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Discovery of a novel polymyxin adjuvant against multidrug-resistant gram-negative bacteria through oxidative stress modulation
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Taotao Lub, Hongguang Hana, Chaohui Wua, Qian Lia, Hongyan Huc, Wenwen Liua, Donglei Shia, Feifei Chend, e, Lefu Lane, f, Jian Lia, b, g, *, Shihao Songa, *, Baoli Lia, *
Acta Pharmaceutica Sinica B | 2025, 15(3) : 1680 - 1695
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Acta Pharmaceutica Sinica B | 2025, 15(3): 1680-1695
• ORIGINAL ARTICLE •
Discovery of a novel polymyxin adjuvant against multidrug-resistant gram-negative bacteria through oxidative stress modulation
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Taotao Lub, Hongguang Hana, Chaohui Wua, Qian Lia, Hongyan Huc, Wenwen Liua, Donglei Shia, Feifei Chend, e, Lefu Lane, f, Jian Lia, b, g, *, Shihao Songa, *, Baoli Lia, *
Affiliations
  • aKey Laboratory of Tropical Biological Resources of Ministry of Education and Hainan Engineering Research Center for Drug Screening and Evaluation, School of Pharmaceutical Sciences, Hainan University, Haikou 570228, China
  • bState Key Laboratory of Bioreactor Engineering, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
  • cHainan Hospital of Chinese PLA General Hospital Laboratory Department, Sanya 5720225, China
  • dShanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
  • eSchool of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China
  • fAnhui Province Key Laboratory of Infectious Diseases, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
  • gKey Laboratory of Xinjiang Phytomedicine Resource and Utilization, Ministry of Education, School of Pharmacy, Shihezi University, Shihezi 832003, China
About Author:

E-mail addresses: (Jian Li)

(Shihao Song)

(Baoli Li).

†

These authors made equal contributions to this work.

Author contributions

This study was initiated and designed by Baoli Li, Shihao Song, and Jian Li. Taotao Lu, Chaohui Wu, and Qian Li synthesized the compounds and evaluated their synergistic activity. Hongguang Han conducted the biological evaluation and mechanism studies. Hongyan Hu, Wenwen Liu, Donglei Shi, Feifei Chen, and Lefu Lan provided reagents, materials, and analysis tools. Data analysis and interpretation were carried out by Baoli Li, Shihao Song, and Jian Li. The manuscript was written by Taotao Lu, Baoli Li, and Shihao Song. All of the authors have read and approved the final manuscript.

doi: 10.1016/j.apsb.2025.01.022
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Antibiotic adjuvants offer a promising strategy for restoring antibiotic sensitivity, expanding antibacterial spectra, and reducing required dosages. Previously, compound 15 was identified as a potential adjuvant for Polymyxin B (PB) against multidrug-resistant (MDR) Pseudomonas aeruginosa DK2; however, its clinical utility was hindered by high cytotoxicity, uncertain in vivo efficacy, and an unclear synergetic mechanism. To address these challenges, we synthesized and evaluated a series of novel benzamide derivatives, with A22 emerging as a particularly promising candidate. A22 demonstrated potent synergistic activity to PB, minimal cytotoxicity, improved water solubility, and broad-spectrum synergism of polymyxins against various clinically isolated MDR Gram-negative strains. In vivo studies using Caenorhabditis elegans and mouse models further confirmed the efficacy of A22. Moreover, A22 effectively suppressed the development of PB resistance in Pseudomonas aeruginosa DK2. Mechanistic investigations revealed that A22 enhances polymyxins activity by inducing reactive oxygen species production, reducing ATP levels, increasing NOX activity, and inhibiting biofilm formation, leading to bacterial death. These findings position A22 as a highly promising candidate for the development of polymyxin adjuvants, offering a robust approach to combating MDR Gram-negative bacterial infections.

Multidrug resistance  /  Gram-negative bacteria  /  Niclosamide  /  Drug repurposing  /  Polymyxin B  /  Adjuvant  /  Broad-spectrum synergism  /  Oxidative stress
Taotao Lu, Hongguang Han, Chaohui Wu, Qian Li, Hongyan Hu, Wenwen Liu, Donglei Shi, Feifei Chen, Lefu Lan, Jian Li, Shihao Song, Baoli Li. Discovery of a novel polymyxin adjuvant against multidrug-resistant gram-negative bacteria through oxidative stress modulation[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (3) : 1680 -1695 . DOI: 10.1016/j.apsb.2025.01.022
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1.　Introduction
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Antibiotic resistance is a significant global public health challenge, complicating the treatment of bacterial infections and leading to increased mortality and morbidity. Recent estimates suggest that approximately 700,000 people die annually from antibiotic-resistant bacterial infections, with projections indicating this number could rise to 10 million by 2050 if no action is taken1. The emergence of bacterial resistance has rendered many traditional antibiotics ineffective, further exacerbating the need for novel treatment strategies.
Antibiotic adjuvants, or “antibiotic potentiators”, typically lack antimicrobial activity or exhibit minimal inhibition of bacterial growth but enhance the efficacy of antibiotics against resistant bacteria2-5. By broadening the spectrum of bacterial coverage and suppressing resistance, adjuvants offer a complementary approach to combating bacterial infections6,7. Currently, only a few β-lactamase inhibitor adjuvants (clavulanate, sulbactam, and tazobactam) are available7, underscoring the urgent need for more diverse adjuvant types.
Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic pathogen that causes acute or chronic infection in immunocompromised patients, such as those with cystic fibrosis (CF), cancer, traumas, burns, chronic obstructive pulmonary disease, sepsis, and ventilator-associated pneumonia, including cases related to COVID-198,9. The treatment of P. aeruginosa infections is particularly challenging due to its pathogen's rapid mutation rate and capacity to develop resistance to multiple antibiotics10. The multidrug-resistant (MDR) P. aeruginosa DK2 strain, first isolated from CF patients in 1973, has since evolved into an independent subspecies, significantly increasing morbidity and mortality rates11. Our results demonstrated that P. aeruginosa DK2 exhibits severe resistance to multiple classes of antibiotics, particularly the last-resort antibiotics Polymyxin B (PB, MIC = 256 μg/mL) and Colistin (MIC >256 μg/mL) (Supporting Information Fig. S1A). Our previous study identified niclosamide (NIC), an anthelmintic drug, as an adjuvant of PB activity against P. aeruginosa DK2 and led to the development of a synergistic compound 15 (Fig. 1)12. However, compound 15 was limited by high cytotoxicity, uncertain in vivo efficacy, and an unclear synergistic mechanism.
In this study, we designed, synthesized, and evaluated a series of derivatives based on compound 15 to improve synergistic potency and druggability, with a focus on derivative A22 (Fig. 1). It demonstrated significant in vitro and in vivo synergistic activity to PB against P. aeruginosa DK2. Additionally, in combination with PB, derivative A22 exhibited broad-spectrum antibacterial activity against other clinical drug-resistant Gram-negative bacteria. Mechanistic studies revealed that derivative A22 enhances PB activity by inducing excessive ROS production, reducing ATP production, increasing NOX activity, and inhibiting biofilm formation, ultimately leading to bacterial death.
2.　Materials and methods
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The clinical P. aeruginosa DK2 isolate was provided by Professor Lars Jelsbak of Technical University of Denmark. A. Baumannii 186 and K. Pneumoniae 674 isolates were provided by Professor of Min Li of Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine. E. Coli 15,017 was provided by Professor of YouJun Feng, Zhejiang University. K. Pneumoniae 15,004, Proteus mirabilis, Enterobacter cloacae 107, Enterobacter asburiae 184, A. baumannii 186 and Serratia arcescens were provided by Professor Hongyan Hu, Hainan Hospital of Chinese PLA General Hospital. P. aeruginosa PAO1 isolate was from ATCC (https://www.atcc.org).
2.1.　 Synthesis and characterization of A1–A30
The details of the synthesis and characterization of A1–A30 were employed in this manuscript and Supporting Information The purity of all derivatives was determined by HPLC >95% (Supporting Information Table S1). The 1H NMR, HRMS and HPLC spectra for each derivative were provided in Supporting Information Figs. S4–S33.
2.2.　 Antibiotic efficacy test
The in vitro antimicrobial activity of all derivatives/antibiotics was evaluated by the micro broth dilution method, as described previously12. In brief, bacterial cultures were diluted with fresh Cation-Adjusted Mueller Hinton broth (CAMHB, BD, CA, USA) to OD600 of 0.001. Derivatives/antibiotics were 2-fold serially diluted in bacterial cultures in the 96-well plate and incubated with equal volumes of inoculum for 18 h at 37 ℃. Then, MIC values were measured by recording the optical density at 600 nm (OD600) of each well using BioTek Synergy 2 (BioTek, VT, USA). MIC values were recorded as the lowest concentration required to achieve 100% growth inhibition compared to growth in untreated wells.
2.3.　 Chessboard assay
In vitro synergetic activity was evaluated in 96-well plates by the checkerboard microdilution method. In brief, bacterial cultures were diluted with fresh CAMHB (BD) to OD600 of 0.001 and were plated in a 96-well culture plate at 100 μL/well. Antibiotics and derivatives to be measured were added to the 96-well plate, and the checkerboard method was used for determination. The concentration range was 0.25–32 μg/mL, and the final concentration range of derivatives was 5–100 μmol/L (continuous 2-fold change). The plates were incubated at 37 ℃ for 18 h and measured by optical density at OD600. The value of FIC was calculated as Eq. (1):
FIC=[MICoftheagentsincombination]/[MICoftheagentalone]
The FICI is the sum of the FICs of the derivatives and antibiotics.
2.4.　 Time-kill assay
The detailed method for time-kill assay was previously described12.
2.5.　 Mammalian cytotoxicity
Cells were seeded in 96-well plates at 10,000 cells per well in a total volume of 100 μL of media containing 10% serum. Serially diluted compounds in 10 μL of media were added to the cells 24 h later. After 1 day of incubation, Cell Counting Kit-8 reagents (CCK8, Yeasen, Shanghai, China) were added, and luminescence was measured according to the manufacturer's instructions. Cell viability was measured by CCK8 assay of 293 T and HUVEC cells line. The data are presented as a percentage of viable cells with vehicle-treated cells set as 100.
2.6.　 Water solubility assay
The derivative was dissolved in HPLC-grade methanol and diluted in a gradient to 0.56, 0.28, 0.14, 0.07, 0.035, 0.018, 0.0088, 0.0044, 0.0022, and 0.0011 mg/mL of the solution. The UV absorption peak area in 254 nm was measured by HPLC to construct the concentration–UV absorption area standard curve. Add an excess of the derivatives into water to form a supersaturated solution. After stirring for 24 h, the UV absorption peak area in 254 nm was measured by HPLC, and then the water solubility value of the derivative was calculated from the previous standard curve.
2.7.　 Plasma protein binding assay
This experiment was outsourced to Pharmalegacy Laboratories Co., Ltd. (Shanghai, China). Briefly, aliquots of 100 μL of blank dialysis buffer were applied to the receiver side of the dialysis chambers. Then 100 μL of the plasma spiked with test compounds was applied to the donor side. Preparing T0 plasma samples for initial concentrations. Aliquot 25 μL of the plasma spiked with test and reference compounds was added to the 96-well sample preparation plate as T0 plasma samples. The samples were added blank buffer. Quench the samples with 200 μL of acetonitrile containing internal standard. Cover the dialysis block with a plastic lid and place the entire apparatus for 5 h at 37 ℃. After 5 h of incubation, aliquots of 25 μL were taken from both the donor and receiver sides of the dialysis apparatus and transferred into new sample preparation plates. Each aliquot was then mixed with an equal volume of the opposite matrix (blank buffer for plasma and plasma for blank buffer). Quench the samples with 200 μL acetonitrile containing internal standard. Vortex all the samples at 500 rpm for 10 min followed by centrifugation at 4000×g for 10 min. The transfer of 100 μL of the supernatants to a new 96-well plate and mix the samples with 200 μL of Milli-Q water. Cover the sample plate and store it in a freezer (−20 ℃) until LC–MS/MS analysis.
2.8.　 hERG channel Qpatch assay
This experiment was commissioned by the ion channel platform of Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). The inhibitory activity of test compounds on hERG potassium channels was tested in CHO-hERG cells, which were cultured in 175 cm2 culture flasks. Cell density was ranged within 2 × 106‒5 × 106 cells/mL. The single-cell high-impedance sealing and whole-cell pattern formation processes are automatically completed by the Qpatch instrument. After acquiring whole-cell recording mode, cells were clamped at −80 mV. The voltage stimulation was applied every 15 s and recorded for 2 min, and the extracellular fluid was administered for 5 min, and then the administration process was started. Each test concentration was given for 2.5 min starting from low concentrations to high concentrations. Cisapride was used as a positive control.
2.9.　 Stability test in liver microsomes assay
Compounds were incubated with human, or mouse liver microsomes and reduced nicotinamide adenine dinucleotide phosphate in 0.05 mol/L phosphate buffer (pH = 7.4) at 37 ℃ for 0–60 min. The reaction was quenched, and the amount of the remaining compound was analyzed using liquid chromatography-tandem mass spectrometry (LC–MS/MS).
2.10.　 The human CYP450 enzymes inhibition assay
This experiment was outsourced to Pharmalegacy Laboratories Co., Ltd. (Shanghai, China). The reaction was initiated by the addition of the coenzyme NADPH (6.5 mmol/L). Prepare probe substrate and HLM/MLM mixture in a 96-well plate according to the appropriate proportion. Probe substrate (318 μL) and human liver microsome mixture were dispensed to the incubation plates. Then add 2 μL of 8 serial concentration levels, and then add 80 μL aliquot of the NADPH regenerating system as 0 min to initiate the reaction. The sample plates were incubated at 37 ℃ and started a timer. The assay plates for testing inhibition of different CYP isoforms were incubated for different periods of time: 10 min for 1A2, 2B6, 2C8, 2C9, 2D6, and 3A4; 40 min for 2C19). At time points, 100 μL incubation solution was transferred to the wells of the corresponding plates containing 300 μL of internal standard solution, respectively, to stop the reaction. After quenching, shake the plates for 10 min and centrifuge the samples at 3000×g for 15 min, and 100 μL of the supernatant was transferred from each well to a 96-well sample plate containing 300 μL of water for LC–MS/MS analysis.
2.11.　 C. elegans liquid medium killing and rescue assay
Wild-type C. elegans (N2) strains were obtained from Caenorhabditis Genetics Center (MN, USA). The age-synchronized L4 larvae were transferred and incubated on peptone-glucose-sorbitol (PGS) agar plates (containing PAO1 strain) for 24 h at 20 ℃. PAO1 trains cells at midlogarithmic phase (OD600∼0.5) were harvested and diluted with S-Buffer to OD600 of 2, then 2 mL of bacterial solution was added to PGS agar plates. Infected N2 worms were washed with M9 buffer, resuspended, and then washed again with M9 buffer (1 L ddH2O, 5 g NaCl, 6 g Na2HPO4. 3 g KH2PO4, and 1 mL 1 mol/L MgSO4). Approximately 20 worms were transferred to 24-well plates (containing compounds to be tested or not, and 0.2 mmol/L floxuridine to prevent the worms from spawning). The number of worms (live or dead) was recorded every day.
2.12.　 Mice infection assay
A murine wound infection model in BALB/c female mice (6–8 week, 20–22 g) was established, and healthy mice were provided by Biotechnology Co., Ltd. (Guangzhou, China). Derivative A22 was dissolved in saline containing 0.1% Tween 80 and PB was prepared in saline. First, mice were anesthetized with 4% chloral hydrate and then shaved on the back. A ∼5 mm wound was created on the back of the mice and then 30 μL of P. aeruginosa DK2 (5 × 108 CFU) was inoculated on the surface of the wound. Surrounded by 48 h of wound infection, the wound was treated with saline, PB alone, or PB in combination with derivative A22, respectively. The drugs were administered twice a day. Then, a homogenate of the skin at the wound site was prepared and the bacterial density was determined CFU counting was obtained on an agar plate. Meanwhile, the wounds and body weights of the mice were monitored daily. After Day 5, the surviving mice were euthanized by cervical dislocation. Wounds were removed and homogenized in sterile PBS for bacterial loading. All experimental procedures were executed according to the protocols approved by the Hainan University Animal Research Committee (SYXK2023-0031).
2.13.　 Resistance assay
The detailed method for resistance assay was previously described12. Briefly, the ability of the combination of PB and derivative A22 to suppress resistance development was determined by serial passaging. P. aeruginosa DK2 with approximately OD = 0.01 was grown in 3 mL of LB broth with a sub-MIC (1/2 MIC) concentration of PB alone or in combination with derivative A22. At 24 h intervals, the cultures were assessed for growth. A bacterial dilution was made by using the bacteria from the sub-MIC concentration of PB (1/2 MIC). Then, the concentration of these bacteria was adjusted to OD = 0.01 and subjected to the next MIC assay. The tubes were incubated at 37 ℃ for 24 h. For the PB and A22 combination group, the concentration of derivative A22 was kept constant at 10 μmol/L throughout the experiment. This serial passaging was repeated for 14 days, and the fold change in the MIC value was determined.
2.14.　 qPCR analysis assay
Bacterial cells were cultured at 37 ℃ in LB broth to the logarithmic-growth phase (OD600 = 1.0). Cells were collected by centrifugation, and total RNA was extracted using an Eastep Super Total RNA Extraction Kit (Hlingene, Shanghai, China). cDNA synthesis was performed using a Hifair III One Step RT-qPCR SYBR Green Kit (Yeasen, Shanghai, China). Primers for RT-qPCR were listed in Supporting Information Table S2. The RT-qPCR (Thermo Fisher, QuantStudio™ 1, MA, USA) experiments were performed on a Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). The relative expression levels of the target genes were calculated using the comparative CT (2–ΔΔCT) method13.
2.15.　 Oxidative stress sensitivity measurement
Dilute the bacteria to 0.01 and treat the bacteria with compounds. When the OD600 of the bacteria reaches 1.0, collect 3 mL of bacteria liquid, and resuspend and centrifuge (JIDI, JIDI-21 R, Guangzhou, China) with 1 mL of sterile 1 × PBS buffer. Then, PBS containing 0.2 mol/L H2O2 was added to resuspend the organisms, and constant temperature was shaken for 15 min at 37 ℃. Afterward, the number of DK2 strains was determined by the gradient dilution coated plate method and plate colony counting method. The suspension of test bacteria was diluted 101–107 times using sterile 1 × PBS, respectively, and 5 μL of the diluted suspension was taken in LB solid medium spot plates, which were incubated at 37 ℃ in an incubator for days 1–2, after the number of colonies on the plates was counted and recorded, and the CFU to analyze the susceptibility to oxidative stress14.
2.16.　 Bacterial ATP level assay
The ATP content in bacteria was detected by Solarbio ATP Content Detection Kit (Solarbio, BC0300, Beijing, China), and the strains were treated with the compounds singly and in combination, when OD600 = 1.0, 2 mL of bacterial liquid was collected and centrifuged, 1 mL of extraction solution was added, and ultrasonically crushed in an ice bath, and then ATP was extracted from bacteria according to the instructions, and put on the ice to be measured. The samples were added into 96-well plates according to the instructions, and after mixing thoroughly, the absorbance value A1 at 340 nm for 10 s was measured immediately, and then the 96-well plates were placed in an incubator at 25 ℃ for 3 min, and then the absorbance value A2 at 3 min for 10 s was measured immediately. The relative ATP levels was calculated15.
2.17.　 Intracellular NADH oxidase assay
The NADH oxidase (NOX) content in bacteria was assayed using the Solarbio NOX activity assay kit (Solarbio, BC0630, Beijing, China). The strains were treated with the compounds singly and in combination, and 1 mL of the bacteria was collected when OD600 = 1.0, and the NOX activity was measured by the extraction method according to the instructions of the kit. Calculate the relative NOX activity16.
2.18.　 Intracellular ROS detection
In this experiment, a Beyotime reactive oxygen detection kit (Beyotime, S0033S, Shanghai, China) was used, bacteria were treated with the drug, and the organisms were collected, DCFH-DA was diluted according to 1:1000, so that the final concentration was 10 μmol/L. The organisms were collected and suspended in diluted DCFH-DA, incubated at 37 ℃ for 20 min; the organisms were resuspended with LB medium, and then centrifuged at 5000 rpm (JIDI-21 R) for 5 min, and washed to remove the DCFH-DA that had not entered into the cells. The organisms were centrifuged at 5000 rpm (JIDI-21 R) for 5 min, washed, and the DCFH-DA that did not enter the cells was removed. 488 nm excitation wavelength and 525 nm emission wavelength were used for the zymography detection17.
2.19.　 Biofilm production assay
Single colonies were inoculated in LB culture solution and cultured overnight at 37 ℃ and 200 rpm (MIULAB, ES-60 E, Hangzhou, China). Determine the OD600 value of the bacterial liquid and dilute the bacterial liquid with LB until OD600 = 0.01. The diluted bacterial solution was treated by compounds alone and combined with polymyxin B respectively. Bacterial cells were inoculated 1:100 in CPG medium in 96-well polystyrene plates. After incubation at 37 ℃ for 24 h, the cells were stained with 0.1% crystal violet (CV) for 30 min. The planktonic cells were removed by several rinses with H2O. The CV-stained bound cells were air dried for 1 h and then dissolved in 95% ethanol, and the optical density at 570 nm (OD570) of the solution was measured to quantify biofilm formation18.
2.20.　 Confocal laser scanning microscopy assay
Cover glass was placed in a six-well plate, and the strain was treated with the compound. The biofilms formed on the coverslips were washed three times with PBS (pH 7.2) and then dried at 60 ℃ for 30 min. The biofilms were stained with a 0.01% acridine orange (Macklin, 65-61-2, Shanghai, China) and ethidium bromide solution (Macklin, 25,535-16-4, Shanghai, China) for 15 min and observed by confocal laser scanning microscopy (Olympus, FV3000, Japan)19.
2.21.　 RNA-seq analysis assay
Total RNA was extracted from P. aeruginosa DK2 by using the Eastep Super Total RNA Extraction Kit. RNA purity was detected by using the Nano Photometer spectrophotometer (Bio-DL, Mico Drop Shanghai, China), RNA concentration was measured by using Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Thermo Fisher, Qubit 2.0, MA, USA), and RNA integrity was assessed by using the RNA Nano 6000 Assay Kit with the Bioanalyzer 2100 system (Agilent Technologies, 2100 system, CA, USA). Sequencing libraries were generated by using the NEB. Next, Ultra Directional RNA Library Prep Kit for Illumina (NEB, E7760S, MA, USA). Finally, the products were purified (Beckman Coulter, AMPure XP system, CA, USA), and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, CA, USA). After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform, and paired-end reads were generated. Trimmed sequence reads were aligned to the P. aeruginosa DK2 genome sequence using Bowtie2-2.2.3, and normalized read counts were compared using HTSeq v0.6.1 as described previously. Then, the FPKM of each gene was calculated based on 26 reads of the gene, and the read count was mapped for each gene20,21.
2.22.　 Statistical analysis
Statistical analyses were performed with GraphPad Prism 8. The data are presented as the means ± standard deviation (SD). Asterisks in the figures indicate corresponding statistical significance.
3.　Results and discussion
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3.1.　 Design and chemistry
Building on the structure–activity relationship (SAR) insights from NIC and compound 15, we identified that the presence of two benzene rings and the 2-hydroxyl and 5-fluoro substitutions on the left benzene ring were crucial for synergistic potency12,22. Modifications, including the introduction of new linkers and substituents on the right benzene ring, were modulated to enhance synergistic activity, reduce cytotoxicity, and improve druggability (Fig. 1).
A range of linker types, including sulfonamide, urea, and hydrazideamide, were introduced to modulate electronic and steric properties. Besides, research has shown that incorporating additional carbon atoms and amino acid linkers can enhance the flexibility and pharmacokinetic profiles of compounds23. Then, various bulky bis-amide linkers were inserted into the benzene rings. Moreover, substituents were strategically introduced at different positions on the right benzene ring. Electron-withdrawing groups (e.g., trifluoromethyl, nitro, etc.) and electron-donating groups (e.g., methoxy, hydroxyl, etc.) were introduced at the ortho, meta, and para positions to modulate electronic properties, enhance synergistic potential and minimize mammalian cytotoxicity. The synthesis of compounds A1–A30 was accomplished as depicted in Schemes 1—4.
Specifically, the synthesis of target derivatives A1–A4 is depicted in Scheme 1. The intermediate M1 was synthesized via a nucleophilic substitution starting from commercially available 5-fluoro-2-methoxybenzenesulfonyl chloride (1). M1 was then reacted with commercially available 2-chloro-1-fluoro-4-nitrobenzene (4) in the presence of potassium carbonate, yielding intermediate M2. Concurrently, commercially available 5-fluoro-2-methoxyaniline (2) was treated with triphosgene and triethylamine to generate isocyanic acid, which was subsequently reacted with 2-chloro-4-nitroaniline (3) to give the urea intermediate M3. Intermediate M4 was obtained through the nucleophilic substitution reaction of 4, followed by condensation with commercially available 5-fluoro-2-methoxybenzoic acid (20) to give the intermediate M5. Additionally, commercially available 2-(5-fluoro-2-methoxyphenyl)acetic acid (5) underwent a condensation with 3 to produce the intermediate M6. Finally, demethylation of the key intermediates, M2, M3, M5, and M6, produced derivatives A1–A4, respectively.
The synthesis of compounds A5–A15 is shown in Scheme 2. Using PCl3 as the acylating agent, various commercially available substituted amino acids (6–16) were coupled with 3 to produce intermediates M7–M17. These intermediates were then deprotected from Fmoc in the presence of piperidine in acetonitrile to generate intermediates M18–M28. Condensation of intermediates M18–M28 with 20 in the presence of EDCI produced M29–M39, which were subsequently demethylated using BBr3 to afford the corresponding derivatives A5–A15.
As shown in Scheme 3, intermediates M40–M41 were prepared by nucleophilic substitution of 4 with various substituted amines, followed by deprotecting the BOC group using trifluoroacetic acid to yield intermediates M42–M43. Condensation of intermediates M42–M43 with 20 in the presence of EDCI and DMAP or HOBt produced intermediates M44–M45, respectively, which were then demethylated using BBr3 to afford the corresponding derivatives A16–A17.
As described in Scheme 4, the direct coupling of commercially available 5-fluoro-2-hydroxybenzoic acid (19) and 2,4-dimethoxyaniline afforded the derivative A18. Derivative A19 was accessed under similar coupling conditions by condensation of 19 with 2-methoxy-4-nitroaniline, followed by demethylation with BBr3 to yield derivative A20. Commercially available 2,4-bis(trifluoromethyl)aniline reacted with sodium nitrite in the presence of SnCl2 and concentrated HCl to give hydrazine intermediate M55. Subsequent condensation of the intermediate M55, or various substituted aniline, with 20 yielded intermediates M46–M54 and M56, respectively, which were then subjected to demethylation using BBr3 to give derivatives A21–A30.
3.2.　 Evaluation of derivatives for antibacterial activity against P. aeruginosa DK2
Adjuvants were typically defined as compounds that lack direct antimicrobial activity or exhibit minimal inhibition of bacterial growth but enhance the inhibitory effect of antibiotics7. Therefore, all target derivatives A1–A30 were evaluated for their in vitro antibacterial activity against P. aeruginosa DK2 using the broth-dilution method. The minimum inhibitory concentration (MIC) was determined as the concentration at which there was approximately 100% reduction in bacterial growth compared to the drug-free control. The antibacterial activity results were tabulated in Tables 1 and 2. As expected, the majority of the derivatives were inactive against P. aeruginosa DK2, with MIC values exceeding 1000 μmol/L. Only compounds A3, A20, A25, and A28–A30 demonstrated moderate antibacterial activity, with MIC values ranging from 50 to 100 μmol/L.
3.3.　 Evaluation of derivatives for synergistic activity to PB against P. aeruginosa DK2 and SAR study
The synergistic activity of derivatives was assessed using the checkerboard microdilution assay. The synergistic effects were evaluated by the fractional inhibitory concentration (FICI), with synergism defined as a FICI ≤ 0.524. The FICI was calculated by summing the ratios with the MIC of each drug in combination to the MIC of the drug when used individually. As shown in Table 1, derivatives A1–A3, incorporating sulfonamide, urea, and hydrazide amide linkers, demonstrated a reduction in synergistic activity (FICI range: 0.108–0.204) compared to the lead compound 15 (FICI = 0.014). Notably, derivative A3 (10 μmol/L), featuring a novel amido hydrazine linker, significantly improved the MIC of PB from 256 to 1 μg/mL, below the clinical sensitivity threshold (MIC ≤2 μg/mL). However, A3 exhibited weak antibacterial activity against P. aeruginosa DK2, with a MIC of 50 μmol/L (16.28 μg/mL), raising concerns about potential drug resistance after long-term usage. Derivatives A4–A17 exhibited synergistic activity against P. aeruginosa DK2 at concentrations of 10–100 μmol/L (FICI = 0.026–0.204). Unfortunately, none surpassed the performance of 15. The results indicated that the introduction of sulfonamide, urea, various diamine, and amino acid linkers led to a reduction in synergetic activity. Additionally, derivatives with bulky linkers (A6, A10–A11) were explored and resulted in a complete loss of synergetic activity (MIC of PB > 32 μg/mL). Overall, the amide linker was found to be crucial for maintaining synergetic activity.
Due to the weak potency observed, further optimization of the linker moiety was not pursued. Subsequently, our focus shifted to examining the impact of substitutions on the right benzene ring (Fig. 1). To explore the effects of introducing various electron-withdrawing and electron-donating groups, derivatives A18–A30 were synthesized and evaluated (Table 2). Initially, derivative A18, featuring a 2′–OCH3-4′–OCH3 substitution, led to a significant loss of synergistic activity. Additionally, the 2′–Cl substituent was replaced with 2′–OCH3 or 2′–OH to yield derivatives A19–A20, both of which exhibited limited potency (FICI = 0.018 and 0.258, respectively). This suggests that the introduction of electron-donating groups adversely affects synergistic activity. Further exploration with various electron-withdrawing substituents (A21–A29), such as –CF3, –CN, –SO2CF3, and –NO2 revealed improved synergistic activity (FICI <0.5) across all derivatives. Notably, electron-withdrawing substitutions at the 2′,4′-position exerted favorable effects on activity. Among these, derivative A22, with bis(trifluoromethyl) substitution, maintained potency (FICI = 0.014) comparable to lead compound 15, enhancing the MIC of PB from 256 to 1 μg/mL, while itself exhibiting no inhibitory activity against P. aeruginosa DK2 (MIC >1000 μmol/L). Conversely, derivative A23, with 3′,5′-bis(trifluoromethyl) substitution, demonstrated lightly reduced synergetic activity (FICI = 0.026) compared to A22. These results suggested that 2′,4′-substitution with electron-withdrawing groups played a crucial role in modulating synergetic activity. Further substitutions, including replacing the 4′-NO2 group with 4′–CN (A21), 4′–CF3 (A24), and 4′–SO2CF3 (A26–A27), as well as replacing 2′–Cl with 2′–CF3 (A25), collectively resulted in a reduction in synergistic activity. Derivatives A28 and A29 demonstrated synergistic activity comparable to compound 15, yet at 100 μmol/L, they exhibited inhibitory activity against P. aeruginosa DK2. Finally, using the pharmacophore confluence strategy, from the most potent derivatives, A3 (linker) and A22 (substitutions), derivative A30 was synthesized. However, it still demonstrated some antibacterial activity, along with a decrease in synergistic activity (FICI = 0.108). Based on these findings, A22 was selected for further investigation to determine its potential to overcome the limitations of lead compound 15 and establish itself as a promising adjuvant candidate.
3.4.　 Derivative A22 as a potent adjuvant from time-kill assay
To further elucidate the synergistic effects of derivative A22 to PB, time-kill curves were determined. The results confirmed that neither A22 (10 μmol/L = 3.67 μg/mL, or 20 μmol/L = 7.34 μg/mL) nor PB (0.5 or 1 μg/mL) significantly affected bacterial growth when used alone. However, a substantial reduction in colony-forming units (CFUs) as early as 2 h when A22 was combined with PB (Fig. 2A and B). Moreover, the combination of A22 (10 or 20 μmol/L) with PB (1 μg/mL) completely inhibited bacterial growth at 36 or 24 h, respectively (Fig. 2B), outperforming the lead compound 15 (Fig. S1B). These results are consistent with the checkerboard assay findings, suggesting that A22 was a potent adjuvant for enhancing the efficacy of PB against P. aeruginosa DK2.
3.5.　 Derivative A22 as a potent broad-spectrum antibiotic adjuvant to polymyxins
To evaluate the broader applicability of A22 in combination with PB against various MDR Gram-negative bacteria, we assessed the FICI values for eight species of bacteria: Acinetobacter baumannii (A. baumanni) 186, Klebsiella pneumoniae (K. pneumoniae) 674, K. pneumoniae 15,004 (resistant to all antibiotics used clinically), Enterobacter asburiae (E. asburiae) 184, Escherichia coli (E. coli) 15,017, Enterobacter cloacae (E. cloacae) 107, Proteus mirabilis, and Serratia arcescens. All these strains are clinically obtained and phenotypically resistant to PB (Fig. S1C). As illustrated in Fig. 2C, derivative A22 significantly synergized PB against these species (FICI ≤0.26), except for Proteus mirabilis. Interestingly, when A22 was combined with colistin (polymyxin antibiotic as well) or other classes of antibiotics, such as tobramycin, piperacillin, meropenem, ciprofloxacin rifampicin and sulfadiazine, and no synergetic activity was observed against P. aeruginosa DK2, except with colistin (Supporting Information Table S3 and Fig. 2D). These findings suggest that A22 is a broad-spectrum adjuvant specifically to polymyxins against Gram-negative bacteria, providing insights into its synergistic mechanism.
3.6.　 Safety, solubility, and plasma protein binding evaluation of derivative A22
To address the cytotoxicity concerns of compound 15, we evaluated the safety profile of derivative A22 using two mammalian cell lines, 293 T and HUVEC. As shown in Table 3, derivative A22 exhibited minimal cytotoxicity (IC50 > 150 μmol/L) in both cell lines, surpassing compound 15 (IC50 = 12.11 μmol/L for 293 T and IC50 = 18.69 μmol/L for HUVEC). Given the potential risk of cardiotoxicity associated with human ether-a-go-go (hERG) suppression25, derivative A22 was tested on the hERG potassium channel patch-clamp assay, with cisapride as a control. Derivative A22 displayed an acceptable affinity for hERG (IC50 = 6.94 μmol/L), which is significantly lower than that of cisapride (IC50 = 0.036 μmol/L, Supporting Information Fig. S2), indicating a lower cardiotoxicity risk.
In addition, A22 exhibited improved water solubility (4 μg/mL) compared to compound 15, as well as high plasma protein binding (PPB) potential (97.94%), suggesting a long maintenance time and stable effect in vivo (Table 3). These results indicate that derivative A22 has a favorable safety profile in vitro and improved bioavailability.
3.7.　 Metabolic stability evaluations of derivative A22
We conducted a stability assay in human liver microsomes (HLM) and mouse liver microsomes (MLM), using verapamil as a control. As presented in Table 4, derivative A22 demonstrated reasonable metabolic stability, with a half-life (t1/2) of 29.09 min in HLM and 54.43 min in MLM, showing 23.96% and 46.31% of the compound remaining after 1 h, respectively. These results suggest that derivative A22 possessed adequate metabolic stability, further supporting its potential as a drug candidate.
3.8.　 The inhibition of human CYP450 enzymes of derivative A22
As shown in Table 5, A22 exhibited moderate inhibition of CYP2C8, CYP2C9, and CYP2D6 with IC50 values of 2.61, 0.29 and 1.88 μmol/L, respectively. Moreover, A22 demonstrated minimal inhibition against CYP1A2, CYP2B6, CYP2C19, and CYP3A with IC50 values of 6.13, >33.00, 20.5, and >33.00 μmol/L, respectively. This profile suggests a low risk of drug–drug interactions from A22.
3.9.　 In vivo synergetic potency of derivative A22
Given the potent synergistic activity, favorable safety profile, and adequate metabolic stability of derivative A22, we further conducted in vivo pharmacodynamic studies. First, Caenorhabditis elegans (C. elegans) was infected with P. aeruginosa PAO1 and treated with the monotherapy or combinational therapy of derivative A22 and PB (Fig. 3A and B). The results demonstrated that neither A22 nor PB had significant therapeutic effects on the survival of the worms. Whereas the combination of 8 μg/mL A22 and 8 μg/mL PB synergistically extended the lifespan of the worms, demonstrating the efficacy of the combination therapy in vivo.
Next, the therapeutic potential of A22 was evaluated in a mouse wound infection model infected with P. aeruginosa DK2. Excisional wounds were created on the dorsal region of the mice on day 1, followed by P. aeruginosa DK2 infection. Treatment began on day 2 post-infection to allow sufficient time for wound colonization (Fig. 3A). As shown in Fig. 3C and D, neither A22 monotherapy (10 mg/kg) nor PB monotherapy (1 mg/kg) exhibited significant antibacterial effects after 7 days of treatment. In stark contrast, the combinational therapy of A22 (10 mg/kg) and PB (1 mg/kg) significantly reduced bacterial loads in the infected wounds compared to control and monotherapy, effectively clearing the infection.
3.10.　 Induction of resistance of derivative A22
The development of drug resistance is a major challenge in the advancement of novel antibiotics. To evaluate the ability of A22 to suppress resistance development to PB in P. aeruginosa DK2 under selective pressure, a resistance induction experiment was conducted. DK2 was cultured daily in the presence of subinhibitory concentrations of PB, with or without combination treatment with A22, and the minimum inhibitory concentration (MIC) of PB was measured for both groups over 14 days to monitor resistance progression. As shown in Fig. 4, the group treated with PB alone (64 μg/mL) displayed a significant increase in resistance over time. The MIC of PB increased 4 folds, from 256 μg/mL at the start to 1024 μg/mL after 14 generations, clearly indicating substantial resistance development. In stark contrast, the group treated with a combination of A22 (10 μmol/L = 3.67 μg/mL) and PB (0.5 μg/mL) exhibited no resistance development, with the MIC of PB remaining stable at 1 μg/mL (combined with 10 μmol/L A22) throughout the 14-day period, matching the MIC of the initial generation (Table 2).
These findings demonstrate that A22 effectively prevents the emergence of PB resistance in P. aeruginosa DK2, aligning with the design rationale for adjuvants to combat bacterial resistance, and highlighting A22's potential as a promising adjuvant for improving the efficacy and longevity of polymyxin-based therapies.
3.11.　 Synergistic mechanism of derivative A22
Previous results from Section 2.5 demonstrated that derivative A22's mechanism of synergism is closely related to the mode of action of polymyxins, which primarily disrupt bacterial membranes by binding to lipopolysaccharides (LPS) on the outer membrane. The disruption of the bacterial outer membrane compromises membrane integrity, leading to increased permeability and subsequent membrane depolarization. This process not only weakens the bacterial cell wall but also perturbs the proton motive force (PMF), which is essential for ATP production and homeostasis in bacteria26,27. The resulting cellular damage from membrane disruption likely triggers a cascade of stress responses, including the activation of oxidative stress pathways28. Given this membrane destabilization and the associated stress, we hypothesized that A22 amplifies polymyxin-induced bactericidal effects by modulating oxidative stress responses in P. aeruginosa DK2.
To investigate this hypothesis, P. aeruginosa DK2 was treated with PB (0.25 or 0.5 μg/mL), the lead compound 15, and derivative A22 (4 or 8 μg/mL). Antioxidant capacity was assessed by observing P. aeruginosa DK2 growth under oxidative stress (H2O2) environments. PB alone did not reduce CFU, whereas treatment with either the lead compound 15 or derivative A22, with or without PB, significantly reduced CFU (Fig. 5A and B and Supporting Information Fig. S3A). These above results indicated derivative A22 significantly decreases the antioxidant capacity of P. aeruginosa DK2. This hypothesis is further supported by ATP level measurements. Monotherapy with 15 or A22 reduced ATP levels in P. aeruginosa DK2, consistent with PMF dissipation, and the reduction in ATP levels was even more pronounced when combined with PB (Fig. 5C and Fig. S3B). Additionally, NOX activity, which reflected ROS generation29, was significantly increased by both 15 and A22, with a further significant increase in combination with PB (Fig. 5D and Fig. S3C).
Direct measurement of ROS levels confirmed these results, with both 15 and derivative A22 significantly elevating ROS levels, and A22 demonstrating a more pronounced effect than 15, especially in combination with PB (Fig. 6A and Fig. S3D). The relationship between oxidative stress and antibiotic resistance in P. aeruginosa is well-established, influencing not only efflux pump gene expression but also biofilm formation, a critical virulence factor often links to antimicrobial resistance30—34.
As shown in Fig. 6B and C, A22 exhibited potent inhibitory activity against biofilm formation in P. aeruginosa DK2 at both 4 and 8 μg/mL, as evidenced by crystal violet staining and confocal laser scanning microscopy (CLSM) images. Notably, the inhibitory effect was significantly enhanced when 8 μg/mL A22 was combined with either 0.25 or 0.5 μg/mL PB, demonstrating a robust synergy in disrupting biofilm-associated bacterial defenses.
Together, these findings suggest that A22 enhances the bactericidal effect of polymyxins by reducing P. aeruginosa DK2's antioxidant capacity, facilitating ROS accumulation, disrupting the PMF, inhibiting biofilm formation, and ultimately leading to bacterial death. This oxidative stress modulation is a key mechanism by which A22 selectively synergizes polymyxins.
3.12.　 Gene expression modulated by derivative A22
To elucidate the impact of derivative A22 on P. aeruginosa DK2, we conducted a transcriptomic analysis using RNA sequencing (RNA-Seq) to compare the gene expression profiles of the A22-treated group with the control group. Differential gene expression analysis revealed that 257 genes were upregulated and 121 genes were downregulated (Log2 fold change ≥2.0, Fig. 7A and Supporting Information Table S4). These differentially expressed genes are involved in various resistance-related functions, including the two-component regulatory system, oxidative phosphorylation, nitrogen metabolism, bacterial chemotaxis, amino acid metabolism, pyruvate metabolism, butyrate metabolism, fatty acid degradation, and ABC transporters (Fig. 7B and Table S4).
To validate the RNA-Seq results, quantitative RT-PCR was performed on selected genes (Fig. 7C). The analyzed genes included those related to the denitrification nir system (nirS, nirM, nirC, nirF, nirN, and nirJ), arginine metabolism (arcA, arcD, and arcC) and oxidative stress related genes (nosZ, bfr, anvM, dnr, and hemF). AnvM acts as an anaerobic toxicity regulator, contributing to oxidative stress response, redox enzyme activity, transcription regulation, exercise, and pathogenicity35. HemF is an oxygen-dependent cytochrome III oxidase that plays a certain role in oxygen metabolism36. The nir system regulates nitrite metabolism pathways and modulates nitric oxide (NO) levels, which are crucial for the increased activity of KatA37.
The transcriptome data also verified our previous experimental results and the antioxidative stress-related genes were also significantly down-regulated after derivative A22 treatment, which further suggests that derivative A22 may synergize PB through the oxidative stress pathway. However, we also found that derivative A22 also affects a number of pathways associated with drug resistance, and it is possible that there are multiple targeting pathways of action for derivative A22 to synergize polymyxins. There is still a long way to go to study the synergistic mechanism of derivative A22, and in the future, we will focus more on the identification and study of direct targets.
4.　Conclusions
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In this study, we successfully synthesized and evaluated a series of novel benzamide derivatives for their ability to synergize polymyxins against MDR P. aeruginosa DK2. Among these, derivative A22 emerged as a particularly promising candidate, demonstrating superior synergistic activity to PB in vitro, while exhibiting no inherent antibacterial activity. Compared to the lead compound 15, A22 showed significantly lower cytotoxicity toward mammalian cells, improved solubility, and exhibited reasonable metabolic stability. Notably, A22 broadly potentiated polymyxins against various clinically isolated MDR Gram-negative strains. Furthermore, A22 demonstrated potent in vivo efficacy in both C. elegans and mouse infection models, further supporting its potential as a safe and effective adjuvant. Resistance assay indicated combination treatments with derivative A22 and PB significantly suppressed resistance development in P. aeruginosa DK2 compared to PB single treatment. Mechanistic studies revealed that A22 effectively reduces antioxidant capacity, diminishes ATP production, dissipates the PMF, increases NOX activity, elevates ROS levels, and inhibits biofilm, leading to a cascade of events that culminates in bacterial death. These findings suggest that A22 is a promising candidate for further clinical development as an adjuvant for polymyxin-based therapies, offering a valuable approach to combating MDR bacterial infections.
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Appendix
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Year 2025 volume 15 Issue 3
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doi: 10.1016/j.apsb.2025.01.022
  • Receive Date：2024-09-12
  • Online Date：2026-09-17
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  • Received：2024-09-12
  • Revised：2024-11-20
  • Accepted：2024-12-23
Affiliations
    aKey Laboratory of Tropical Biological Resources of Ministry of Education and Hainan Engineering Research Center for Drug Screening and Evaluation, School of Pharmaceutical Sciences, Hainan University, Haikou 570228, China
    bState Key Laboratory of Bioreactor Engineering, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
    cHainan Hospital of Chinese PLA General Hospital Laboratory Department, Sanya 5720225, China
    dShanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
    eSchool of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China
    fAnhui Province Key Laboratory of Infectious Diseases, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
    gKey Laboratory of Xinjiang Phytomedicine Resource and Utilization, Ministry of Education, School of Pharmacy, Shihezi University, Shihezi 832003, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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