docking was performed using GNINA and PLIP (see Methods). The protein (PDB ID: 7P2Y) (22) was assigned protonation states using the H++ server, (23) compounds were protonated at pH 7 using Open Babel, (24) and both were prepared for docking using Autodock Tools. Docking was performed using GNINA with a fixed grid box centered around cAsp60. Poses from GNINA were then input into PLIP 2.3.0 to extract interaction level data.

The strong inhibitors WSA 290, 289, 275, 288, and 305 all formed salt-bridges between their secondary or tertiary amines and cAsp60 (Table S2). The conformations adopted also allowed for hydrogen bonding with the backbones of cPhe53, cGly57 (WSA 290), cMet64 (WSA 275), and aLeu257 (WSA 305). Additionally, cIle65, cLeu71, aIle242, aLeu245, aIle246, and aLeu257 formed a hydrophobic binding pocket that consistently interacts with these compounds. The enriched binding pocket for high affinity compounds is shown in Figure 3A, where residue-ligand contacts were computed using PLIP on the chosen GNINA poses for each compound. This binding pocket overlaps substantially with that predicted for our previous quinoline scaffold (Figure 1A), supporting a conserved binding mode centered around the cAsp60-mediated salt-bridge and a hydrophobic cavity across both chemotypes.

ACS Omega 2025, 10, 39, 45823–45839: Figure 3. (A) The putative binding site in the ac interface of A. baumannii ATP synthase (PDB ID: 7P2Y) is shown at the interface of subunit a (left, pale cyan) and the c-ring (right, light blue). Residues in the binding pocket are colored by enrichment score, defined here as frequency of binding to strong inhibitors divided by frequency of binding to weak inhibitors, where strong inhibitors are those with IC50 < 500 ng/mL and weak inhibitors are those with IC50 > 5000 ng/mL. The residues shown as sticks are those with enrichment >1 (i.e., frequent interaction with strong inhibitors) and cAsp60 (a key salt-bridge forming residue). (B) Computed binding poses of compounds WSA 276 (cyan), WSA 278 (yellow), and WSA 280 (magenta) in the methyl piperazine series with 4-dimethylamino, 4-methoxyphenyl, and 4-methylphenyl western fronts. (C) Computed binding poses of compounds WSA 288 (cyan), WSA 289 (magenta), and WSA 302 (yellow) in the cyclopentyl piperidine series with 4-methoxyphenyl, 4-methylphenyl, and phenyl western fronts, respectively. (D) Computed binding poses of compounds WSA 297 (yellow), WSA 298 (cyan), WSA294 (orange), and WSA296 (purple) of the benzyl pyrrolidine series, with 2,4-fluoromethoxyphenyl, 4-fluorphenyl, 4-phenyl, and 4-methoxyphenyl C4 substituents respectively.

Conclusions

As discussed, development of new antibiotics that overcome bacterial resistance in MDR AB is a critical need globally due to the rising rate of bacterial infections caused by this pathogen. Bacterial bioenergetics are underexplored drug targets that if properly and selectively inhibited could provide a robust area for antibiotic development, helping to slow the rate of resistance emergence. While the quinoline scaffolds derived from the antitubercular ATP synthase inhibitor BDQ have been the standard for inhibitor development of this type to date, limitations in molecular design and synthesis require new scaffolds to be explored.

Through biochemical and antibacterial evaluation of our trisubstituted pyridine library we have demonstrated that the traditional quinoline core is not required for ATP synthase inhibition in AB, opening a new area of drug development from a more readily available starting material. Specifically, we explored the SAR of AB ATP synthase inhibition and found that enzyme inhibition is driven by both molecular size and interaction with key residues in the proposed binding site of the ac interface of F_o. However, despite the increase in enzyme inhibition compared to the traditional quinoline series, antibacterial activity was limited, and the most potent compounds were cytotoxic against mammalian cells indicating that there is room for improvement using strategic molecular design. Four pyridines, WSA 276, WSA 288, WSA 289, WSA 298, with variations at C2 and C4 but similar overall molecular size and flexibility demonstrated potent antibacterial activity against both susceptible and MDR AB clinical isolates due to selective inhibition of AB ATP synthase in ng/mL concentrations. Additionally, this set had the most favorable preliminary toxicity ratio when dosed in combination with colistin, making them the basis for future studies to improve activity and selectivity of the class.