Skip to main content
NCBI home page
JACS Au logoLink to JACS Au
. 2026 Oct 11;6(10):5949–5960. doi: 10.1021/jacsau.6c01288

Modular Triethylene Glycol Decouples Antimicrobial Potency from Cytotoxicity in Membrane-Active Drugs

Marc Seewald †,‡, Marla Stamen †,‡, Jian Li §, Sven Engelkamp †,‡, Jan-Simon Behnke †,‡, Ralf Lucassen ∥, Katharina Borowick †,‡, Marie Weilburg †,‡, Virginia Wycisk †,‡, Tom Fielitz ⊥, Tzu-Chien Wu ⊥, Matthias Hartlieb ⊥, Frieder Schaumburg #, Dirk Bockmühl ∥, Tobias Raisch §,*, Leonhard Hagen Urner †,‡,*
PMCID: PMC13714845  PMID: 42953173

Abstract

Cationic membrane-active drugs are an important focus of antibiotic research, yet their potential is often limited by poor selectivity. Here, we establish steric protection of cations by modular triethylene glycol as a chemical design framework to decouple antimicrobial potency from cytotoxicity. Using antipsychotics, a class of widely prescribed membrane-active drugs, we show that lipophilicity and rigidity jointly govern antimicrobial potency and mammalian cell cytotoxicity. Membrane perturbation emerged as a key determinant of bioactivity and revealed physicochemical windows that decouple antimicrobial potency and cytotoxicity. Our findings spotlight octanol as a design motif for steric protection of cations that emerged from systematic tuning of side chain lipophilicity and mimics a known metabolite motif. As exemplified with the model drug chlorpromazine, this modification can maintain water solubility, improve membrane engagement, reduce mammalian cell cytotoxicity, enhance antibacterial potency, and amplify polymyxin amplification across Gram-negative bacteria, including drug-resistant clinical isolates. Our findings indicate that membrane remodeling associated with polymyxin resistance in Escherichia coli correlates with a collateral vulnerability to cationic amphiphiles, suggesting a physicochemically tractable consequence of resistance. These findings establish a chemical design framework for small-molecule cationic amphiphiles with improved physicochemical properties, membrane engagement, and bioactivity that can be difficult to achieve by traditional alkylation.

Keywords: Cationic Amphiphile, Cytotoxicity, β-Lactam Resistance, Polymyxin Resistance, Escherichia coli

graphic file with name au6c01288_0007.webp

graphic file with name au6c01288_0005.webp

1. Introduction

Biological membranes are universal cellular interfaces. Strategies to control membrane interactions in small molecules are of broad interest in chemical biology and medicine, − including the search for antimicrobials. − The increasing prevalence of β-lactam-resistance in Gram-negative bacteria poses a significant challenge in human health. , Availability of treatment options relies on the development of new antimicrobials, β-lactamase inhibitors, , and the use of membrane-active, last-resort antibiotics, such as polymyxins. The use of polymyxins is limited by dose-dependent nephro- and neurotoxicity and the emergence of resistance. , Strategies that reduce polymyxin dosage while maintaining its efficiency are crucially needed.

Cationic amphiphiles are membrane-active molecules that can sensitize bacteria to antibiotics. Cationic amphiphilic drugs provide an attractive entry point. They combine established pharmacology with synthetic accessibility. Antipsychotic drugs, such as phenothiazines, represent a historically privileged subclass. Tracing back to antimicrobial dye chemistry, this lineage gave rise to chlorpromazine (CPZ), the first widely used antipsychotic. , CPZ and other antipsychotics have been repurposed as antimicrobial agents and adjuvants for antibiotics against drug-resistant pathogens. ,, Their utility as antimicrobials is constrained by close coupling between antibacterial potency and mammalian cytotoxicity.

Membrane features governing uptake of amphiphiles, i.e., hydrophobic and electrostatic interactions, are conserved across species. Physicochemical properties promoting membrane engagement commonly enhance antibacterial activity and mammalian cytotoxicity, creating a fundamental design challenge for cationic amphiphiles. , Selectivity is a relative property. ,− Chemical design parameters to improve bioactivity of amphiphiles are an enormously valuable asset in antibiotic research.

Steric protection of cations by alkylation with hydrocarbon chains recently emerged as a strategy to improve bioactivity of cationic amphiphiles, including drugs. However, the aqueous solubility of hydrocarbon chains decreases with increasing chain length. Poor solubility correlates with poor bioavailability and limits utility of hydrocarbon chains in small-molecule design. Short oligoethylene glycol chains provide water-soluble counterparts to linear hydrocarbon chains and have enabled simultaneous improvements in antibacterial potency and cytotoxicity of cationic antibacterial polymers. This led us to the question if modular triethylene glycol , can improve physicochemical properties and bioactivity of small-molecule amphiphiles. Using antipsychotic drugs as models, we combine orthogonal biophysical techniques, , antimicrobial susceptibility testing , and cell viability assays , to uncover physicochemical property windows that decouple antimicrobial potency from cytotoxicity and improve polymyxin amplification. Surprisingly, complementary potency of cationic amphiphiles and polymyxins in Escherichia coli suggests membrane remodeling linked to polymyxin resistance as a design opportunity for amphiphiles against this increasingly important resistance phenotype. ,

2. Results and Discussion

2.1. Steric Protection of Cation in CPZ by Triethylene Glycol Partially Decouples Potency from Cytotoxicity

Improving the antimicrobial potency of cationic amphiphiles while minimizing cytotoxicity remains a central goal in antimicrobial research. ,,,,, Bacterial membranes expose anionic lipids at the cell surface, whereas mammalian plasma membranes present charge-neutral lipids at the outer leaflet and anionic lipids on the cytoplasmic leaflet. , Steric protection of cations in amphiphiles attenuates electrostatic attraction to membrane surfaces. , The stronger negative surface potential of bacterial membranes partly compensates for this effect, serving as a starting point to decouple antimicrobial potency from cytotoxicity.

To test this concept, we used CPZ as our initial model and quaternized its terminal amine with triethylene glycol. Hydrocarbon chains longer than 12 carbon atoms sterically protect quaternary ammonium groups through backfolding. , However, their poor aqueous solubility limited application to small-molecule amphiphiles, as illustrated by our poorly soluble heptyl CPZ derivative (Figure S1). In contrast, triethylene glycol-modified CPZ derivative 1 was highly water-soluble (≥50 mg·mL–1). Furthermore, ethylene glycol units adopt gauche conformations, which promotes backfolding , and qualified triethylene glycol as a motif to investigate steric protection of cations.

To evaluate how steric protection in CPZ derivative 1 altered membrane engagement, we analyzed its potency to perturb membrane models (Figure A–C). The hydrochloride of CPZ (CPZ*HCl) is a cationic amphiphile that has been shown to weakly associate with lipid bilayers. , Triethylene glycol increases the overall polarity of CPZ, which should reduce its partitioning into lipid membranes. , Langmuir–Blodgett experiments revealed lower surface pressures for CPZ derivative 1 compared to CPZ*HCl, indicating reduced lipid monolayer insertion (Figure B). Compared to CPZ*HCl, its derivative 1 led to lower median effective concentration (EC50) required to permeabilize calcein-loaded vesicles derived from E. coli cytoplasmic membrane lipids and phosphatidylcholine (Figure C). ,

1.

1

Open in a new tab

Steric protection of cation in CPZ by triethylene glycol partially decouples antimicrobial potency from cytotoxicity. (A) Sterical protection of the cation in CPZ and accompanied property changes. (B) Langmuir–Blodgett isotherms showing decrease in surface pressure of lipid monolayer upon steric protection of cation in CPZ derivative 1. (C) Vesicle permeabilization assay showing fluorescence of calcein released from vesicles against concentration of CPZ derivatives. EC50 values are shown to compare membrane permeabilizing potency. (D) MIC data and (E) IC50 values of CPZ derivatives (bar charts). Data are shown as average from three independent biological repeats (n = 3). IC50 values are shown as mean with standard deviation (±SD).

Model membranes cannot fully recapitulate the structural complexity of cellular envelopes. To test how altered membrane engagement correlates with antimicrobial potency and cytotoxicity, ,, we compared minimal inhibitory concentrations (MICs) , and half-maximal inhibitory concentrations (IC50) across bacteria and mammalian cell models. , First, we determined MIC values in seven high-priority ESKAPEE pathogens from the priority list of the World Health Organization (Figure D). Quaternization of CPZ with triethylene glycol resulted in an average 4.4-fold increase in MIC across bacterial strains (Figure D). Given that MIC values were determined in 2-fold dilution steps with an uncertainty of ± one dilution step, changes below 8-fold are not considered significant under our conditions. The antimicrobial potency of CPZ*HCl was mildly reduced after steric protection, with the associated increase in MIC of derivative 1 remaining within the experimental uncertainty of the 2-fold dilution method (Figure D).

Second, we evaluated cytotoxicity in skin (HaCaT), liver (HEP3B), and kidney cell models (HEK293T) commonly used for in vitro toxicity assessment of cationic amphiphiles. − Cytotoxicity of CPZ was significantly reduced upon quaternization with triethylene glycol, as indicated by an average 30-fold increase in IC50 (Figure E). Steric protection of the cation in CPZ derivative 1 with triethylene glycol reduced membrane engagement and partially decoupled antimicrobial potency from cytotoxicity.

2.2. Membrane Perturbation Correlates with Antimicrobial Activity

We next sought to identify a key biophysical parameter governing antibacterial activity. The breadth of chemical strategies reported for membrane-active antimicrobials ,,,,, made it difficult to prioritize design options for CPZ derivative 1. Demonstrating a correlation between membrane perturbation and antibacterial potency was decisive to confirm a mechanistic parameter for compound optimization. Using E. coli K12 MG1655 as an established model for mode of action studies, , we dissected whether antimicrobial activity of CPZ derivative 1 involved increased intracellular retention of an efflux pump substrate, bilayer destabilization, and envelope damage. These membrane-related mechanisms of action are commonly assessed in cationic amphiphiles. ,,,,,

CPZ*HCl has been shown to increase the intracellular retention of efflux pump substrates in bacteria. , To determine whether this property is retained upon quaternization with triethylene glycol, we compared intracellular retention of ethidium bromide, a widely used substrate of bacterial multidrug efflux pumps. We observed comparable fluorescence levels of intracellularly retained ethidium bromide for CPZ*HCl and its derivative 1 (Figure A). Steric shielding did not abolish the ability of the compound to increase intracellular retention of the efflux pump substrate (Figure A).

2.

2

Open in a new tab

Antimicrobial membrane activity of CPZ derivative 1. (A) Normalized fluorescence of intracellular ethidium bromide that was retained in E. coli in the presence of CPZ*HCl and derivative 1, compared to inactive efflux (+) and active efflux (−) controls (bar chart). Normalized mean fluorescence is reported as mean ± SD (n = 3). (B) Diffusion coefficients and normalized fluorescence values obtained from calcein-loaded vesicles at different concentrations of CPZ derivative 1. The inset shows zeta potentials of vesicles measured at concentrations well below and near the EC50 of CPZ derivative 1 (bar chart). Data are presented as mean ± SD (n = 3). Representative cryo-electron micrographs of vesicles exposed to increasing concentrations of CPZ derivative 1 are shown on the right. (C) Effect of CPZ derivative 1 on bacterial morphology. Representative cryo-electron micrographs of untreated E. coli and cells treated with CPZ derivative 1 at MIC. Bar charts compare length and width of untreated and treated E. coli. Data are presented as mean ± SD (n = 60 cells).

While CPZ*HCl is an inhibitor of the AcrAB-TolC multidrug efflux pump, the E. coli genome encodes up to 20 efflux pumps. Many bacterial efflux pumps are powered by the proton motive force. , Increased intracellular retention of ethidium bromide may arise not only from direct inhibition of efflux pumps but also from disruption of the energetic driving force that supports efflux. However, CPZ derivative 1 interfered with hydrophobic fluorescence reporter assays used for this purpose. Therefore, we leveraged model lipid bilayers as an orthogonal, dye-independent approach to assess membrane perturbation, a process linked to proton motive force dissipation. ,

Cationic amphiphiles interact with membranes through electrostatic attraction to anionic phospholipids. To confirm electrostatic interactions with lipid bilayers, we performed zeta potential measurements on vesicles at concentrations well below the EC50 and near the EC50 of CPZ derivative 1. As expected, the zeta potential increased from −29 to −7.4 mV (Figure B), consistent with progressed membrane insertion near the median effective concentration to permeabilize vesicles.

Dynamic light scattering and cryo-electron microscopy supported vesicle destabilization by decreasing diffusion coefficients and disappearance of vesicles caused by CPZ derivative 1 near EC50 (Figure B) (Figure S2). EC50 values from vesicle assays were comparable to MIC values, supporting a correlation between changes in molecular structure, membrane perturbation and bacterial growth inhibition.

To examine how membrane perturbation manifests at the cellular level, we investigated bacterial morphology. Cryo-electron microscopy of untreated E. coli revealed morphologically intact cells with an average length of 2–4 μm and a width of 1 μm, consistent with previous results (Figure C) (Figure S3). Treatment with CPZ derivative 1 at MIC levels increased the length of E. coli cells to 4–6 μm (Figure C). In contrast, cell width remained unchanged and membranes appeared morphologically intact (Figure C) (Figure S4). Consistent with the absence of overt membrane disruption, protein leakage remained comparable to untreated controls, indicating no lytic cell damage under the employed conditions (Figure S5). The 2-fold increase in cell length upon treatment with CPZ derivative 1 suggests impaired cell division. , Similar changes in bacterial morphology have been reported to happen with CPZ*HCl and other phenothiazines. , CPZ*HCl has been shown to inhibit multiple membrane-bound metabolic proteins, leading to dissipation of the proton motive force, ,, depletion of intracellular ATP, , as well as perturbation of central carbon metabolism, transcription and translation.

Our integrated research approach is correlative and unable to resolve whether observed changes in bacterial growth inhibition and morphology result directly from membrane perturbation or downstream cellular responses. However, intact membrane integrity and associated functions are vital for divisome activity. The membrane-perturbing potency of CPZ derivative 1 provides a plausible explanation for the observed cellular elongation, supporting a relevant parameter for optimizing antimicrobial potency.

2.3. A Physicochemical Window for Decoupling Antimicrobial Potency from Cytotoxicity

To identify property windows that decouple antimicrobial potency from cytotoxicity, we examined structurally related antipsychotics differing in lipophilicity and rigidity (Figure A). Both properties govern membrane engagement. , To systematically change these parameters, we extended our compound library by hydrohalide acid salts of 1-(naphtalen-1-ylmethyl)­piperazine (NAP), citalopram (CIT), and imipramine (IMI) (Figure A). These antipsychotics are widely used drugs with known antimicrobial properties (Figure A). We included dimethyldodecylamine (DDA) to evaluate the role of backbone flexibility (Figure A). To determine antibacterial potency and cytotoxicity with increased throughput, we compared MIC values in E. coli K12 MG1655 and IC50 values in HaCaT cells (Figure A). Relative changes in lipophilicity and rigidity of hydrophobic backbones were assessed by means of clogP values and number of rotatable bonds (ΣRB) (Figure A). ,

3.

3

Open in a new tab

Lipophilicity and rigidity in our amphiphiles jointly determine coupling between antimicrobial potency and cytotoxicity. (A) Molecular structures of functionalized antipsychotic drugs and surfactant DDA. Relative changes in lipophilicity and rigidity in hydrophobic backbones are indicated by trends in clogP and sum of rotatable bonds (ΣRB backbone). MIC and IC50 values of acid salts (HX) and derivatives quaternized with triethylene glycol (E3) obtained in E. coli and HaCaT cells. Highest tested concentrations without inhibition are indicated with asterisks (*). (B) Side chains in quaternized CPZ derivatives, their MIC and IC50 values (n = 3). (C) MIC values and (D) IC50 data obtained from high-priority bacterial strains and mammalian cell models (bar chart) (n = 3). IC50 values are reported as mean ± SD.

Within our compound series, we observed less lipophilic and more rigid scaffolds, such as NAP and CIT (c log P ≤ 4; ΣRB 2–5), exhibited substantial losses in antibacterial potency despite improved cytotoxicity compared to their acid salts (Figure A). The more flexible and lipophilic DDA derivative showed increased cytotoxicity without gains in antimicrobial activity compared to its acid salt (Figure A). Rigid scaffolds with intermediate lipophilicity, such as CPZ and IMI (c log P 4–4.2; ΣRB 3–4), preserved antimicrobial potency alongside reduced cytotoxicity compared to their acid salts (Figure A).

These results suggest that the coupling between antimicrobial potency and cytotoxicity can be chemically modulated by varying the balance between lipophilicity and rigidity under the employed experimental conditions.

Turning to the more potent CPZ derivative 1, we asked if increasing the lipophilicity of the shielding chain improves decoupling of antimicrobial potency from cytotoxicity. In line with our mechanistic studies, we observed an increase in lipid bilayer permeabilizing potency in CPZ diethylene glycol derivative (2) and CPZ octanol derivative (3) compared to derivative 1 (Figure S6). Interestingly, this trend correlated with favorable biological activity. A mono­(ethylene glycol)-substituted control derivative of CPZ displayed an intermediate MIC in E. coli MG1655 (0.5 mM) but a lower IC50 in HaCaT cells (21 ± 0.5 μM) compared to derivatives 1 and 3. Thus, shortening the alcohol-containing side chain did not further improve relative selectivity compared to CPZ derivative 3 (Table S1). This indicated that the superior decoupling of antibacterial potency and cytotoxicity in CPZ derivative 3 reflected a defined window of shielding-chain length and lipophilicity.

Turning to the more potent CPZ derivative 3, we observed higher relative potency against E. coli K12 MG1655 and lower relative cytotoxicity against HaCaT cells compared to CPZ*HCl (Figure B). To assess the transferability of these observations, we evaluated CPZ derivative 3 across ESKAPEE strains and mammalian cell models. Notably, the CPZ derivative 3 caused an average 18-fold decrease in MIC across ESKAPEE strains and an average 7-fold increase in IC50 across mammalian cell lines relative to CPZ*HCl (Figure C–D). This led us to observe increases in in vitro selectivity indices of approximately 5-fold for almost all combinations of ESKAPEE bacterial strains and mammalian cell lines (Table S2). Despite strain-dependent variations in the magnitudes of these changes, our findings confirm that increasing the lipophilicity of the shielding chain can provide a handle to decouple antimicrobial potency from cytotoxicity.

Remarkably, the CPZ derivative 3 incorporated an octanol chain that we identified post hoc as a plausible cytochrome P450-mediated metabolite of alkylated cationic amphiphiles containing linear hydrocarbon chains. − Despite its increased lipophilicity, CPZ derivative 3 combined water solubility, enhanced antimicrobial potency, and reduced cytotoxicity (Figure C–D). This property profile was inaccessible through conventional alkylation in CPZ, suggesting linear alkyl chains with terminal hydroxyl groups provide a design strategy for membrane-active amphiphiles with improved biological performance. , However, the improved balance between antibacterial potency and cytotoxicity in nucleated cell models did not extend to erythrocytes. The minimum concentration required for hemolysis decreased with increasing lipophilicity of the side chain from CPZ derivatives 1 to 3 (Table S3). This is consistent with previous observations made from cationic amphiphilic peptides demonstrating that hemolytic activity and cytotoxicity can respond differently to amphiphile structure and hydrophobicity. , Furthermore, we interpreted physicochemical consequences of modifying the cation in CPZ as literature-supported correlative model to explain trends in antibacterial potency and cytotoxicity, warranting future investigations of the underlying mechanisms at the level of cellular membranes.

Consistently, across bacterial species, CPZ derivative 3 was more potent against Gram-positive bacteria compared to Gram-negative bacteria, in line with the established role of outer bacterial membranes as barriers against cationic amphiphiles (Figure C) (Table S4). Notably, the CPZ derivative 3 exhibited MIC values of 16 μM for Enterococcus faecium and 1.5 μM for Staphylococcus aureus (Figure C) (Table S4). This potency approaches or even surpasses that of recently reported dendritic oligoglycerol-based cationic surfactants, which displayed remarkably low MIC values against Staphylococcus aureus near 10 μM. Interestingly, this improvement led us to observe remarkable increases in strain-specific in vitro selectivity indices compared to CPZ derivative 1 (Table S2).

Current strategies to improve antimicrobial potency and cytotoxicity of amphiphiles focus on chemical strategy to improve relative selectivity, which includes the design of multivalent cationic amphiphiles, bactericidal urea crown ethers, , LPS-targeting peptides, and chimeras of inner and outer membrane-targeting antibiotics. Alkylation of cations with hydrocarbon chains is also widely used to increase relative selectivity, − which led recently to new vancomycin lead structures. However, as exemplified with our CPZ derivatives, alkylation of cations with hydrocarbon chains can reduce water solubility, which correlates with poor bioavailability and complicates the analysis of biological effects (Figure S1). Modular triethylene glycol served as a design handle that improved solubility, membrane engagement, antimicrobial potency, and cytotoxicity in CPZ.

2.4. Octanol Mimics a Metabolite and Causes Functional Advantages in Polymyxin Amplification

Since productive membrane engagement emerged as determinant of antibacterial activity, we finally asked whether modular triethylene glycol improves amplification of polymyxins. To evaluate how steric protection influences polymyxin amplification, we compared CPZ derivatives across a panel of E. coli strains spanning a representative range of colistin susceptibility reported by the EUCAST (Figure A). Consistent with MIC values observed on ESKAPEE strains, the CPZ derivative 3 had lower MIC values compared to CPZ*HCl and derivative 1 across E. coli strains (Figure B).

4.

4

Open in a new tab

Complementary potency and amplification with colistin. (A) Colistin MIC values in E. coli reported by EUCAST and E. coli strains from this study indicated. (B) Concentration of CPZ derivatives used for amplification (0.5× MIC) relative to the MIC of colistin against E. coli strains. Trends are visualized with dashed lines. (C) Amplification factors of colistin caused by CPZ derivatives at 0.5× MIC. (D) MIC values of colistin in laboratory E. coli strains and β-lactam-resistant clinical isolates before and after amplification. (E) MIC values of colistin for other Gram(−) strains before and after amplification. (F) Comparison of mean MIC across Gram(−) strains, number of Gram(−) strains showing amplification, and mean colistin MIC after amplification with CPZ derivatives ± SD.

Unexpectedly, the antimicrobial susceptibility profiles of our adjuvants and colistin were perfectly complementary (Figure B). E. coli strains with lower relative MIC values for colistin showed higher relative MIC values for adjuvants (Figure B). Notably, colistin-resistant E. coli B IMG 1702 had lower relative MIC values for adjuvants (Figure B). Comparable results with polymyxin B confirm that complementary potency extends to other polymyxins (Figure S7).

Colistin-resistance is mediated by lipid A modifications in lipopolysaccharides, like phosphatidyl amine and/or 4-amino-4-deoxy-l-arabinose, lowering electrostatic attraction to polymyxins. Polymyxin-resistance has been shown to correlate with reduced structural integrity of bacterial outer membranes, , which correlated with increased susceptibility to cationic amphiphiles in our study (Figure B). In addition, E. coli B strains have been shown to express elevated levels of OmpF porin proteins, resulting in increased outer membrane permeability that may also enhance susceptibility to cationic amphiphiles.

We observed similar trends in complementary potency for DDA derivatives (Figure S8). Relative changes in susceptibility across susceptible and polymyxin-resistant E. coli extend to cationic amphiphiles beyond CPZ.

To test bacterial cell wall integrity as determinant for antimicrobial potency, we performed control experiments using E. coli K12 ΔTolC. Deletion of TolC in this strain increases outer membrane permeability. Comparable MIC values of our adjuvants in E. coli K12 ΔTolC and the polymyxin-resistant strain supported the hypothesis that reduced cell wall integrity increased antimicrobial potency (Figure B) (Figure S8).

To test whether complementary potency profiles extend to polymyxin amplification, we quantified amplification factors of colistin caused by CPZ*HCl, 1, and 3 across our E. coli panel (Figure C). Amplification was mainly significant in strains with higher relative MIC values to CPZ-based adjuvants, while no significant amplification occurred mainly in strains that were more sensitive to our adjuvants, i.e., E. coli K12 ΔTolC and B IMG 1702 (Figure C) (Table S5).

To ascertain if amplification requires measurable antimicrobial activity of both compounds, we performed checkerboard assays. For polymyxin-susceptible E. coli K12 MG1655, we observed mild, concentration-dependent synergies (Figure S9). The presence of the compound at higher concentrations enhanced the activity of the other at lower concentrations. In contrast, checkerboard analysis on polymyxin-resistant E. coli B IMG 1702 revealed no interactions (Figure S9). Similar interaction patterns were reported for established antibiotic combinations, for example, pairs of β-lactam and aminoglycoside antibiotics. Loss of activity of one antimicrobial reagent has been shown to reduce the interaction, consistent with the absence of detectable interactions in our colistin-resistant strain. Similar trends among CPZ derivative 1 and CPZ*HCl suggested mild synergy requires adjuvant and colistin to retain measurable antibacterial activity, regardless of the motif used for steric protection of the cation (Figure S9). In line with these observations, we rarely observed significant amplification for antibiotics targeting peptidoglycan cross-linking (vancomycin, ampicillin, penicillin), protein biosynthesis (erythromycin, tetracycline), or genome replication (ciprofloxacin) across E. coli strains (Figures S10–S12). Collectively, these findings underline membrane activity as a mechanistic requirement for polymyxin amplification by CPZ-based adjuvants.

To examine morphological consequences, we turned to cryo-electron microscopy. Polymyxin has been shown to disrupt E. coli cell surface morphology, including protrusions. Under our amplification conditions, E. coli K12 MG1655 displayed a morphology that closely resembled that induced by CPZ derivative 1 alone (Figure S13). Amplification remained associated with cellular elongation rather than overt envelope disruption under employed conditions.

To test if polymyxin amplification enabled by our most potent CPZ derivative 3 is transferable to clinically relevant bacteria, we investigated a proof-of-concept panel of ten E. coli bloodstream isolates collected at the University Hospital Münster. Encouragingly, we observed similar amplification trends among ß-lactam-sensitive isolates; aminopenicillin-/ureidopenicillin-resistant isolates; and extended β-lactamase-producing, third-generation cephalosporin-resistant isolates (Figure D). The CPZ derivative 3 reduced colistin MIC values to lower nanogram-per-milliliter concentrations across strains (<10 ng·mL–1) (Figure D). Comparable amplification was observed for other Gram-negative species, including Acinetobacter baumannii, Klebsiella pneumoniae, and Enterobacter hormaechei (Figure E). The CPZ derivative 3 exhibited significantly increased antimicrobial potency, enabled an increased number of amplifiable Gram-negative strains, and significantly reduced colistin concentrations required to inhibit bacterial growth upon amplification (Figure F). Increasing the lipophilicity of modular triethylene glycol for steric protection of the cation enabled functional gains in polymyxin amplification.

3. Conclusion

In summary, our findings establish steric protection of cations by modular triethylene glycol as a design framework for improving membrane engagement, antibacterial potency, and cytotoxicity in cationic antipsychotic drugs. Contrary to the prevailing view that membrane engagement couples antimicrobial potency to mammalian cytotoxicity, we identified physicochemical windows in which lipophilicity and rigidity jointly enabled control over membrane engagement in antipsychotic drugs. Within these windows, steric protection of cations by modular triethylene glycol enabled us to increase or reduce membrane engagement. Surprisingly, we identified octanol, a plausible cytochrome P450 metabolite, − as a simple-to-apply design motif to increase membrane engagement and decouple antibacterial potency from cytotoxicity. Quaternization with octanol of CPZ maintained water solubility, improved membrane engagement, antimicrobial potency, cytotoxicity, and polymyxin amplification, which was not possible by conventional alkylation.

Interestingly, membrane remodeling associated with polymyxin-resistance in E. coli correlated with increased susceptibility to cationic antipsychotic drugs and surfactants. This observation aligns with previous reports on collateral sensitivity involving resistance-associated remodeling of outer-membrane lipopolysaccharides and impaired proton motive force-dependent drug efflux. , In our work, polymyxin-resistance in E. coli correlated with increased susceptibility to cationic amphiphiles that resemble the canonical molecular architectures of head–tail surfactants. This molecular design space has expanded remarkably over the past decade, from soft and multicationic quaternary ammonium compounds to dendritic amphiphiles, , in which antibacterial activity can be tuned by modulating the hydrophilic–lipophilic balance, charge topology, molecular shape, and self-assembly. ,, Together, these findings create an opportunity to establish molecular design principles that exploit resistance-associated membrane remodeling in antibiotic-resistant bacteria as a physiochemically tractable vulnerability.

More broadly, the ability to modulate membrane engagement through steric protection of cations in polymers and antipsychotic drugs points to a broader design opportunity for extending this strategy across distinct classes of cationic amphiphiles. The ability of our modified CPZ derivatives to amplify last-resort polymyxin antibiotics further connects this molecular design approach to emerging concepts in securing and leveraging the utility of polymyxins against Gram-negative bacteria, including chimeric peptidomimetic antibiotics, synthetic lipopeptide analogues, , truncated peptides derived from polymyxin (SPR741), and turnercyclamycin.

The observation that decoupling of antibacterial potency and cytotoxicity did not extend to erythrocytes highlights the need to define how physicochemical properties of head–tail surfactants govern membrane disruption across distinct membrane architectures (Table S3). Despite this membrane-context dependence, substantial improvements in relative in vitro safety indices were achieved for individual bacterial strains, including Staphylococcus aureus (Table S2) and polymyxin-resistant E. coli B 1702 (Table S6). Next logical steps to understand the transferability of strain-specific improvements include testing the efficiency in in vivo infection models, , resistance emergence, , mechanistic analysis of cell membrane selectivity and cytotoxicity, , as well as antibacterial activity tests across geographically diverse clinical isolate collections. , Steric protection of cations by modular ethylene glycol provides a route toward decoupling antimicrobial potency from cytotoxicity in membrane-active amphiphiles.

Supplementary Material

au6c01288_si_001.pdf (4.4MB, pdf)

Acknowledgments

ChatGPT was used to improve grammar and readability. Work in the lab of L.H.U. was supported by the Ministry of Culture and Science of the German State of North Rhine-Westphalia (NRW return program), the Fonds der Chemischen Industrie (Material Cost Allowance), and the “Netzwerke 2021” program, an initiative of the Ministry of Culture and Science of the State of North Rhine-Westphalia (CANcerTARgeting, NW21–062C). This work was supported by a Humboldt Research Fellowship for Postdoctoral Researchers (to J.L). Work in the lab of D.B. was self-funded through department reserves (Faculty of Life Sciences; Rhein Waal University of Applied Sciences). T.-C. W., T.F., and M.H. gratefully acknowledge funding by the DFG (project numbers 445804074 & 527477869). The authors further acknowledge support by the University Research Focus “Sustainable Materials Design” of the University of Potsdam.

Glossary

Abbreviations

CPZ*HCl

chlorpromazine hydrochloride

CPZ

chlorpromazine

E. coli

Escherichia coli

Gram­(−)

Gram-negative

SD

standard deviation

MIC

minimal inhibitory concentration

IC50

half-maximal inhibitory concentrations

EC50

median effective concentration to permeabilize vesicles

NAP

1-(naphtalen-1-ylmethyl)­piperazine

CIT

citalopram

IMI

imipramine

DDA

dimethyldodecylamine

ESKAPEE

Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumniae, Acinobacter baumannii, Pseudomonas aeruginosa, Enterobacter subspecies, E. coli

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.6c01288.

  • Synthesis and characterization of amphiphiles, procedure for Langmuir–Blodgett, procedure for vesicle calcein-leakage assay, procedure for dynamic light scattering, zeta potential, broth microdilution procedure, procedure for intracellular ethidium bromide retention, procedures for cryo-electron microscopy of vesicles and bacteria, hemolysis, polymyxin amplification procedure, procedure for checkerboard analysis, and in vitro selectivity comparison CPZ derivatives 2 and 3 (Table S1), in vitro selectivity indices across ESKAPEE panel (Table S2), IC50 and HC10 summary (Table S3), MIC summary (Table S4), summary of amplification factors of colistin caused by CPZ derivatives across E. coli strains (Table S5), in vitro selectivity indices across lab E. coli panel (Table S6), statistical test data summary (Tables S7–S9), bacterial strain information (Table S10), antibiograms of clinical isolates (Tables S11–S13), strain-specific OD600-cfu·mL–1 relations for inoculations (Table S14), pipetting scheme for vesicle calcein-leakage assay (Figure S15), 1H and 13C NMR spectra of synthesized compounds (Figure S16–S45), and high-resolution mass spectrometry data of synthesized compounds (Figures S46–S60) (PDF)

∇.

M.S., J.L., and S.E. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Published as part of JACS Au special issue “Molecular Editing in Natural Products and Drug Molecules”.

References

  1. Watson H.. Biological membranes. Essays Biochem. 2015;59:43–69. doi: 10.1042/bse0590043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Marie E., Sagan S., Cribier S., Tribet C.. Amphiphilic Macromolecules on Cell Membranes: From Protective Layers to Controlled Permeabilization. J. Membr. Biol. 2014;247:861–881. doi: 10.1007/s00232-014-9679-3. [DOI] [PubMed] [Google Scholar]
  3. Wu X., Hu J.-J., Yoon J.. Cell Membrane as A Promising Therapeutic Target: From Materials Design to Biomedical Applications. Angew. Chem. Int. Ed. 2024;136:e202400249. doi: 10.1002/anie.202400249. [DOI] [PubMed] [Google Scholar]
  4. Seewald M., Nielinger L., Alker K., Behnke J.-S., Wycisk V., Urner L. H.. Detergent Chemistry Modulates the Transgression of Planetary Boundaries including Antimicrobial Resistance and Drug Discovery. Angew. Chem. Int. Ed. 2024;63:e202403833. doi: 10.1002/anie.202403833. [DOI] [PubMed] [Google Scholar]
  5. Epand R. M., Walker C., Epand R. F., Magarvey N. A.. Molecular mechanisms of membrane targeting antibiotics. Biochim. Biophys. Acta, Biomembr. 2016;1858:980–987. doi: 10.1016/j.bbamem.2015.10.018. [DOI] [PubMed] [Google Scholar]
  6. Melo M. N., Ferre R., Castanho M. A. R. B.. Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations. Nat. Rev. Microbiol. 2009;7:245–250. doi: 10.1038/nrmicro2095. [DOI] [PubMed] [Google Scholar]
  7. Cardona S. T., Rahman A. S. M. Z., Nachcoff J. N.. Innovative perspectives on the discovery of small molecule antibiotics. npj Antimicrob. Resist. 2025;3:19. doi: 10.1038/s44259-025-00089-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Naghavi M., Vollset S. E., Ikuta K. S.. et al. Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050. Lancet. 2024;404:1199–1226. doi: 10.1016/S0140-6736(24)01867-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Sati H., Carrara E., Savoldi A., Hansen P., Garlasco J., Campagnaro E., Boccia S., Castillo-Polo J. A., Magrini E., Garcia-Vello P.. et al. The WHO Bacterial Priority Pathogens List 2024: a prioritisation study to guide research, development, and public health strategies against antimicrobial resistance. Lancet Infect. Dis. 2025;25:1033–1043. doi: 10.1016/S1473-3099(25)00118-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Miethke M., Pieroni M., Weber T., Brönstrup M., Hammann P., Halby L., Arimondo P. B., Glaser P., Aigle B., Bode H. B.. et al. Towards the sustainable discovery and development of new antibiotics. Nat. Rev. Chem. 2021;5:726–749. doi: 10.1038/s41570-021-00313-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Boyd S. E., Livermore D. M., Hooper D. C., Hope W. W.. Metallo-β-Lactamases: Structure, Function, Epidemiology, Treatment Options, and the Development Pipeline. Antimicrob. Agents Chemother. 2020;64:10–128. doi: 10.1128/AAC.00397-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heil E. L., McCreary E. K.. REVISITing treatment of metallo-β-lactamases. Lancet Infect. Dis. 2025;25:144–146. doi: 10.1016/S1473-3099(24)00561-9. [DOI] [PubMed] [Google Scholar]
  13. Yang S., Wang H., Zhao D., Zhang S., Hu C.. Polymyxins: recent advances and challenges. Front. Pharmacol. 2024;15:1424765. doi: 10.3389/fphar.2024.1424765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mohapatra S. S., Dwibedy S. K., Padhy I.. Polymyxins, the last-resort antibiotics: Mode of action, resistance emergence, and potential solutions. J. Biosci. 2021;46:85. doi: 10.1007/s12038-021-00209-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Liang L., Li Y., Wang L., Wang W., Zhang Y., Zhao H., Wang Y., Lyu L., Li J., Zhou D.. et al. Fitness costs of mobilised colistin resistance gene 3 (mcr-3): systematic review, epidemiological study, and functional analysis. eBioMedicine. 2025;120:105923. doi: 10.1016/j.ebiom.2025.105923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mehta D., Saini V., Aggarwal B., Khan A., Bajaj A.. Unlocking the bacterial membrane as a therapeutic target for next-generation antimicrobial amphiphiles. Mol. Asp. Med. 2021;81:100999. doi: 10.1016/j.mam.2021.100999. [DOI] [PubMed] [Google Scholar]
  17. Liu Y., Tong Z., Shi J., Li R., Upton M., Wang Z.. Drug repurposing for next-generation combination therapies against multidrug-resistant bacteria. Theranostics. 2021;11:4910–4928. doi: 10.7150/thno.56205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Boyd-Kimball D., Gonczy K., Lewis B., Mason T., Siliko N., Wolfe J.. Classics in Chemical Neuroscience: Chlorpromazine. ACS Chem. Neurosci. 2019;10:79–88. doi: 10.1021/acschemneuro.8b00258. [DOI] [PubMed] [Google Scholar]
  19. Ban T. A.. Fifty years chlorpromazine: a historical perspective. Neuropsychiatr. Dis. Treat. 2007;3:495–500. doi: 10.2147/NDT.A922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ohlow M. J., Moosmann B.. Phenothiazine: the seven lives of pharmacology’s first lead structure. Drug Discovery Today. 2011;16:119–131. doi: 10.1016/j.drudis.2011.01.001. [DOI] [PubMed] [Google Scholar]
  21. Grimsey E. M., Gromsey E. M., Fais C., Marshall R. L., Ricci V., Ciusa M. L., Stone J. W., Ivens A., Malloci G., Ruggerone P., Vargiu A. V.. Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump. mBio. 2020;11:e00465-20. doi: 10.1128/mbio.00465-00420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Faraji N., Abavisani M., Ebadpour N., Karav S., Kesharwani P., Sahebkar A.. Double-Duty Drugs: Repositioning Antipsychotics to Combat Bacterial Infections. CNS Neurosci. Ther. 2026;32:e70724. doi: 10.1002/cns.70724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grimsey E. M., Piddock L. J. V.. Do phenothiazines possess antimicrobial and efflux inhibitory properties? FEMS Microbiol. Rev. 2019;43:577–590. doi: 10.1093/femsre/fuz017. [DOI] [PubMed] [Google Scholar]
  24. van Meer G., Voelker D. R., Feigenson G. W.. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 2008;9:112–124. doi: 10.1038/nrm2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mingeot-Leclercq M.-P., Décout J.-L.. Bacterial lipid membranes as promising targets to fight antimicrobial resistance, molecular foundations and illustration through the renewal of aminoglycoside antibiotics and emergence of amphiphilic aminoglycosides. Med. Chem. Commun. 2016;7:586. doi: 10.1039/C5MD00503E. [DOI] [Google Scholar]
  26. Liang Y., Zhang Y., Huang Y., Xu C., Chen J., Zhang X., Huang B., Gan Z., Dong X., Huang S.. et al. Helicity-directed recognition of bacterial phospholipid via radially amphiphilic antimicrobial peptides. Sci. Adv. 2024;10:eadn9435. doi: 10.1126/sciadv.adn9435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Glukhov E., Stark M., Burrows L. L., Deber C. M.. Basis for Selectivity of Cationic Antimicrobial Peptides for Bacterial Versus Mammalian Membranes. J. Biol. Chem. 2005;280:33960–33967. doi: 10.1074/jbc.M507042200. [DOI] [PubMed] [Google Scholar]
  28. Herzog I. M., Fridman M.. Design and synthesis of membrane-targeting antibiotics: from peptides- to aminosugar-based antimicrobial cationic amphiphiles. MedChemComm. 2014;5:1014–1026. doi: 10.1039/C4MD00012A. [DOI] [Google Scholar]
  29. Moretti A., Weeks R. M., Chikindas M., Uhrich K. E.. Cationic Amphiphiles with Specificity against Gram-Positive and Gram-Negative Bacteria: Chemical Composition and Architecture Combat Bacterial Membranes. Langmuir. 2019;35:5557–5567. doi: 10.1021/acs.langmuir.9b00110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zhou C., Wang Y.. Structure-activity relationship of cationic surfactants as antimicrobial agents. Curr. Opin. Colloid Interface Sci. 2020;45:28–43. doi: 10.1016/j.cocis.2019.11.009. [DOI] [Google Scholar]
  31. Yuan J., Wang J., Li X., Zhang Y., Xian J., Wang C., Zhang J., Wu C.. Amphiphilic small molecule antimicrobials: From cationic antimicrobial peptides (CAMPs) to mechanism-related, structurally-diverse antimicrobials. Eur. J. Med. Chem. 2023;262:115896. doi: 10.1016/j.ejmech.2023.115896. [DOI] [PubMed] [Google Scholar]
  32. Ntallis C., Martin N. I., Edwards A. M., Weingarth M.. Bacterial cell envelope-targeting antibiotics. Nat. Rev. Microbiol. 2026;24:183–196. doi: 10.1038/s41579-025-01247-x. [DOI] [PubMed] [Google Scholar]
  33. Deng Z., Zhang R., Gong J., Zhang Z., Zhang L., Qiu Z., Alam P., Zhang J., Liu Y., Zhao Z.. et al. Unveiling the Role of Alkyl Chain in Boosting Antibacterial Selectivity and Cell Biocompatibility. JACS Au. 2025;5:675–683. doi: 10.1021/jacsau.4c00915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van Groesen E., Mons E., Kotsogianni I., Arts M., Tehrani K. H. M. E., Wade N., Lysenko V., Stel F. M., Zwerus J. T., de Benedetti S.. et al. Semisynthetic guanidino lipoglycopeptides with potent in vitro and in vivo antibacterial activity. Sci. Transl. Med. 2024;16:eabo4736. doi: 10.1126/scitranslmed.abo4736. [DOI] [PubMed] [Google Scholar]
  35. Ferguson A. L., Denebedetti P. G., Panagiotopoulos A. Z.. Solubility and Molecular Conformations of n-Alkane Chains in Water. J. Phys. Chem. B. 2009;113:6405–6414. doi: 10.1021/jp811229q. [DOI] [PubMed] [Google Scholar]
  36. Lipinski C. A., Lombardo F., Dominy B. W., Feeney P. J.. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 1997;23:3–25. doi: 10.1016/S0169-409X(96)00423-1. [DOI] [PubMed] [Google Scholar]
  37. Yun M., Lee J., Shin S. S., Park S.-J., Choi J., Seo J., Lee J.-H.. Ethylene glycol derivative-pendanted quaternary ammonium antibacterial polymers with low cytotoxicity. Eur. Polym. J. 2024;206:112772. doi: 10.1016/j.eurpolymj.2024.112772. [DOI] [Google Scholar]
  38. Saunders, W. B. Methanol, Ethylene Glycol, and Other Toxic Alcohols. In Haddad and Winchester’s Clinical Management of Poisoning and Drug Overdose, 4th ed.; Shannon, M. W. ; Borron, S. W. ; Burns, M. J. , Eds.; Elsevier, 2007; Chapter 32; pp 605–633 9780721606934. [Google Scholar]
  39. Yang X., Ye G., Tran K., Liu Y., Cao J., Dong J., Portale G., Liu G., Liu J., Zhang P.. et al. Impact of Oligo­(Ethylene Glycol) Side Chains on the Thermoelectric Properties of Naphthalenediimide–Dialkoxybithiazole Polymers. ACS Mater. Lett. 2024;6:1207–1215. doi: 10.1021/acsmaterialslett.4c00068. [DOI] [Google Scholar]
  40. Clausell A., Busquets M. A., Pujol M., Alsina A., Cajal Y.. Polymyxin B–lipid interactions in Langmuir–Blodgett monolayers of Escherichia coli lipids: A thermodynamic and atomic force microscopy study. Biopolymers. 2004;75:480–490. doi: 10.1002/bip.20165. [DOI] [PubMed] [Google Scholar]
  41. Seewald M., Behnke J.-S., Wu H., Wagner M.-C., Cordes T., Raisch T., Urner L. H., Wycisk V.. Detergent Conjugation Decouples Chlorpromazine Solubility from Antibacterial Activity against Bacteria Relevant for Gut Microbiota. Bioconjugate Chem. 2026;37:881–887. doi: 10.1021/acs.bioconjchem.6c00016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wiegand I., Hilpert K., Hancock R. E. W.. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 2008;3:163–175. doi: 10.1038/nprot.2007.521. [DOI] [PubMed] [Google Scholar]
  43. Singh A. K., Seewald M., Schade B., Zoister C., Haag R., Urner L. H.. Chemical linkers switch triglycerol detergents from bacterial protein purification to mild antibiotic amplification. Commun. Chem. 2025;8:70. doi: 10.1038/s42004-025-01477-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wycisk V., Behnke J.-S., Nielinger L., Seewald M., Weisner J., Binsch M., Wagner M.-C., Raisch T., Urner L. H.. Synthesis of Asymmetric Ionic Hybrid Detergents enables Micelles with Scalable Properties including Cell Compatibility. Chem. Eur. J. 2024;30:e202401833. doi: 10.1002/chem.202401833. [DOI] [PubMed] [Google Scholar]
  45. Wesseling C. M. J., Martin N. I.. Synergy by Perturbing the Gram-Negative Outer Membrane: Opening the Door for Gram-Positive Specific Antibiotics. ACS Infect. Dis. 2022;8:1731–1757. doi: 10.1021/acsinfecdis.2c00193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Duffey M., Jumde R. P., da Costa R. M. A., Ropponen H.-K., Blasco B., Piddock L. J. V.. Extending the Potency and Lifespan of Antibiotics: Inhibitors of Gram-Negative Bacterial Efflux Pumps. ACS Infect. Dis. 2024;10:1458–1482. doi: 10.1021/acsinfecdis.4c00091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Li F., Weir M. D., Xu H. H. K.. Effects of Quaternary Ammonium Chain Length on Antibacterial Bonding Agents. J. Dent. Res. 2013;92:932–938. doi: 10.1177/0022034513502053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Hanheiser N., Jiang Y., Zoister C., Dimde M., Achazi K., Nie C., Li Y., Haag R., Singh A. K.. Modular Synthesis of Dendritic Oligo-Glycerol Cationic Surfactantsfor Enhanced Antibacterial Efficacy. Angew. Chem. Int. Ed. 2025;64:e202425069. doi: 10.1002/anie.202425069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Lehr R. E., Kaul P. N.. Chlorpromazine Metabolism IV: Quaternization as a key to determination of picomoles of chlorpromazine and other tertiary amine drugs. J. Pharm. Sci. 1975;64:950–953. doi: 10.1002/jps.2600640611. [DOI] [PubMed] [Google Scholar]
  50. McDonnell M. T., Xu H., Keffer D. J.. Ab Initio Molecular Dynamics Simulations of an Excess Proton in a Triethylene Glycol–Water Solution: Solvation Structure, Mechanism, and Kinetics. J. Phys. Chem. B. 2016;120:5223–5242. doi: 10.1021/acs.jpcb.6b02445. [DOI] [PubMed] [Google Scholar]
  51. Jindal A., Vasudevan S.. Molecular Conformation and Hydrogen Bond Formation in Liquid Ethylene Glycol. J. Phys. Chem. B. 2020;124:9136–9143. doi: 10.1021/acs.jpcb.0c06324. [DOI] [PubMed] [Google Scholar]
  52. Tenforde T. S., Yee J. P., Mel H. C.. Electrophoretic detection of reversible chlorpromazine · HCl binding at the human erythrocyte surface. Biochim. Biophys. Acta, Biomembr. 1978;511:152–162. doi: 10.1016/0005-2736(78)90310-3. [DOI] [PubMed] [Google Scholar]
  53. Wimley W. C., White S. H.. Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struct. Mol. Biol. 1996;3:842–848. doi: 10.1038/nsb1096-842. [DOI] [PubMed] [Google Scholar]
  54. White S. H., Wimley W. C.. Hydrophobic interactions of peptides with membrane interfaces. Biochim. Biophys. Acta, Biomembr. 1998;1376:339–352. doi: 10.1016/S0304-4157(98)00021-5. [DOI] [PubMed] [Google Scholar]
  55. Nasr G., Greige-Gerges H., Elaissari A., Khreich N.. Liposomal membrane permeability assessment by fluorescence techniques: Main permeabilizing agents, applications and challenges. Int. J. Pharm. 2020;580:119198. doi: 10.1016/j.ijpharm.2020.119198. [DOI] [PubMed] [Google Scholar]
  56. Tormena N., Pilizota T., Voïtchovsky K.. A Minimalist Model Lipid System Mimicking the Biophysical Properties of Escherichia coli’s Inner Membrane. Langmuir. 2025;41:12301–12310. doi: 10.1021/acs.langmuir.5c01138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Landersdorfer C. B., Nation R. L.. Limitations of Antibiotic MIC-Based PK-PD Metrics: Looking Back to Move Forward. Front. Pharmacol. 2021;12:770518. doi: 10.3389/fphar.2021.770518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Ramata S. A., Boroduskis M., Vorobjeva V., Ancans J.. Cell and tissue culture-based in vitro test systems for evaluation of natural skin care product ingredients. Environ. Exp. Biol. 2013;11:159–177. [Google Scholar]
  59. Seguin R. P., Herron J. M., Lopez V. A., Dempsey J. L., Xu L.. Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450. Chem. Res. Toxicol. 2019;32:4266–4278. doi: 10.1021/acs.chemrestox.9b00293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Nunes B., Cagide F., Borges F., Simões M.. Antimicrobial activity and cytotoxicity of novel quaternary ammonium and phosphonium salts. J. Mol. Liq. 2024;401:124616. doi: 10.1016/j.molliq.2024.124616. [DOI] [Google Scholar]
  61. Chueca B., Berdejo D., Gomes-Neto N. J., Pagán R., García-Gonzalo D.. Emergence of Hyper-Resistant Escherichia coli MG1655 Derivative Strains after Applying Sub-Inhibitory Doses of Individual Constituents of Essential Oils. Front. Microbiol. 2016;7:273. doi: 10.3389/fmicb.2016.00273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. French S., Guo A. B. Y., Ellis M. J., Deisinger J. P., Johnson J. W., Rachwalski K., Piquette Z. A., Lluka T., Zary M., Gamage S.. et al. A platform for predicting mechanism of action based on bacterial transcriptional responses identifies an unusual DNA gyrase inhibitor. Cell Rep. 2024;43:114053. doi: 10.1016/j.celrep.2024.114053. [DOI] [PubMed] [Google Scholar]
  63. Piddock L. J. V., Jin Y.-F., Ricci V., Asuquo A. E.. Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli . J. Antimicrob. Chemother. 1999;43:61–70. doi: 10.1093/jac/43.1.61. [DOI] [PubMed] [Google Scholar]
  64. Nishino K., Yamasaki S., Nakashima R., Zwama M., Hayashi-Nishino M.. Function and Inhibitory Mechanisms of Multidrug Efflux Pumps. Front. Microbiol. 2021;12:10.3389/fmicb.2021.737288. doi: 10.3389/fmicb.2021.737288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Lomovskaya O., Zgurskaya H. I., Totrov M., Watkins W. J.. Waltzing transporters and ’the dance macabre’ between humans and bacteria. Nat. Rev. Drug Discovery. 2007;6:56–65. doi: 10.1038/nrd2200. [DOI] [PubMed] [Google Scholar]
  66. Henderson P. J. F., Maher C., Elbourne L. D. H., Eijkelkamp B. A., Paulsen I. T., Hassan K. A.. Physiological Functions of Bacterial “Multidrug” Efflux Pumps. Chem. Rev. 2021;121:5417–5478. doi: 10.1021/acs.chemrev.0c01226. [DOI] [PubMed] [Google Scholar]
  67. Machado D., Fernandes L., Costa S. S., Cannalire R., Manfroni G., Tabarrini O., Couto I., Sabatini S., Viveiros M.. Mode of action of the 2-phenylquinoline efflux inhibitor PQQ4R against Escherichia coli . PeerJ. 2017;5:e3168. doi: 10.7717/peerj.3168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Elliott A. G., Huang J. X., Neve S., Zuegg J., Edwards I. A., Cain A. K., Boinett C. J., Barquist L., Lundberg C. V., Steen J.. et al. An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria. Nat. Commun. 2020;11:3184. doi: 10.1038/s41467-020-16950-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Mohiuddin S. G., Nguyen T. V., Orman M. A.. Pleiotropic actions of phenothiazine drugs are detrimental to Gram-negative bacterial persister cells. Commun. Biol. 2022;5:217. doi: 10.1038/s42003-022-03172-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Cabrera J. E., Jin D. J.. The distribution of RNA polymerase in Escherichia coli is dynamic and sensitive to environmental cues. Mol. Microbiol. 2003;50:1493–1505. doi: 10.1046/j.1365-2958.2003.03805.x. [DOI] [PubMed] [Google Scholar]
  71. den Blaauwen T., Hamoen L. W., Levin P. A.. The divisome at 25: the road ahead. Curr. Opin. Microbiol. 2017;36:85–94. doi: 10.1016/j.mib.2017.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Navarro P. P., Vettinger A., Ananda V. Y., Llopis P. M., Allolio C., Bernhardt T. G., Chao L. H.. Cell wall synthesis and remodelling dynamics determine division site architecture and cell shape in Escherichia coli . Nat. Microbiol. 2022;7:1621–1634. doi: 10.1038/s41564-022-01210-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Amaral L., Lorian V.. Effects of chlorpromazine on the cell envelope proteins of Echerichia coli. Antimicrob. Agents Chemother. 1991;35:1923–1924. doi: 10.1128/AAC.35.9.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Yang B., Tong Z., Shi H., Wang Z., Liu Y.. Bacterial proton motive force as an unprecedented target to control antimicrobial resistance. Med. Res. Rev. 2023;43:1068–1090. doi: 10.1002/med.21946. [DOI] [PubMed] [Google Scholar]
  75. Caldara M., Marmiroli N.. Antimicrobial Properties of Antidepressants and AntipsychoticsPossibilities and Implications. Pharmaceuticals. 2021;14:915. doi: 10.3390/ph14090915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Urner L. H., Schade B., Schulze M., Folmert K., Haag R., Pagel K.. Switchable Solubility of Azobenzene-Based Bolaamphiphiles. ChemPhysChem. 2019;20:1690–1697. doi: 10.1002/cphc.201900334. [DOI] [PubMed] [Google Scholar]
  77. Daina A., Michielin O., Zoete V.. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017;7:42717. doi: 10.1038/srep42717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Hardwick J. P.. Cytochrome P450 omega hydroxylase (CYP4) function in fatty acid metabolism and metabolic diseases. Biochem. Pharmacol. 2008;75:2263–2275. doi: 10.1016/j.bcp.2008.03.004. [DOI] [PubMed] [Google Scholar]
  79. Edson K. Z., Rettie A. E.. CYP4 Enzymes As Potential Drug Targets: Focus on Enzyme Multiplicity, Inducers and Inhibitors, and Therapeutic Modulation of 20- Hydroxyeicosatetraenoic Acid (20-HETE) Synthase and Fatty Acid ω- Hydroxylase Activities. Curr. Top. Med. Chem. 2013;13:1429–1440. doi: 10.2174/15680266113139990110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Nguyen R., Seguin R. P., Ross D. H., Chen P., Richardson S., Liem J., Lin Y. S., Xu L.. Development and Application of a Multidimensional Database for the Detection of Quaternary Ammonium Compounds and Their Phase I Hepatic Metabolites in Humans. Environ. Sci. Technol. 2024;58:6236–6249. doi: 10.1021/acs.est.3c10845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Chigozie V. U., Esimone C. O.. Bacterial secondary metabolites as resistance-modifying adjuvants: microbial origins, molecular mechanisms, and translational relevance. Front. Microbiol. 2026;17:1779022. doi: 10.3389/fmicb.2026.1779022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Arora H. S., Lev K., Robida A., Velmurugan R., Chandrasekaran S.. A Metabolism-Informed Neural Network Identifies Pathways Influencing the Potency and Toxicity of Antimicrobial Combinations. npj Drug Discovery. 2026;3:11. doi: 10.1038/s44386-026-00042-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Rodrigues de Almeida N., Han Y., Perez J., Kirkpatrick S., Wang Y., Sheridan M. C.. Design, Synthesis, and Nanostructure-Dependent Antibacterial Activity of Cationic Peptide Amphiphiles. ACS Appl. Mater. Interfaces. 2019;11:2790–2801. doi: 10.1021/acsami.8b17808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Greco I., Molchanova N., Holmedal E., Jenssen H., Hummel B. D., Watts J. L., Håkansson J., Hansen P. R., Svenson J.. Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides. Sci. Rep. 2020;10:13206. doi: 10.1038/s41598-020-69995-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Delcour A. H.. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta, Proteins Proteomics. 2009;1794:808–816. doi: 10.1016/j.bbapap.2008.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Herschede S. R., Gneid H., Dent T., Jaeger E. B., Lawson L. B., Busschaert N.. Bactericidal urea crown ethers target phosphatidylethanolamine membrane lipids. Org. Biomol. Chem. 2021;19:3838–3843. doi: 10.1039/D1OB00263E. [DOI] [PubMed] [Google Scholar]
  87. Maslowska-Jarzyna K., Ojah E. O., Korczak M., Chmielewski M. J., Busschaert N.. Combining Anion Transport and Phospholipid Binding for Improved Antibacterial Activity of Diamidocarbazoles. ACS Omega. 2025;10:53557–53563. doi: 10.1021/acsomega.5c09348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Luther A., Urfer M., M Z., Müller M., Wang S.-Y., Mondal M., Vitale A., Hartmann J.-B., Sharpe T., Lo Monte F.. et al. Chimeric peptidomimetic antibiotics against Gram-negative bacteria. Nature. 2019;576:452–458. doi: 10.1038/s41586-019-1665-6. [DOI] [PubMed] [Google Scholar]
  89. Uppu D. S. S. M., Konai M. M., Baul U., Singh P., Siersma T. K., Samaddar S., Vemparala S., Hamoen L. W., Narayana C., Haldar J.. Isosteric substitution in cationic-amphiphilic polymers reveals an important role for hydrogen bonding in bacterial membrane interactions. Chem. Sci. 2016;7:4613–4623. doi: 10.1039/C6SC00615A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. Niu Y., Wang M., Cao Y., Nimmagadda A., Hu J., Wu Y., Cai J., Ye X.-S.. Rational Design of Dimeric Lysine N-Alkylamides as Potent and Broad-Spectrum Antibacterial Agents. J. Med. Chem. 2018;61:2865–2874. doi: 10.1021/acs.jmedchem.7b01704. [DOI] [PubMed] [Google Scholar]
  91. Dhumal D., Maron B., Malach E., Lyu Z., Ding L., Marson D., Laurini E., Tintaru A., Ralahy B., Giogio S.. et al. Dynamic self-assembling supramolecular dendrimer nanosystems as potent antibacterial candidates against drug-resistant bacteria and biofilms. Nanoscale. 2022;14:9286–9296. doi: 10.1039/D2NR02305A. [DOI] [PubMed] [Google Scholar]
  92. Schumann A., Cohn A. R., Gaballa A., Wiedmann M.. Escherichia coli B-Strains Are Intrinsically Resistant to Colistin and Not Suitable for Characterization and Identification of mcr Genes. Mircob. Genet. 2023;11:e00894–00823. doi: 10.1128/spectrum.00894-23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. Feng S., Liang W., Li J., Cheng Y., Zhou D., Liang L., Lin D., Li Y., Zhao H., Du H.. et al. MCR-1-dependent lipid remodelling compromises the viability of Gram-negative bacteria. Emerg. Microbes. Infect. 2022;11:1236–1249. doi: 10.1080/22221751.2022.2065934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  94. Liang L., Zhong L.-L., Wang L., Zhou D., Li Y., Li J., Chen Y., Liang W., Wei W., Zhang C.. et al. A new variant of the colistin resistance gene MCR-1 with co-resistance to β-lactam antibiotics reveals a potential novel antimicrobial peptide. PLoS Biol. 2023;21:e3002433. doi: 10.1371/journal.pbio.3002433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Han M.-J., Lee S. Y., Hong S. H.. Comparative Analysis of Envelope Proteomes in Escherichia coli B and K-12 Strains. J. Microbiol. Biotechnol. 2012;22:470–478. doi: 10.4014/jmb.1110.10080. [DOI] [PubMed] [Google Scholar]
  96. Zhu S., Alexander M. K., Paiva T. O., Rachwalski K., Miu A., Xu Y., Verma V., Reichelt M., Dufrêne Y. F., Brown E. D., Cox G.. The inactivation of tolC sensitizes Escherichia coli to perturbations in lipopolysaccharide transport. iScience. 2024;27:109592. doi: 10.1016/j.isci.2024.109592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  97. Fatsis-Kavalopoulos N., Sánchez-Hevia D. L., Andersson D. I.. Beyond the FIC index: the extended information from fractional inhibitory concentrations (FICs) J. Antimicrob. Chemother. 2024;79:2394–2396. doi: 10.1093/jac/dkae233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  98. Fatsis-Kavalopoulos N., Roelofs L., Andersson D. I.. Potential risks of treating bacterial infections with a combination of β-lactam and aminoglycoside antibiotics: A systematic quantification of antibiotic interactions in E. coli blood stream infection isolates. eBioMedicine. 2022;78:103979. doi: 10.1016/j.ebiom.2022.103979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  99. Borrelli C., Douglas E. J. A., Riley S. M. A., Lemonidi A. E., Larrouy-Maumus G., Lu W.-J., Bonev B. B., Edwards A. M., Hoogenboom B. W.. Polymyxin B lethality requires energy-dependent outer membrane disruption. Nat. Microbiol. 2025;10:2919–2933. doi: 10.1038/s41564-025-02133-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  100. Lázár V., Singh G. P., Spohn R., Nagy I., Horváth B., Hrtyan M., Busa-Fekete R., Bogos B., Méhi O., Csörgő B.. et al. Bacterial evolution of antibiotic hypersensitivity. Mol. Syst. Biol. 2013;9:MSB201357. doi: 10.1038/msb.2013.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  101. Lázár V., Martins A., Spohn R., Daruka L., Grézal G., Fekete G., Számel M., Jangir P. K., Kintsesm B., Csörgő B.. et al. Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides. Nat. Microbiol. 2018;3:718–731. doi: 10.1038/s41564-018-0164-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  102. Crnčević D., Krce L., Brkljača Z., Cvitković M., Brčić S. B., Čož-Rakovac R., Odžak R., Šprung M.. A dual antibacterial action of soft quaternary ammonium compounds: bacteriostatic effects, membrane integrity, and reduced in vitro and in vivo toxicity. RSC Adv. 2025;15:1490–1506. doi: 10.1039/D4RA07975B. [DOI] [PMC free article] [PubMed] [Google Scholar]
  103. Seferyan M. A., Saverina E. A., Frolov N. A., Detusheva E. V., Kamanina O. A., Arlyapov V. A., Ostashevskaya I. I., Ananikov V. P., Vereshchagin A. N.. Multicationic Quaternary Ammonium Compounds: A Framework for Combating Bacterial Resistance. ACS Infect. Dis. 2023;9:1206–1220. doi: 10.1021/acsinfecdis.2c00546. [DOI] [PubMed] [Google Scholar]
  104. Zhang N., Dhumal D., Kuo S. H., Lew S. Q., Patil P. D., Taher R., Vaidya S., Galanakou C., Elkihel A., Oh M. W.. et al. Targeting the phosphatidylglycerol lipid: An amphiphilic dendrimer as a promising antibacterial candidate. Sci. Adv. 2024;10:eadn8117. doi: 10.1126/sciadv.adn8117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  105. Roberts K. D., Zhu Y., MAK A., Han M.-L., Wang J., Yu H. H., Horne A. S., Pinson J.-A., Rudd D., Voelcker N. H.. et al. A synthetic lipopeptide targeting top-priority multidrug-resistant Gram-negative pathogens. Nat. Commun. 2022;13:1625. doi: 10.1038/s41467-022-29234-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  106. Ramirez D., Ramirez D. M., Arora R., Arthur G., Schweizer F.. Noncytotoxic polymyxin derivatives enhance antibiotic action against multidrug-resistant Gram-negative bacteria. Antimicrob. Agents Chemother. 2025;69:e00712–00725. doi: 10.1128/aac.00712-25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  107. Corbett D., Wisea A., Langleya T., Skinnera K., Trimbya E., Birchalla S., Doralia A., Sandiforda S., Williamsa J., Warna P.. et al. Potentiation of Antibiotic Activity by a Novel Cationic Peptide: Potency and Spectrum of Activity of SPR741. Antimicrob. Agents Chemother. 2017;61:10–128. doi: 10.1128/aac.00200-00217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  108. Lim A. L., Miller B. W., Fisher M. A., Haygood M. G., Barrows L. R., Schmidt E. W.. Differential membrane lipid disruption by lipopeptide antibiotics, colistin and turnercyclamycins. Nat. Commun. 2026;17:1880. doi: 10.1038/s41467-026-68681-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  109. Eckburg P. B., Lister T., Walpole S., Keutzer T., Utley L., Tomayko J., Kopp E., Farinola N., Coleman S.. Safety, Tolerability, Pharmacokinetics, and Drug Interaction Potential of SPR741, an Intravenous Potentiator, after Single and Multiple Ascending Doses and When Combined with β-Lactam Antibiotics in Healthy Subjects. Antimicrob. Agents Chemother. 2019;63:10–128. doi: 10.1128/AAC.00892-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  110. Yu B., Choudhury M. R., Yang X., Benoit S. L., Womack E., Lyles K. V. M., Acharya A., Kumar A., Yang C., Pavlova A.. et al. Restoring and Enhancing the Potency of Existing Antibiotics against Drug-Resistant Gram-Negative Bacteria through the Development of Potent Small-Molecule Adjuvants. ACS Infect. Dis. 2022;8:1491–1508. doi: 10.1021/acsinfecdis.2c00121. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

au6c01288_si_001.pdf (4.4MB, pdf)

Articles from JACS Au are provided here courtesy of American Chemical Society

RESOURCES