YZ462 exhibits potent antibacterial activity against methicillin-resistant Staphylococcus aureus through bacterial membrane disruption

Abstract

Background

The global rise of antibiotic resistance presents a critical public health challenge, with Staphylococcus aureus being a leading cause of hospital- and community-acquired infections. Methicillin-resistant S. aureus (MRSA) is particularly concerning due to its high prevalence, multidrug resistance, and association with severe morbidity and mortality. Developing novel small-molecule antimicrobial agents with reduced resistance susceptibility is essential to combat these infections.

Results

From our in-house compound library, we identified YZ462, a heteroaromatic-aryl scaffold compound, demonstrating potent antibacterial activity and low toxicity. YZ462 showed strong anti-MRSA effects through bacterial membrane disruption, increased permeability, and induction of endogenous ROS production. In vitro, it effectively killed both log-phase and stationary-phase MRSA while inhibiting biofilm formation and disrupting established biofilms. In vivo studies revealed significant reduction of bacterial loads and improved survival rates in infected animal models. Importantly, the major S. aureus membrane component cardiolipin (CL) directly interacted with YZ462 and attenuated its antibacterial activity, suggesting CL as a potential molecular target.

Conclusions

Our study establishes YZ462 as a promising small-molecule candidate for MRSA treatment, featuring a unique mechanism of membrane disruption and ROS induction. These results confirm its potent antimicrobial activity and demonstrate therapeutic potential against MRSA infections.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04475-6.

Introduction

Antibiotics have made an indispensable contributions to combating infectious diseases. However, the irrational use of antimicrobial agents has accelerated the spread of drug resistance, posing a serious threat to public health. Staphylococcus aureus, an opportunistic pathogen, is one of the most common bacteria colonizing humans, primarily residing in the mouth, skin, nasopharynx, external ear canal, conjunctiva and urogenital tract. Invasive S. aureus infections can occur when host immunity is compromised, anatomical positioning changes, or microbial flora becomes imbalanced [1]. Methicillin-resistant S. aureus (MRSA) poses a serious global threat due to its association with high morbidity and mortality, representing a major public health challenge-particularly in resource-limited regions such as Africa [2, 3]. The resistance mechanisms of S. aureus to penicillin include: (1) β-lactamase-mediated hydrolysis of penicillin’s β-lactam ring; (2) mutations in penicillin-binding proteins that abolish or reduce binding activity [4]; and (3) cell wall thickening that reduces permeability [5]. Vancomycin, a glycopeptide antibiotic originally isolated from Actinomyces Nocardia in East China, was approved by the U.S. FDA in 1958 for clinical use [6]. Initially limited due to toxicity (particularly nephrotoxicity), its application expanded in the 1980 s as MRSA prevalence and resistance escalated [7]. Vancomycin became the last-line defense against MRSA infections [8]. However, the global rise of MRSA and overuse of vancomycin have led to the emergence of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA), alarming clinicians and microbiologists [9, 10]. Additionally, S. aureus biofilms-colonizing medical devices and shielding bacteria from antibiotics-further complicate treatment [11]. Thus, developing novel antimicrobial agents or therapies against MRSA, VRSA, multidrug-resistant strains, and biofilms is an urgent priority for strategic reserves.

Currently reported antibacterial agents and therapies primarily focus on: (1) repurposing approved drugs; (2) combining antibiotics with adjuvants; (3) utilizing plant extracts and their modifications; (4) developing antimicrobial nanomaterials; and (5) synthesizing novel compounds and antimicrobial peptides [12–14]. Antimicrobial agents that selectively target cell walls or membranes are less prone to rapid resistance development [15, 16]. The cell wall is a relatively thick, tough, and elastic structure located outside the bacterial inner membrane. It functions to maintain intracellular osmotic pressure and protect bacterial cells, and its synthesis is a highly conserved process. Consequently, antimicrobial drugs that inhibit cell wall synthesis have remained one of the research hotspots in the field of antibacterial development [17]. In particular, cationic amphiphilic small-molecules open a door to the new mode of action of bactericidal agents by depolarizing and disturbing the bacteria membrane [18]. Fatty acid-lysine conjugates, a new class of membrane-targeting agents, offer a promising alternative to conventional antibiotics for treating MRSA infections [19]. The optimized dodecanoyl lysine analogue demonstrated efficacy as a broad-spectrum antibacterial agent with low toxicity, capable of eradicating MRSA biofilms and persisters, as evidenced by its success in ex vivo skin infection models [20]. Consequently, developing small-molecule antimicrobial drugs targeting cell walls or membranes represents a promising therapeutic strategy.

Small-molecule compounds with innovative structures can avoid cross-resistance, serving as one of the prominent directions to alleviate bacterial resistance [21]. Therefore, the new main ring structure screening of new natural and synthetic compounds has a unique role in the anti-infection of drug-resistant bacteria [22]. Here, we developed compound YZ462 on the basis of a heteroaromatic-aryl scaffold (Fig. 1A). The antimicrobial activity of YZ462 was assessed under physiological conditions, and comparisons with the activities of methicillin and vancomycin were conducted in a series of subsequent experiments. Furthermore, the bioactivities of YZ462 against MDR pathogens, biofilms, and persisters were carefully evaluated. For the damage mechanism analysis, membrane damage and membrane potential differences were observed in YZ462-treated bacteria. In addition, its efficacy and safety were determined after topical application ex vivo and in vivo in sepsis infection models.

Fig. 1.

A Chemical structure of YZ462. B Time-kill kinetics of YZ462 against S. aureus ATCC6538. C Resistance development of S. aureus ATCC6538 to YZ462 and norfloxacin. D Cytotoxicity assessment of YZ462 in HeLa, A549, and Vero E6 cells. E Hemolytic activity of YZ462 on rabbit erythrocytes. All experiments (B-E) were performed in triplicate (n = 3 biologically independent replicates), and data are presented as mean ± standard deviation (SD), statistical analyses were conducted using Student’s t-test for comparisons between groups (*p < 0.05, **p < 0.01, vs. 1 × MIC)

Materials and methods

Bacterial strains, growth and reagents

S. aureus ATCC6538 strains were obtained from the Shanghai Bioresource Collection Center. The clinical isolates of MRSA were obtained from the Second Affiliated Hospital of Anhui Medical University. S. aureus cultures were grown aerobically at 37℃ in 2 × YT liquid media with rotary shaking at 220 rpm. Methicillin sodium salt (S82852), vancomycin (Y25829) and dimethyl sulfoxide (DMSO, S26063) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Propidium iodide (PI, KGA1813-50) was purchased from KeyGEN BioTECH (Jiangsu, China). DiSC₃(5) (HY-D0085) and ethidium bromide (EtBr, HY-D0021) were purchased from MedChemExpress. Crystal violet (G1059), 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCF-DA, ID3130), daptomycin (ID0090), melittin (IM1270) and 2.5% glutaraldehyde (P1126) were purchased from Beijing Solarbio Science & Technology Co., Ltd. The small-molecule compound YZ462 was dissolved in DMSO at a concentration of 10 mg/mL and stored long-term at −80℃. When treating S. aureus with daptomycin, CaCl₂ was consistently supplemented to a final concentration of 2.5 mM.

Susceptibility determination

The MICs were determined via the broth microdilution method according to CLSI guidelines. Mid-log-phase cultures were diluted in 2 × YT broth to ~ 10⁵ CFU/mL. The drug concentrations in the first rows of the 96-well plate were set at 64 µg/mL and then measured via a 2-fold dilution scheme. Methicillin and vancomycin were chosen as the positive controls to evaluate the effects of the compounds shown in this study. Finally, after a 96-well plate containing a diluted mixture was incubated at 37℃ for 18 h, the concentration of the drugs in a well with no apparent bacterial growth served as the MIC value. The determination of the MICs of different compounds was independently repeated 3 times, and the average value was calculated.

Time-killing assay

To analyze the kinetics of bacterial cell killing, log-phase-grown S. aureus cells (OD₆₀₀ = 0.3) were treated with YZ462 or antibiotics at the indicated concentrations in test tubes and shaken at 220 rpm at 37℃. At different times of drug exposure, a 100 µL aliquot of the bacterial suspension was taken from each tube, and a 10-fold ratio was diluted in normal saline. The diluted bacterial suspension was spread onto 2 × YT drug-free agar plates followed by incubation at 37℃ for overnight. The numbers of bacterial colonies on each plate were counted. For the experiment examining the protective effect of reactive oxygen species (ROS) scavengers (5% or 7.5% DMSO, 0.25 mM bipyridyl + 100 mM thiourea) or Chloramphenicol (Cm, 60 µg/mL) on YZ462-mediated bactericidal activity, exponentially growing S. aureus cells were pretreated with sub-inhibitory concentrations of ROS scavengers or Cm for 10 min, followed by treatment with a defined concentration of YZ462. Bacterial survival was monitored by CFU enumeration at regular time intervals. The MIC values of DMSO and BT are provided in Table S1.

Resistance development assay

The ability of the compound to cause resistance development among pathogenic bacteria was evaluated by first determining the MIC value via S. aureus ATCC6538. The bacteria were subsequently inoculated at a ratio of 1:100, and the drug concentration in the tube was 1/2 × MIC. On the following day, the bacteria were serially added to twofold compound gradients in a 96-well plate, after which an MIC assay was performed. Following the determination of the new MIC, the bacteria were taken and diluted at a ratio of 1:100 into a new test tube at 1/2 × MIC, beginning the second growth cycle. The above operation was repeated after the end of each growth cycle (20–24 h), and the experiment was performed for 10 cycles to determine the changes in the MICs of the compounds.

Cytotoxicity assay

The cell counting kit-8 (CCK-8) assay was used to evaluate the cytotoxicity of YZ462 against the A549, VeroE6, and HeLa cell lines as previously described [23]. Briefly, cells were seeded in 96-well plates at a density of 5,000 cells/well in 100 µL culture medium and incubated for 24 h prior to treatment with varying concentrations of YZ462. Ten microlitres of CCK-8 solution (APExBIO, K1018) was added to each well after treatment for 24 h. The plates were incubated in an incubator for 1–4 h, and then the absorbance at 450 nm was determined by a Multiskan FC (Thermo Scientific).

Hemolysis test analysis

Fresh sterile blood was taken from the rabbit artery and centrifuged at 400 × g for 10 min. The supernatant was discarded, and the samples were resuspended in the same volume of 1 × PBS. The erythrocytes were resuspended in PBS after being washed twice, and all of the above operations were carried out on ice. Then, 150 µL of the suspension was added to each well of a 96-well plate, and different concentrations of YZ462 were diluted 2-fold to achieve the final concentration used in this experiment. Triton X-100 (1%, v/v) and 1 × PBS were used as positive controls and negative controls, respectively. The mixture was then incubated at 37℃ for 1 h. Finally, the mixture was centrifuged at 200 × g for 10 min to obtain the supernatant, after which 100 µL of the supernatant was transferred to a new 96-well plate to measure the absorbance at 570 nm. Hemolysis was calculated via the following equation of (OD_Sample−OD_{Negative control})/(OD_{Positive control}−OD_{Negative control}) ×100%.

Bactericidal activity against persisters

This assay was performed as described previously with modifications [24, 25]. To obtain persisters, overnight cultured S. aureus ATCC6538 was diluted in fresh 2 × YT broth and grown at 37℃ until it reached the exponential phase (OD₆₀₀ = 0.4–0.6). The cells were then washed three times and resuspended in 2 × YT broth to a final concentration of 1 × 10⁹ CFU/mL. After inducing persisters with ciprofloxacin (10 × MIC, 6 h, 37℃), cells were washed and treated with YZ462 (5 × or 10 × MIC) to assess its antibacterial activity. At the indicated times, the CFUs were counted by plating serial dilutions of cultures washed with 1 × PBS on 2 × YT drug-free agar plates.

Biofilm eradication assay

The disruption effect of YZ462 on mature biofilms of S. aureus ATCC 6538 and MRSA 153 was assessed using a crystal violet staining assay, as previously described [26, 27]. The inocula of S. aureus ATCC6538 or MRS153 were diluted by 2 × YT medium to 1 × 10⁵ CFU/mL in a 96-well microplate and incubated at 37℃ for 24 h. The concentrations (32 × MIC, 16 × MIC, 8 × MIC, 4 × MIC, 2 × MIC and 1 × MIC) of YZ462 were prepared in the 96-well microplate, whereas vancomycin (32 × MIC) was used as the control drug, and 1 × PBS was used as the negative control. After incubation for 24 h at 37℃, the suspensions were removed, and the wells were rinsed with 200 µL of 1 × PBS to remove free-floating bacteria. After air drying, the biofilms formed by adherent cells at the bottom of the wells were stained with 0.1% crystal violet (200 µL). After incubation at room temperature for 30 min, the wells were thoroughly washed with 1 × PBS. The plate was then destained with 96% ethanol (200 µL) and incubated for 15 min. The absorbance of the solution was read with a microplate reader at 570 nm.

ROS measurement

The ROS levels of S. aureus ATCC6538 and MRS153 were detected with H₂DCF-DA as the probe. S. aureus ATCC6538 and MRS153 were incubated to the logarithmic stage, and then H₂DCF-DA was added and diluted at 1000:1 (final concentration of 10 µM) and incubated in the dark for 20 min. After incubation, the mixture was centrifuged at 8000 rpm for 5 min and suspended in 1× PBS. The mixture was subsequently resuspended in 2 × YT medium after two washes. YZ462 at a 10× MIC was added to the suspension, which was subsequently incubated at 220 rpm 37℃. Samples were taken every 20 min, centrifuged at 6500 × g for 2 min, suspended in ultrapure water, and detected with CytoFLEX, and a total of 100,000 cells were detected.

Scanning electron microscopy

Log-phase S. aureus ATCC6538 cultures were exposed to YZ462 (5 × MIC) for 1.5–3 h. Moreover, drug-free bacterial cultures were used as a negative control. The cultures were subsequently washed with saline and fixed overnight by suspension in 2.5% glutaraldehyde. The fixed cultures were dehydrated in a gradient from 30% to 100% ethanol. The samples were dried in a CO₂ critical point dryer for 2.5 h, a layer of metal was plated, and the samples were observed via scanning electron microscopy (SEM) (Hitachi S-4800, Japan).

PI or SYTOX green staining analysis

Propidium iodide (PI), a dye capable of reflecting the membrane damage, was used in our experiments to verify the extent of membrane destruction. Exponentially growing cultures of S. aureus ATCC6538 or MRS153 were treated with YZ462 or DMSO in 2 × YT broth for 2 h at 37℃ with shaking at 220 rpm. PI (5 µg/mL) plus DAPI (5 µg/mL) or SYTOX Green (5 µM) alone was added, and the mixture was incubated in the dark for 20 min before flow cytometry.

Fluorescence determination of ethidium bromide

The bacteria were inoculated at a ratio of 100:1 at 220 rpm and 37℃ until the OD₆₀₀ reached 0.3. The bacteria were then placed in a centrifuge tube and centrifuged at 4℃ and 6500 × g for 5 min. The supernatant was discarded, the bacteria were washed with 1 × PBS once and centrifuged at 6500 × g again for 3 min, the supernatant was discard, and the bacteria were resuspended in an equal volume of PBS to mix the bacterial mixture evenly. Later, EtBr was added to the 1 × PBS suspension to achieve a final concentration of 2 µg/mL. After mixing, 200 µL of mixed mixture was immediately added to each well of a 96-well plate. Measurements were performed on a CLARIOstar PLUS microplate reader using the following parameters: excitation at 540 ± 20 nm, emission at 590 ± 20 nm, with four consecutive measurement cycles of 2 min each. Following the initial measurements, test compounds were added to designated wells under light-protected conditions at room temperature, including: YZ462 (1–16 µg/mL in serial dilutions), vancomycin (16 µg/mL), Triton X-100 (1%), and DMSO. Fluorescence readings were subsequently acquired every 2 min for 30 min. DMSO as the negative control and Triton X-100 as the positive control.

Membrane depolarization assays

To evaluate the disruption ability of the membrane potential, we introduced DiSC₃(5) as a probe. In brief, a single colony of S. aureus ATCC6538 or MRS153 was picked overnight incubation at 220 rpm at 37℃ before inoculation in fresh sterile 2 × YT broth. When the optimal density was OD₆₀₀ = 0.3, the bacteria were centrifuged at 8000× rpm for 5 min and washed twice before being resuspended in fresh 2 × YT broth. After treatment with YZ462 or vancomycin at the indicated concentrations for certain time intervals (0, 10, 20, 40, 80, 120, and 180 min), samples were taken and aspirated from the 96-well plates. DiSC₃(5) was added at a final concentration of 0.4 µM and incubated at 37℃ in the dark for 60 min. KCl was added at 100 mM before measurement. The fluorescence intensity was recorded at 622 ± 20/670 ± 20 nm.

Antibacterial activity of the mixtures of YZ462 with cell membrane or cell wall components

To assess the effects of the cell membrane or cell wall components on the antibacterial activity of YZ462, YZ462 was mixed with phosphatidylglycerol (PG), phosphatidylethanolamine (PE), cardiolipin (CL), and peptidoglycan. Bacterial suspensions (S. aureus ATCC6538 and MRS153 at 10⁵ CFU/mL) in 96-well plates containing various concentrations of YZ462 were mixed with PG, PE, CL, and peptidoglycan dissolved in sterile DMSO-sterile water. After incubation with 2 × YT broth at 37℃ for 18 h, the MICs of the mixtures were determined, and the fold changes were calculated compared with those of the MICs without the cell membrane or cell wall components.

RNA sequencing

Mid-log phase S. aureus ATCC6538 was treated with YZ462 (5 × MIC) for 2 h. Total RNA was extracted via QIAzol Lysis Reagent (Qiagen) and purified via RNeasy Micro Kit MinElute Spin Columns (Qiagen). The rRNA was removed, and the library was constructed via the Illumina TruseqTM RNA Sample Prep Kit. RNA sequencing was performed via the Illumina NovaSeq 6000 platform. Transcript and gene expression levels were quantified by the number of mapped reads on the gene by string tie software (http://cufflinks.cbcb.umd.edu/). RNA sequencing and data analysis were performed by Sanshu Biotechnology (www.sanshubio.com). The genes and correlated functions were annotated according to the UniProt database, and the selected results are listed in supplemental Table S2−10.

Galleria mellonella larvae model for antimicrobial efficacy evaluation

To evaluate the antibacterial ability of YZ462 in vivo, Galleria mellonella larvae ranging from 0.3 to 0.5 g were chosen as the hosts of S. aureus MRS153. Before the test, a single colony of S. aureus MRS153 was incubated overnight at 37℃ and 220 rpm before reinoculation in fresh sterile 2×YT broth. When the OD₆₀₀ was approximately 0.3, the mixture of medium and bacteria was centrifuged at 6500 × g for 5 min before resuspension and dilution in saline, with viable cells of 1 × 10⁶ CFU/mL. A total of 100 G. mellonella larvae were selected, and 25 larvae were included in each of the groups as follows: the controls were injected with saline, the experimental groups were injected with YZ462 at different concentrations (2 or 4 mg/kg), and vancomycin (8 mg/kg) was injected as a positive control. The G. mellonella larvae were injected with 10 µL of bacterial suspension in the left proleg, followed by injection of the same volume of the compound in the right proleg at intervals of 1 h. After administration, the 10 larvae were placed in the dark, and the survival rate was recorded every 24 h for 7 d. Another 15 larvae were severed after 24 h, ground, and serially diluted in saline before being plated on the 2 × YT medium for bacterial counting.

Mouse abdominal infection model for antimicrobial efficacy evaluation

The toxicity of YZ462 was evaluated in 12 healthy C57BL/6 N female mice (weighing 19–22 g, 5–6 weeks old). All the mice used in this study were divided into 3 groups: a control group without drug injection and experimental groups that were injected with YZ462 (25 or 50 mg/kg). The weights of the mice were recorded every 24 h for 7 d.

S. aureus MRS153 was selected as the model pathogen for establishing the infection model. Fresh overnight cultures of strains were diluted 1:100 (v/v) in 2 × YT broth and incubated at 37℃ and 220 rpm until the OD₆₀₀ reached 0.5. For the survival rate assay, mice were randomly allocated into groups (n = 6) and intraperitoneally administered 100 µL of bacterial suspension (5 × 10⁹ CFU/mL in PBS). One hour after injection, YZ462 (5, 10, or 20 mg/kg) was administered via the tail vein. The mice were closely monitored, and their survival or death was recorded for 7 d. For the organ loading experiment, the mice were randomly divided into 4 groups (n = 4). The mice were infected with 100 µL of bacterial suspension (5 × 10⁸ CFU/mL) via intraperitoneal injection. After 1 h, the mice were treated with YZ462 (5, 10, or 20 mg/kg) or vancomycin (20 mg/kg) through the tail vein, and an equal volume of saline was used as a negative control. The bacterial loads (kidneys, liver, and spleen) were calculated after treatment for 24 h. All mice used in the above experiments were supplied by Beijing Charles River Laboratories. Euthanasia was performed on all mice in this study by cervical dislocation. We hereby confirm that all procedures involving experimental animals, including housing, infection model establishment, and euthanasia, were strictly performed in compliance with the ethical guidelines of Henan University’s Institutional Animal Care and Use Committee.

Statistical analysis

All experiments were performed with at least three independent biological replicates. Data are presented as the mean ± standard deviation (SD) unless otherwise indicated. Statistical significance was defined as *p < 0.05, **p < 0.01 and ***p < 0.001 for all analyses. Data processing and visualization were conducted using GraphPad Prism 9.0.

Results

YZ462 rapidly mediates bacterial death and has low hemolytic activity

To obtain novel small-molecular compounds with high potency against S. aureus that are different from conventional antibacterial agent skeletons, we screened a large number of compounds from our in-house library, and YZ462, which has profound antibacterial ability with a heteroaromatic-aryl scaffold, was identified (Fig. 1A). Compound YZ462 was obtained according to the procedure described in supplementary Scheme S1. Our study revealed that YZ462 had excellent antibacterial activity against S. aureus ATCC6538 strain and clinical MRSA isolates, with MICs ranging from 1 to 2 µg/mL (Table 1). The killing kinetics study revealed that YZ462 exerts concentration-dependent bactericidal activity, effectively suppressing S. aureus growth at 1 × MIC while inducing rapid bacterial death at concentrations above 2 × MIC, with enhanced killing efficacy at higher doses (Fig. 1B). In addition, the time-dependent lethality of YZ462 was confirmed, which was evidenced by a significant drop in the survival of S. aureus ATCC6538 with longer incubation times at the same concentration (Fig. 1B). After 180 min of treatment at 8 × MIC, the survival rate dropped to below 0.1% (Fig. 1B). To further investigate the bactericidal activity of YZ462, we evaluated the killing kinetics of YZ462 against S. aureus MRS153 in a cell line model. The results indicate that the number of CFUs of S. aureus MRS153 was evidently reduced by YZ462 treatment in the RAW264.7 cell line infection model (Fig. S1). The above results indicate that the bactericidal activity of YZ462 exhibits a clear dependence on both time and concentration.

Table 1.

MICs of antibiotics or compounds used in this study

Strains	MIC (µg/ml)
	Met	Van	Dap	Nor	YZ462
S. aureus ATCC6538	1	0.5	0.125	1	1
S. aureus MRS1	16	1	0.25	/	2
S. aureus MRS2	4	0.5	0.125	/	1
S. aureus MRS3	4	1	0.125	/	2
S. aureus MRS4	8	1	0.125	/	2
S. aureus MRS5	1	1	0.125	/	1
S. aureus MRS6	1	1	0.125	/	2
S. aureus MRS7	8	0.5	0.125	/	2
S. aureus MRS8	4	0.5	0.125	/	1
S. aureus MRS9	8	1	0.125	/	1
S. aureus MRS10	16	1	0.25	/	1
S. aureus MRS153	16	1	0.125	/	1
S. aureus ATCC6538 Norr	1	0.5	0.125	256	1
S. albus	4	1	0.25	/	1
E. coli BW25113	/	/	/	/	≧ 128
K. pneumoniae	/	/	/	/	≧ 128
A. baumannii	/	/	/	/	≧ 128

Met methicillin, Van vancomycin, Dap daptomycin, Nor norfloxacin, / no data.

Avoiding the development of drug resistance is one of the important criteria for developing new antimicrobial agents [28]. To investigate the potential for resistance evolution in S. aureus against YZ462, we assessed S. aureus ATCC6538 resistance in the presence of continuous treatment with subinhibitory concentrations of YZ462. To our delight, no significant difference was observed in the MIC of YZ462 when S. aureus ATCC6538 was serially passaged under subinhibitory concentrations of YZ462, whereas exposure to norfloxacin led to a rapid rise in MIC fold change after just three passages (Fig. 1C).

In addition, to evaluate the safety profile of YZ462, we evaluated its effects on cell viability and erythrocyte integrity. The IC₅₀ of YZ462 exceeded 38.08 µM (17.6 µg/mL) in all three tested mammalian cell lines (HeLa, A549, and VeroE6), indicating low cytotoxicity (Fig. 1D). Remarkably, YZ462 exhibited minimal hemolytic activity against rabbit erythrocytes, with an exceptionally high IC₅₀ of 6368.1 µM (Fig. 1E). Furthermore, in vivo toxicity studies revealed no acute toxicity in mice following a single intraperitoneal dose of 100 mg/kg (data not shown), and no significant body weight changes were observed over a 7-day monitoring period (Fig. S2). In summary, these results collectively demonstrate that YZ462 possesses a strong bactericidal effect alongside a favorable safety profile, highlighting its potential as a promising drug candidate against MRSA.

YZ462 demonstrates stable antibacterial activity against MRSA and multidrug-resistant bacterial isolates

Clinical isolates exhibit distinct physiological characteristics and stress responses compared to standard laboratory strains, particularly under antimicrobial pressure. Given the rising prevalence of MRSA in clinical settings, we evaluated the rapid bactericidal activity of YZ462 against resistant strains, including MRSA, multidrug-resistant S. albus, and norfloxacin-resistant S. aureus (S. aureus ATCC6538 Nor^r). The results showed that neither vancomycin nor daptomycin exhibited rapid bactericidal activity during the 150 min treatment period (Fig. 2A). Unlike vancomycin and daptomycin, both methicillin and YZ462 displayed rapid bactericidal effects against S. aureus ATCC6538, classifying them as fast-acting agents (Fig. 2A). Strikingly, YZ462 exhibited superior killing kinetics within the first 100 min compared to methicillin against both S. aureus ATCC6538 and multidrug-resistant S. albus (Fig. 2A and B). To further assess YZ462’s efficacy, we tested it against the norfloxacin-resistant strain ATCC6538 Nor^r, which was isolated from earlier resistance development assays (Fig. 1C). The results showed that treatment with 5 × MIC YZ462 resulted in comparable bactericidal activity against both the wild-type ATCC6538 and its Nor^r variant (Fig. 2C). Additionally, to evaluate the antibacterial efficacy of YZ462 against MRSA, we measured the survival rates of MRSA isolates (S. aureus MRS1, MRS10, and MRS153) following treatment with YZ462. The results indicate that these isolates proliferated rapidly in the presence of 8 µg/mL methicillin, their survival rates dropped significantly upon YZ462 treatment (Fig. 2D-F). Taken together, the above findings show that YZ462 is characterized by high bactericidal efficacy and a lack of cross-resistance with methicillin.

Fig. 2.

Time-kill kinetics of YZ462 against drug-resistant Staphylococcus strains. A Exponentially growing S. aureus ATCC6538 cells were treated with YZ462 (5 × MIC), methicillin (5 × MIC), daptomycin (10 × MIC) or vancomycin (10 × MIC) for the indicated durations. B Exponentially growing multidrug-resistant S. albus cells were exposed to YZ462 (5 × MIC) or methicillin (5 × MIC) over time. C Exponentially growing S. aureus ATCC6538 and norfloxacin-resistant strains were treated with YZ462 (5 × MIC). D-F Clinical methicillin-resistant S. aureus isolates (MRS1, MRS10, and MRS153) were treated with YZ462 (5 × MIC) or methicillin (0.5 × MIC). All the experiments were performed as three biologically independent experiments, and the mean ± SD is shown, statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, vs. vancomycin or methicillin control)

YZ462 demonstrates rapid eradication of persister cells and biofilms

Bacteria at different growth stages may exhibit varying bactericidal effects of antimicrobial agents due to differences in their physiological states [29]. To evaluate the bactericidal efficacy of YZ462 against stationary-phase bacteria, we treated overnight cultures of S. aureus ATCC6538 with varying concentrations of YZ462. The results indicate that under YZ462 treatment, the survival rate of S. aureus significantly decreased, dropping to as low as 0.01% especially when treated with an 8 × MIC concentration (Fig. 3A). Persister cells develop tolerance to multiple antibiotics due to reduced metabolic activity [30]. To evaluate the efficacy of YZ462 against antibiotic-induced persister cells, we first generated a persister population of S. aureus ATCC6538 by pretreated with ciprofloxacin at 10× MIC for 6 h to induce the persister state. Subsequently, the survival rate of the persister cells were examined following exposure to YZ462. Notably, YZ462 exhibited potent activity against ciprofloxacin-induced persister cells, resulting in complete eradication (>99.99%) at concentrations of 5 × and 10 × MIC (Fig. 3B). In contrast, the persister population displayed a high level of tolerance to treatment with 10 × MIC ciprofloxacin alone (Fig. 3B).

Fig. 3.

Anti-persister and antibiofilm activities of YZ462. A Time-kill kinetics of YZ462 against stationary-phase S. aureus ATCC6538. Overnight cultures were adjusted to OD₆₀₀ = 0.3 in fresh medium before treatment with indicated concentrations of YZ462. B Persister cell eradication assay. Overnight cultures were pretreated with ciprofloxacin (10 × MIC, 6 h) to enrich persister cells, followed by treatment with YZ462 (5 × or 10 × MIC) or ciprofloxacin (10 × MIC) for 6 h in PBS. C, D Biofilm eradication activity of YZ462 against S. aureus ATCC6538 (C) and S. aureus MRS153 (D) biofilms, with vancomycin as positive control. All experiments were performed with three biological replicates, and data are presented as mean ± SD, statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

Biofilms represent a major clinical challenge as a form of persistent bacterial infection, often leading to refractory infections [31]. To investigate YZ462’s anti-biofilm potential, we performed comprehensive biofilm disruption assays. Using crystal violet staining, we quantified the remaining biofilm biomass after 24 h YZ462 treatment. Our results revealed that YZ462 effectively disrupted preformed biofilms of both S. aureus ATCC6538 and S. aureus MRS153 in a concentration-dependent manner (Fig. 3C and D). To further characterize YZ462’s biofilm eradication capacity, we employed CFU enumeration to assess its bactericidal activity against biofilm-embedded cells. Notably, YZ462 treatment resulted in a dramatic reduction in viable bacterial counts within the biofilm matrix, demonstrating its potent activity against both the structural and cellular components of biofilms (Fig. S3A and B). These results indicate that YZ462 is effective against both persister cells and biofilms, demonstrating its broad-spectrum efficacy against multiple forms of antibiotic tolerance.

Role of ROS in YZ462’s bactericidal activity

Extensive research has established the critical role of ROS in mediating the lethal effects of antimicrobial agents [32]. To investigate whether ROS contribute to YZ462’s bactericidal activity, we pretreated S. aureus ATCC6538 and MRS153 with sub-inhibitory concentrations of ROS scavengers, including DMSO and a combination of thiourea with 2, 2’-bipyridine (BT). Notably, these treatments significantly protected bacterial cells from YZ462-induced killing (Fig. 4A and B, S4A and B, and Table S1), implicating ROS involvement in its mechanism of action. Further supporting this hypothesis, we observed that pretreatment with chloramphenicol, a protein synthesis inhibitor known to block endogenous ROS production, substantially attenuated YZ462’s bactericidal activity (Fig. 4A and B). This finding aligns with established models of ROS-dependent bacterial killing [33]. To directly assess ROS accumulation, we employed H₂DCF-DA fluorescence probes during YZ462 treatment. Time-course analysis demonstrated a rapid and sustained elevation of intracellular ROS levels in both S. aureus ATCC6538 and MRS153 upon YZ462 exposure, whereas no significant increase was observed in the DMSO-treated control group (Fig. 4C, D and Fig. S5). These results suggest that ROS likely play a critical role in the antibacterial activity of YZ462.

Fig. 4.

ROS-dependent bactericidal mechanism of YZ462. A, B Attenuation of YZ462 lethality by chloramphenicol and BT (bipyridyl plus thiourea) in exponentially growing (A) S. aureus ATCC 6538 and (B) S. aureus MRS153. Cells were treated with YZ462 (5×MIC) alone or in combination with chloramphenicol (60 µg/mL) or BT (0.25 mM bipyridyl + 100 mM thiourea). All experiments were performed with three biological replicates, and data are presented as mean ± SD, statistical significance was determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). C, D ROS production in S. aureus ATCC 6538 (C) and S. aureus MRS153 (D) following YZ462 treatment (10 × MIC). Cultures were preloaded with 5 µM H₂DCF-DA for 10 min, treated with YZ462, and sampled every 20 min for flow cytometry analysis after PBS washing

YZ462 exerts selective membrane-disruptive effects on bacterial cells

Given YZ462’s rapid bactericidal activity and its lack of susceptibility to resistance development, we hypothesized that it may target fundamental cellular structures. To assess this, we first evaluated membrane disruption using PI uptake via flow cytometry. As shown in Fig. 5A and Fig. S6, YZ462 treatment induced a significant increase in PI fluorescence in both S. aureus ATCC6538 and MRSA153, whereas ciprofloxacin (a non-membrane-targeting antibiotic [34]) had no effect. In contrast, YZ462-treated HEK293T and HeLa cells exhibited negligible membrane permeability changes, even after 24 h (Fig. S7). To further confirm membrane damage, we performed fluorescence microscopy on PI- and DAPI-stained bacteria. YZ462-treated S. aureus strains exhibited dual staining (PI and DAPI), whereas untreated cells were only DAPI-positive (Fig. 5B), indicating severe membrane disruption. Additionally, SYTOX Green, a membrane-impermeant nucleic acid stain, exclusively labeled YZ462-treated bacteria, further supporting membrane integrity loss (Fig. 5B). We next assessed membrane depolarization using DiSC₃(5), a potential-sensitive dye. YZ462 induced dose-dependent dissipation of the bacterial membrane potential, while vancomycin and untreated controls had no effect (Fig. 6A and B). To further quantify membrane damage, we employed EtBr, a DNA-intercalating dye that fluoresces upon entering compromised cells. Treatment with increasing YZ462 concentrations led to time- and dose-dependent fluorescence enhancement in both S. aureus strains (Fig. 6C and D), confirming progressive membrane disruption.

Fig. 5.

YZ462 disrupts bacterial membrane integrity. A Flow cytometric analysis of PI uptake in S. aureus ATCC 6538 and MRS153 following treatment with YZ462 (5 × MIC). B Fluorescence microscopy images of S. aureus ATCC 6538 and MRS153 stained with DAPI, PI, and SYTOX Green after YZ462 treatment (5×MIC), with DMSO-treated cells as negative control. All experiments were performed in triplicate with consistent results

Fig. 6.

YZ462 enhances membrane permeability and disrupts membrane potential. A, B Real-time monitoring of inner membrane potential changes in S. aureus ATCC6538 (A) and MRS153 (B) using DiSC₃(5) probe. The blank control was bacteria without drug treatment, and melittin was used as a positive control. C, D EtBr uptake assays demonstrating YZ462-induced cytoplasmic membrane permeability in S. aureus ATCC 6538 (C) and MRS153 (D). 0.1% Triton X-100 served as positive control, with untreated cells as negative control. Data represent mean ± SD from three independent biological replicates

To further characterize YZ462’s membrane-damaging effects, we examined morphological changes in S. aureus ATCC6538 using SEM. Untreated bacterial cells displayed intact, smooth surfaces with typical spherical morphology (Fig. 7A). In contrast, exposure to YZ462 induced significant structural alterations: after 1.5 h of treatment, cells exhibited marked swelling and increased diameter (***p < 0.001, Fig. 7B), suggesting cytoplasmic expansion due to membrane permeabilization. Prolonged treatment (3 h) led to severe membrane disruption, with visible surface deformities and loss of cellular integrity (Fig. 7C). These observations corroborate our biochemical assays, confirming that YZ462 directly compromises bacterial membrane stability.

Fig. 7.

YZ462 induces morphological alterations and membrane-targeting activity in S. aureus. A, B SEM images of S. aureus ATCC 6538 following 1.5 h (A) and 3 h (B) treatment with YZ462, with untreated cells as control. Scale bars: 1 μm (upper), 500 nm (lower). C Quantitative analysis of bacterial cell diameter after 1.5 h YZ462 treatment. Data represent mean ± SD (***p < 0.001, two-tailed Student’s t-test). D, E Modulation of YZ462 antibacterial activity against S. aureus ATCC6538 (D) and MRS153 (E) by exogenous membrane components. Chequerboard microdilution assays were performed with peptidoglycan, PG, PE, and CL. All experiments were performed in triplicate with consistent results

YZ462 specifically binds phosphatidylethanolamine and Cardiolipin

Given YZ462’s potent membrane-disrupting activity, we investigated its molecular targets in S. aureus membranes. Using S. aureus ATCC6538 and MRSA153 as model strains, we systematically evaluated potential interactions between YZ462 and key membrane/wall components. Notably, supplementation with exogenous peptidoglycan, the major gram-positive cell wall component, did not alter YZ462’s MIC (Fig. 7D and E), excluding peptidoglycan as a potential target. Strikingly, supplementation with PE or CL, critical bacterial membrane phospholipids, reduced YZ462’s potency in a concentration-dependent manner (Fig. 7D and E). In contrast, supplementation with PG, another major membrane component, did not inhibit YZ462’s antibacterial activity (Fig. 7D and E). To establish direct binding, we performed isothermal titration calorimetry (ITC), which confirmed specific interactions between YZ462 and both PE and CL (Fig. S8). As CL is a major component of the S. aureus cell membrane, these findings collectively demonstrate that CL serves as a putative molecular target mediating the bactericidal effects of YZ462 in S. aureus.

YZ462 triggers oxidative damage and metabolic dysfunction in bacterial cells

Although YZ462 exerts potent membrane-disrupting effects, the bacterial response to this stress remains unclear. To elucidate this, we performed transcriptomic analysis of S. aureus ATCC6538 following 2 h YZ462 treatment. Compared to the DMSO control, YZ462 induced significant transcriptional changes, with 457 genes upregulated and 680 genes downregulated (> 2-fold, Fig. 8A). Gene Ontology (GO) enrichment analysis revealed that YZ462 treatment broadly perturbed key physiological processes, including membrane transport, amino acid metabolism, carbohydrate metabolism, and nucleotide metabolism (Fig. 8B). Notably, differentially expressed genes (DEGs) were enriched in pathways related to ABC transporters, DNA repair, antioxidant defense, and cell wall biogenesis (Fig. 8C and Fig. S9). The altered expression of ABC transporter genes, primarily localized to the bacterial membrane, likely reflects compensatory responses to membrane damage. Similarly, dysregulation of cell wall-associated genes correlated with the observed morphological abnormalities in YZ462-treated cells. Importantly, upregulation of antioxidant genes (e.g., thioredoxin, alkyl hydroperoxide reductase) suggests YZ462 induces oxidative stress, consistent with ROS accumulation. Collectively, these transcriptomic changes paint a picture of S. aureus mounting a multifaceted emergency response to YZ462-induced membrane disruption and associated metabolic stress.

Fig. 8.

Transcriptomic profiling reveals YZ462’s multifaceted mechanism of action in S. aureus. A Volcano plot displaying differentially expressed genes (DEGs) in S. aureus ATCC 6538 following YZ462 treatment (log₂FC > 1). B Gene Ontology (GO) enrichment analysis of DEGs, highlighting significantly affected biological processes.C Functional categorization of upregulated genes involved in: ABC transporter systems, DNA damage repair pathways, Oxidative stress response, Cell wall biogenesis and modification

In vivo efficacy assessment of YZ462 against S. aureus using a Galleria mellonella infection model

To evaluate YZ462’s antimicrobial efficacy in vivo, we established a G. mellonella larva infection model with S. aureus MRSA153. Infected larvae were treated with either saline (control), vancomycin (positive control), or YZ462. Survival rates were monitored for 7 days (n = 10 per group), while bacterial loads were quantified via CFU counts at 24 h post-infection (n = 15 per group; Fig. 9A and B). YZ462 treatment significantly improved larval survival compared to saline controls, with all saline-treated larvae succumbing to infection within 4 days (Fig. 9C). Notably, YZ462 reduced bacterial burdens by > 2-log units within 24 h (Fig. 9D), demonstrating rapid bactericidal activity. At therapeutic concentrations, YZ462 showed no observable toxicity in uninfected larvae (LD₅₀ > 512 µg/mL). These results demonstrate YZ462’s potent in vivo efficacy against MRSA infections, even in this simple invertebrate model, highlighting its therapeutic potential for treating drug-resistant staphylococcal infections.

Fig. 9.

In vivo antimicrobial activity of YZ462 in a G. mellonella infection model. A, B Schematic illustration of the experimental design for evaluating the therapeutic efficacy of YZ462 in G. mellonella larvae infected with S. aureus MRS153. C Survival curves of G. mellonella larvae (n = 10 per group) following infection with S. aureus MRS153 and treatment with YZ462 (2 or 4 mg/kg). Bacterial inoculation was performed in the left posterior proleg, while YZ462 was administered in the right posterior proleg. The log-rank (Mantel-Cox) test with 95% confidence interval was used, and p < 0.05 was considered statistically significant. D Quantitative analysis of bacterial burden in G. mellonella larvae (n = 15 per group) after YZ462 treatment. Data are presented as mean ± SD. Statistical significance was determined by two-tailed Student’s t-test (***p < 0.001)

In vivo efficacy of YZ462 against S. aureus in a murine infection model

Building upon the promising antimicrobial activity of YZ462 observed in vitro and in G. mellonella larvae infection models, we next evaluated its therapeutic efficacy in mammalian systems. Using a well-established murine sepsis model, C57BL/6 N mice were intraperitoneally challenged with a lethal dose of S. aureus MRS153 (Fig. 10A). Treatment with a single intraperitoneal dose of either YZ462 or vancomycin was initiated 1 h post-infection, followed by 7 days survival monitoring. The untreated control group exhibited 100% mortality within 48 h, demonstrating the high virulence of S. aureus MRS153. YZ462 treatment showed dose-dependent protection, with even low doses significantly improving survival rates compared to controls. While 10 mg/kg YZ462 showed inferior efficacy to vancomycin at the same dose, escalating the YZ462 dose to 20 mg/kg achieved remarkable protection, yielding a 90% survival rate (Fig. 10B). To further characterize YZ462’s antimicrobial activity, we employed a sublethal infection model. Bacterial burden analysis 48 h post-treatment revealed that YZ462 significantly reduced MRSA colonization in all examined organs (kidney, liver, and spleen) compared to untreated controls (Fig. 10C-E). Collectively, these findings established that YZ462 as a promising anti-MRSA therapeutic candidate with demonstrated efficacy in mammalian infection models.

Fig. 10.

Therapeutic efficacy of YZ462 in a murine peritonitis-sepsis model infected with S. aureus MRS153. A Experimental design for evaluating the in vivo efficacy of YZ462 in a S. aureus MRS153-induced peritonitis-sepsis model. B Survival analysis of C57BL/6 N mice (n = 6 per group) following intraperitoneal challenge with a lethal dose of S. aureus MRS153 and treatment with YZ462 (5, 10, or 20 mg/kg) or vancomycin (20 mg/kg). The log-rank (Mantel-Cox) test with 95% confidence interval was used, and p < 0.05 was considered statistically significant. C Bacterial burden in the liver, spleen, and kidneys of infected mice (n = 4 per group) after treatment with YZ462 or vancomycin. Data represent mean ± SD, statistical significance was assessed by two-tailed Student’s t-test (*p < 0.05)

Discussion

MRSA is a highly virulent, multidrug-resistant pathogen responsible for severe infections, including pneumonia, bacteremia, endocarditis, and skin/soft tissue, bone, and joint infections, often leading to fatal outcomes [35]. Recognized as a predominant cause of nosocomial infections, MRSA poses a formidable clinical challenge due to its extensive antibiotic resistance. Current first-line treatments-such as vancomycin, teicoplanin, linezolid, tedizolid, and ceftaroline-remain effective but are increasingly threatened by the emergence of resistant strains, including vancomycin-intermediate S. aureus (VISA), heterogeneous VISA (hVISA), and fully vancomycin-resistant S. aureus (VRSA) [5, 36, 37]. This alarming trend underscores the urgent need for novel anti-MRSA therapeutics. In this study, we report YZ462, a novel heteroaromatic-aryl derivative exhibiting potent antimicrobial activity against MRSA. Through comprehensive in vitro and in vivo evaluations, we demonstrate that YZ462 effectively combats MRSA infections. Mechanistic studies reveal that YZ462 exerts its antibacterial effects by disrupting bacterial membrane integrity, interacting with key phospholipids such as PE and CL, and collapsing membrane potential. Our findings establish heteroaromatic-aryl derivatives as a promising new class of antibiotics with significant potential for clinical development against MRSA.

Bacterial resistance, tolerance, persister formation, and biofilm development represent major challenges in antibiotic therapy, often leading to treatment failure in clinical settings. Among these, persisters-metabolically dormant cells linked to chronic infections-are particularly problematic, as they evade antibiotic killing even in the absence of genetic resistance [24, 38]. In S. aureus, persister formation is closely associated with ATP depletion [39], suggesting that antimicrobial efficacy may be influenced by bacterial metabolic states: attenuated respiration reduces drug activity, while accelerated respiration enhances it [40]. Notably, stationary-phase S. aureus cells exhibit a persister-like phenotype, rendering them highly refractory to conventional antibiotics [25, 41]. Biofilms further exacerbate antibiotic resistance, serving as a protective barrier that enhances bacterial survival in diverse environments. Clinically, biofilms are implicated in ~ 80% of chronic and recurrent infections, making antibiofilm activity a critical criterion for novel antimicrobial development. Given these challenges, there is an urgent need for innovative anti-infective agents capable of overcoming resistance mechanisms. Recent advances include chemically synthesized small molecules [42], antimicrobial peptides [43], and natural products [25] with potent activity against S. aureus persisters and biofilms. In this study, we demonstrated that YZ462 exhibits broad-spectrum efficacy against MRSA and multidrug-resistant S. albus, while also showing remarkable activity against mature biofilms and persisters. Importantly, YZ462 displays a lower propensity for resistance development compared to norfloxacin, positioning it as a promising next-generation antimicrobial candidate.

Clinically available fast-acting bactericidal antibiotics are primarily classified into three major categories: β-lactams, quinolones, and aminoglycosides. These agents exert their lethal effects through mechanisms that include induction of redox-related physiological alterations and endogenous ROS production [29, 33]. Our investigations revealed that YZ462 similarly mediates rapid S. aureus killing while enhancing microbial ROS generation. The bactericidal activity of such antibiotics can be attenuated by pretreatment with either bacteriostatic agents (e.g., chloramphenicol) or ROS scavengers (including thiourea plus 2,2’-bipyridine or DMSO) [44, 45]. This observation aligns with established mechanisms wherein chloramphenicol’s inhibition of protein synthesis prevents ROS-dependent antimicrobial activity [46, 47]. As ROS production is intrinsically linked to respiratory metabolism, chloramphenicol concomitantly suppresses both the ROS surge and lethality induced by fluoroquinolones [46, 48]. In our study, we observed that YZ462’s bactericidal action was partially, but not completely, inhibited by both ROS scavengers and bacteriostatic antibiotics. These findings suggested that ROS generation contributes significantly to YZ462’s mechanism of action. The incomplete inhibition of lethality may reflect either: (1) irreversible cellular damage occurring prior to scavenger intervention; (2) the existence of additional, ROS-independent killing mechanisms. This dual-mode action potentially enhances YZ462’s therapeutic value by reducing susceptibility to resistance development through single-pathway mutations.

Targeting bacterial cell membranes represents a highly promising antimicrobial approach, as evidenced by the clinical success of membrane-acting agents like colistin and daptomycin. These frontline antibiotics demonstrate potent bactericidal activity through their selective membrane disruption mechanisms, underscoring the therapeutic value of this targeting strategy [49, 50]. Recent advances in antimicrobial discovery have identified numerous membrane-targeting agents, including synthetic small molecules, natural products, and antimicrobial peptides, that exhibit both potent bactericidal activity and reduced propensity for resistance development [16, 23, 43, 51, 52]. To elucidate YZ462’s mechanism of action, we systematically evaluated its membrane-disrupting effects using multiple complementary approaches: (1) PI uptake assays demonstrating membrane integrity loss, (2) membrane potential depolarization measurements, and (3) permeability assays. While these membrane perturbations represent primary cellular damage, our data reveal that ROS-mediated cytotoxicity constitutes the predominant bactericidal mechanism. Specifically, YZ462 treatment triggered significant ROS accumulation, and the observed attenuation of its killing efficacy by ROS scavengers provides compelling evidence for ROS-dependent bacterial death. Our findings suggested that YZ462’s primary mechanism of action involves direct interaction with bacterial cell membranes. This conclusion is supported by two key observations: (1) the selective inhibition of YZ462’s antimicrobial activity by PE and CL, but not by PG or peptidoglycan, and (2) the compound’s demonstrated membrane-disrupting effects. These results strongly indicate that YZ462 exerts its antibacterial activity through specific binding to PE and CL components of bacterial membranes. Although PE and CL are conserved membrane components in both gram-positive and gram-negative bacteria, YZ462 demonstrated selective activity against gram-positive pathogens. This specificity suggested that additional structural factors, particularly the distinctive outer membrane and lipopolysaccharide layer of gram-negative bacteria, may restrict YZ462’s access to its membrane targets. Although our in vitro ITC experiments have demonstrated direct interactions between YZ462 and PE/CL, further validation is required to determine whether these molecular interactions are functionally linked to YZ462’s bactericidal activity. As CL is a major constituent of the S. sureus cell membrane, we will perform genetic knockout of non-essential genes involved in CL biosynthesis to further validate whether CL serves as the direct target of YZ462. This reverse genetic approach will provide definitive evidence for the specific interaction between YZ462 and CL. SEM analysis demonstrated a significant increase in the diameter of bacterial cells following a 1.5 h treatment with YZ462. Given that one of the primary roles of the bacterial cell wall is to maintain cellular morphology, the observed morphological alteration strongly suggested that YZ462 substantially compromises cell wall integrity. These findings indicate that YZ462 exerts a disruptive effect on the bacterial cell wall, likely contributing to its antibacterial mechanism.

Antimicrobial agents that either embed within the bacterial membrane or target both the membrane and cell wall precursors appear particularly promising. These compounds demonstrate rapid and potent bactericidal activity while likely exhibiting reduced propensity for resistance development, as their molecular targets are structurally constrained and evolutionarily difficult to modify without compromising bacterial viability [53]. Teixobactin, a novel antibiotic targeting lipid II via a unique dual-binding mechanism, forms supramolecular fibrils that disrupt bacterial membranes while avoiding eukaryotic toxicity, offering a promising template for next-generation antimicrobial design [54]. Gram-negative bacteria possess a protective double-membrane structure, consisting of lipid-rich inner and outer membranes, which acts as a formidable barrier against many antimicrobial agents. This intrinsic permeability limitation likely contributes to the reduced efficacy of YZ462 against gram-negative bacteria compared to its potent activity against gram-positive strains. Consequently, further structural optimization of YZ462 to enhance its penetration and antibacterial activity against gram-negative pathogens represents a promising avenue for future research.

G. mellonella larvae represent a well-established infection model due to their susceptibility to a broad spectrum of pathogenic bacteria, offering a convenient and ethically favorable system for preliminary antimicrobial evaluation [55–58]. Motivated by the promising in vitro activity of YZ462, we assessed its efficacy in this invertebrate model. Treatment with YZ462 significantly reduced bacterial burden and enhanced larval survival, demonstrating potent in vivo antibacterial activity. To further validate these findings, we employed a murine abdominal infection model. Consistent with the G. mellonella results, YZ462 administration led to rapid clearance of bacterial loads in multiple organs and substantially improved survival rates in mice. The robust therapeutic efficacy observed across both animal models highlights YZ462’s strong potential as a novel anti-MRSA therapeutic candidate.

Conclusion

In this study, we demonstrated that heteroaromatic-aryl derivatives exhibit potent antibacterial activity against clinically significant pathogens, including MRSA. Among these compounds, YZ462 displays exceptional efficacy, exhibiting a low MIC against MRSA while simultaneously demonstrating multifaceted anti-infective properties: biofilm disruption, persister cell eradication, and inhibition of intracellular survival. Importantly, YZ462 significantly improved survival outcomes in infected animal models. These findings establish heteroaromatic-aryl derivatives as a promising novel scaffold for antibacterial development, offering new therapeutic opportunities against drug-resistant staphylococcal infections.

Abbreviations

CFU

Colony-forming unit

CLSI

Clinical and laboratory standards institute guideline

CL

Cardiolipin

DiSC3(5)

3,3'-Dipropylthiadicarbocyanine iodide

DMSO

Dimethyl sulfoxide

H

Hour

H2DCF-DA

2',7'-Dichlorodihydrofluorescein diacetate

Min

Minute

MIC

Minimum inhibitory concentration

MRSA

Methicillin resistant Staphylococcus aureus

PI

Propidium iodine

PG

Phosphatidylglycerol

PE

Phosphatidylethanolamine

S. aureus

Staphylococcus aureus

VRSA

Vancomycin resistant Staphylococcus aureus

ITC

Isothermal titration calorimetry

Authors’ contributions

WK. G., YY. Z., and YQ.Y. designed and organized the trial and wrote and revised the manuscript. WY. L. and KX. W. conducted the experiments. F. Z., WW. Z., JQ. L. and XF. L. revised the manuscript. Y.Z., B. H., and X. W. analyzed the data. QM. L Designed, organized, and directed the manuscript. All the authors have read and approved the final manuscript.

Funding

The present study is supported by the Henan Province Key Research and Development and Promotion Project (242102310023; 252102310431); and Provinces and ministries jointly building key projects of the Henan Provincial Health Commission (SBGJ202402084); The Key Scientific Research Projects of Colleges and Universities in Henan Province (25A350010); Natural Science Foundation of Henan (232300420184); Kaifeng Science and Technology Development Program Projects (2303007).

Data availability

All data analyzed in this study are included in the published article and its supplementary information files, and further inquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

Ethics approval for the study was obtained from the First Affiliated Hospital of Henan University Ethics Committee and ethics number 2024-03-017 was assigned to the study, and informed consent was acquired for clinical and biological information. All laboratory mice were supplied by Beijing Charles River Laboratories, a specialized provider of research animals. All research conducted in this study complies with the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.