A mixture of N-pivaloyl indoles 1 (0.2 mmol), 3-phenyl-1,4,2-dioxazol-5-one (0.6 mmol), [RuCl₂(p-cymene)]₂ (5 mol%), AgSbF₆ (20 mol%), PivOH (3 equiv), and HFIP (2 ml) was added in a 5 mL glass tube, which was stirred at room temperature for 24 h (Sheng et al., 2021). The reaction was stopped, and it was mixed with water and dichloromethane. The reaction mixture was extracted three times with dichloromethane. The combined organic layer was washed twice with a small amount of water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was evaporated under a vacuum, and the residue was purified by flash column chromatography on silica gel (eluting with petroleum ether-ethyl acetate) to provide the desired product 2. A mixture of 7-amido indoles 2 (0.2 mmol), diphenyl diselenide (0.22 mmol), t-BuOK (0.4 mmol), and DMF (2 ml, 0.1 M) was added in a 5 mL glass tube, which was stirred at room temperature for 0.5 h. When the reaction was completed, the next steps were the same as those described above to obtain the desired product SYG-180-2-2 (Figure 1). The full name of SYG-180-2-2 is N-(3-(phenylselanyl)-1-pivaloyl-1H-indol-7-yl) benzamide.

Next, 7-amido indoles 2 (0.2 mmol), Pd (TFA)₂ (5 mol%), AgOAc (0.6 mmol), and PivOH (1.2 mmol) were added into a 12 mL screw capped tube with 2 mL of benzene at room temperature. The reaction mixture was allowed to warm up to 110°C and stirred for 4 h. When the reaction was completed, the next steps were the same as those described above to obtain the desired product SYG-180-7 (Supplementary Figure 1). The full name of SYG-180-7 is N-(2-phenyl-1-pivaloyl-1H-indol-7-yl) benzamide.

Bacterial strains used in this study are described at Table 1. Methicillin-resistant S. aureus strains JP5023 and JP4856 were isolated from patients with different infection sites at the First Affiliated Hospital of Wenzhou Medical University. On the basis of their ability to form potent biofilm, we used them to carry out biofilm research. We used Trypticase soy broth (TSB, BD Biosciences, Franklin Lakes, NJ, United States) medium without antibiotics to culture all strains at 37°C with shaking at 220 rpm.

Human normal alveolar epithelial cells BEAS-2B were a gift from the Clinical Transformation Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine and cultured in Dulbecco’s Modified Eagle’s Medium [DMEM, Thermo Fisher Biochemical Products (Beijing) Co., Ltd.].

SYG-180-2-2 was diluted with dimethyl sulfoxide (DMSO, Biosharp, Beijing, China) to the concentration of 20 mg/ml. The MIC values of SYG-180-2-2 against JP5023 and JP4856 were determined by the microtiter broth dilution method (van Hal et al., 2011). The colonies were cultured for 16–18 h and directly extracted to prepare a 0.5 MacFarland turbidity standard bacterial suspension, and then diluted with cation-adjusted Mueller-Hinton broth (CAMHB) 1:100. A total of 100 μl of medium containing SYG-180-2-2 (1–128 μg/ml) and 100 μl of suspension were added into a 96-well microfilter plate. In the experiment, we used DMSO as a control. After that the plate was incubated for 16–18 h at 37°C. All assays were performed in triplicate. The minimum concentration at which no bacterial growth was observed by the naked eye was defined as the MIC.

Methicillin-resistant S. aureus strains were grown in TSB for 4–6 h and made into a bacterial suspension with a turbidity of 0.5 MacFarland standard. Then we performed 1:100 dilution into TSB medium containing SYG-180-2-2, so that the final concentrations of the medium were 4 and 8 μg/ml. No drug was added as a positive control, TSB was the negative control. An equivalent volume of DMSO to the 4 and 8 μg/ml SYG-180-2-2 samples was used as a control in the experiment in order to exclude the influence of solvent on bacterial growth. A 200 μl mixed liquor was added to a sterile bioscreen honeycomb plate. We used an automatic microbial growth curve analyzer (OY Growth Curves, Finland) to measure OD₆₀₀ every 1 h for 24 h and obtain a growth curve according to the measured values. The test was performed in triplicate.

Overnight-cultured MRSA strains JP5023 and JP4856 were diluted 1:100 in different drug concentrations (0–32 μg/ml) with TSB containing 0.5% glucose (TSBG), and each concentration was added to three parallel wells in 96-well microplates. After incubation for 24 h, the wells were washed carefully three times with 200 μl of phosphate-buffered saline [PBS, Sangon Biotech (Shanghai) Co., Ltd.]. Removing unattached bacteria, biofilms were fixed with 200 μl of 99% methanol for 15 min and stained with 200 μl of 1% crystal violet for 8 min (Chaieb et al., 2011). The excess dye was gently washed off the wells with running water until the water was colorless. The absorbance was measured at 600 nm after adding 30% acetic acid.

Strains were incubated by TSBG in 20 mm glass-bottomed cell culture dishes (NEST, Wuxi, China). After 24 h, we washed the dishes twice with PBS to remove floating cells and then added SYTO-9 (0.02%, Thermo Fisher Scientific, Waltham, MA, United States) and PI (0.067%, Thermo Fisher Scientific, Waltham, MA, United States) to stain biofilms for 30 min in the dark. After staining, samples were scanned by CLSM (TCS SP5; Leica, Wetzlar, Germany) using a 63 × oil immersion objective lens directly.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, also called MTT, is reduced to the water-insoluble blue-purple formazan by amber dehydrogenase in the mitochondria of living cells. Formazan is dissolved by DMSO, and then its absorbance can indirectly reflect the number of living bacteria. We used MTT to detect the biofilm metabolism activity. In brief, overnight-cultured MRSA strains JP5023 and JP4856 were diluted 1:100 with TSBG containing 4 μg/ml of SYG-180-2-2 in 96-well plates, wells without SYG-180-2-2 were control. Each condition was tested in three replicate wells. Plates were incubated at 37°C for 6, 12, 24, and 48 h, respectively. We removed the supernatant and washed the wells twice with PBS. Then, 100 μl of TSBG containing 0.25 mg/ml MTT (Beijing Solarbio Science and Technology Co., Ltd.) was added into each well and incubated at 37°C for 0.5 h in dark. Subsequently, the supernatant was discarded and 100 μl of DMSO was added to wells to dissolve biofilms, and then the optical density of the wells was measured at OD₄₉₀.

The experimental method was slightly modified according to the previously described method (Wang et al., 2021). Briefly speaking, after MRSA strains were cultured overnight in TSB containing 2% glucose, 100 μl of the overnight culture was added to 96-well plates. Subsequently, the equal volume of TSB including SYG-180-2-2 and SYG-180-7 was added, respectively, to realize the desired final concentration of 4 μg/ml. The plates were incubated at 37°C for 4 h. After this, the plate was washed with PBS to discard the floating cells and the absorbance was measured at 600 nm. SYG-180-7 was severed as a control compound to exclude the possibility that the hydrophobicity of the compound itself inhibits the interaction between MRSA and the sold surface.

For polysaccharide intercellular adhesin (PIA) detection, we diluted the overnight culture 1:100 in 3 ml TSBG containing a concentration of 4 μg/ml SYG-180-2-2 into a six-well plate at 37°C for 24 h, wells without SYG-180-2-2 served as the control. Planktonic cells were removed and washed with PBS, then biofilms were resuspended with 500 μl of 0.5 M EDTA [PH 8.0, Sangon Biotech (Shanghai) Co., Ltd.] using a scraper. Cells were incubated at 100°C for 5 min and centrifuged at 12,000 rpm for 2 min. Then, 40 μl of supernatant was added to 20 μl of proteinase K (20 mg/ml) at 37°C for 2 h. A total of 10 μl of the treated PIA sample was spotted onto the polyvinylidene fluoride (PVDF) membrane which was activated by methanol. The membrane was kept moist and smooth during the spotting process. After drying, the membrane was blocked with 3.5% bovine serum albumin (BSA) (Biosharp, Beijing, China) in PBS with 0.1% Tween 20 (PBST) [Sangon Biotech (Shanghai) Co., Ltd.] at 4°C overnight, and incubated at 37°C with Wheat Germ Agglutinin-HRP (WGA-HRP) conjugate for 1 h at a Universal Antibody Diluent (New Cell and Molecular Biotech Co., Ltd.) of 1:5,000. The membrane was washed thoroughly three times with PBST and detected using enhanced chemiluminescence (ECL) (Affinity Bio, San Francisco, CA, United States).

For extracellular DNA (eDNA) detection, MRSA strains were cultured in six-well plates as described above. After incubation at 37°C for 24 h, the eDNA was extracted as previously described (Rice et al., 2007). The amount of eDNA was measured using a UV Nanodrop 2000 (ThermoFisher Scientific Ltd.). The experiment was repeated three times.

We followed the manufacturer’s instructions [(Spin Column Bacteria Total RNA Purification Kit and Sangon Biotech (Shanghai) Co., Ltd.] for RNA extraction. Briefly, MRSA strains were cultured in TSB with and without SYG-180-2-2 at 37°C for 16 h. The bacterial mass was collected by centrifugation and suspended in lysozyme (20 mg/ml) and lysostaphin (1 mg/ml) at 37°C for 1 h. Then total RNA was extracted and cDNA was synthesized using a Primescript™ RT reagent Kit with gDNA Eraser (Takara, Tokyo, Japan).

Quantitative real-time PCR (qPCR) was performed using the Fast Start DNA Master SYBR Green II Mixture (Takara, Tokyo, Japan) and QuantStudio^® 5 Applied Biosystems (ABI) Fluorescence quantitative PCR instrument (Thermo Fisher Scientific). The reaction used the DNA sequence of gyrB as an internal reference and was performed in a 20 μl reaction volume per well. Table 2 shows the primer pairs used for RT-PCR. The cycling conditions were 95°C for 30 s, followed by 40 cycles, with 1 cycle consisting of 95°C for 5 s and 60°C for 34 s. The cycle threshold (Ct) measurements were calculated by the QuantStudio™ Design and Analysis SE software version 1.6.0. First, the relative expression levels of biofilm-related genes treated with and without SYG-180-2-2 were normalized to the gyrB reference gene to obtain ΔCt₁ and ΔCt₂, respectively. ΔΔCt was acquired by subtracting ΔCt₂ from ΔCt₁. Then we used the relative quantification method (2^–ΔΔCt) to analyze the transcription level of the target gene in the sample with SYG-180-2-2. Three replicates were performed for each condition.

The Cell Counting Kit-8 (CCK-8) solution was used to evaluate the proliferation and cytotoxicity of SYG-180-2-2 to BEAS-2B (Yu et al., 2017). WST-8 is reduced by cellular dehydrogenase to an orange formazan product which can dissolve in culture medium. The amount of formazan product is directly proportional to the number of living cells. In short, the cells were seeded in a 96-well plate at different cells/well for 12 h. Then we discarded the supernatant and added a final concentration of 4 μg/ml of SYG-180-2-2 DMEM containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, United States) and 1% penicillin/streptomycin solution (sterile), wells without SYG-180-2-2 were used as a positive control. After 24 h, the supernatant was removed and wells were washed twice with PBS. Then 100 μl of DMEM and 10 μl of cck8 were added to wells at 37°C for 1–2 h. Finally, 450 nm absorbance was measured. Three independent experiments were carried out.

Statistical analysis was performed using GraphPad Prism (version 8.0). Multiple t-tests were used for the growth curve. Biofilm formation assessment was analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test. Unpaired two-tailed t-tests were used for the other experiments. P values < 0.05 were considered statistically significant. *P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001. All figures were presented as mean ± standard deviation.

SYG-180-2-2 was a white solid (96.0527% purity) after purification by chromatography (elution: 35% EtOAc in petroleum ether) with a melting point of 230–231°C. SYG-180-2-2 was characterized by nuclear magnetic resonance (NMR, Figure 2) spectroscopy and high-resolution mass spectrometry (HRMS, Figure 3), obtaining the following results: ¹H NMR (600 MHz, DMSO-d₆) δ 11.51 (brs, 1H), 10.20 (brs, 1H), 8.07 (d, J = 7.3 Hz, 2H), 7.74 (d, J = 2.6 Hz, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H), 7.43 (d, J = 7.5 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.20–7.18 (m, 4H), 7.14–7.08 (m, 2H) ppm;¹³C NMR (151 MHz, DMSO-d₆) δ 165.51, 134.61, 133.53, 132.62, 131.45, 130.92, 130.38, 128.98, 128.20, 128.01, 127.81, 125.52, 123.57, 119.89, 116.82, 116.14, and 95.33 ppm; ⁷⁷Se NMR (115 MHz, DMSO-d₆) δ 205.33 ppm; HRMS: calc. for C₂₁H₁₇N₂OSe⁺ [M + H]⁺: 393.05061, found: 393.05005.

SYG-180-7 was a white solid (100% purity) after purification by chromatography (elution: 15% EtOAc in petroleum ether) with a melting point of 125–126°C. SYG-180-7 was characterized by nuclear magnetic resonance (NMR, Supplementary Figure 2) spectroscopy and high-resolution mass spectrometry (HRMS, Supplementary Figure 3), obtaining the following results: ¹H NMR (600 MHz, CDCl₃) δ 8.27 (brs, 1H), 8.04 (d, J = 7.3 Hz, 2H), 7.97 (d, J = 7.8 Hz, 1H), 7.60–7.53 (m, 5H), 7.48 (d, J = 7.8 Hz, 1H), 7.45 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.4 Hz, 1H), 7.29–7.26 (m, 1H), 6.77 (s, 1H), 0.75 (s, 9H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 193.1, 164.8, 139.9, 134.1, 133.0, 131.9, 130.5, 129.1, 128.8, 128.7, 128.6, 128.1, 127.1, 123.0, 122.1, 119.6, 117.9, 106.2, 46.6, and 27.6 ppm. HRMS (ESI) m/z: [M + H]⁺ calc. for C₂₆H₂₅N₂O₂: 397.1916; found, 397.1914.

The minimum inhibitory concentration (MIC) values of SYG-180-2-2 against MRSA JP5023 and JP4856 were >128 μg/ml. According to the growth curve we drew, the amount of MRSA strain JP4856 in the late logarithmic growth period was consistent at the subinhibitory concentration of 4 μg/ml. But, at 4 μg/ml, MRSA strain JP5023 grew more slowly than bacteria in the control wells at 4–11 h, and the growth was consistent in the late logarithmic phase (Figure 4). The high concentration of SYG-180-2-2 (8 μg/ml) inhibited the growth of MRSA JP5023 (Supplementary Figure 4).

Bacterial biofilms are difficult to eradicate and resistant to antibacterial drugs (Tan et al., 2012). We used semi-quantitative biofilm to detect the effect of subinhibitory concentrations of SYG-180-2-2 on MRSA biofilm. Treatment with SYG-180-2-2 at a concentration of 4 μg/ml decreased the JP5023 and JP4856 biofilm by 82.9 ± 2.3 and 71.9 ± 6.8%, respectively, when compared with the untreated group (Figure 5). Similarly, treatment with SYG-180-2-2 at concentrations of 8, 16, and 32 μg/ml had a significant reduction effect on biofilms. These results showed that sub-MICs of SYG-180-2-2 (4, 8, 16, and 32 μg/ml) were not affected by dose. We observed in the bacterial biofilm treated with SYG-180-2-2 by CLSM at 4 μg/ml that the density of the biofilm was lower and sparser (Figure 6), compared with the untreated group.

Under the action of a subinhibitory concentration of SYG-180-2-2 (4 μg/ml), the bacterial metabolic activity of the strains was measured in four time points by MTT staining. Reduction of the metabolic activity of JP5023 strain in the presence of SYG-180-2-2 at 4 μg/ml after 6, 12, 24, and 48 h were 47.2 ± 0.6, 53.3 ± 3.6, 40.3 ± 6.6, and 57.6 ± 9.9%, respectively, when compared to the untreated groups. At the same time, reduction of the metabolic activity of JP4856 strain after 6, 12, 24, and 48 h of SYG-180-2-2 treatment with 4 μg/ml were 71.0 ± 2.7, 64.8 ± 3.5, 57.0 ± 8.5, and 71.2 ± 7.3%, respectively, compared to the control (Figure 7).

We observed the effect of a subinhibitory concentration of SYG-180-2-2 (4 μg/ml) on the initial adhesion stage of MRSA biofilm by an attachment assay. The results showed that SYG-180-2-2 significantly suppressed the adhesion of JP5023 and JP4856 to the solid surface at 4 μg/ml by 37.6 ± 9.3 and 21.4 ± 5.1%, respectively (Figure 8). Meanwhile, SYG-180-7 showed no significant difference in cell adhesion, when compared with the untreated strains (Figure 8).

In order to study the effect of SYG-180-2-2 on the biofilm matrix of MRSA, the release of PIA and eDNA was detected. Compared with the untreated group, the production of PIA with SYG-180-2-2-treated strains was decreased significantly (Figure 9), however, there was no significant difference in eDNA (Supplementary Figure 5).

The transcript levels of biofilm-related genes treated with the concentration of 4 μg/ml of SYG-180-2-2 were determined using RT-PCR to clarify the effect of SYG-180-2-2 on the formation of biofilm. In general, the results showed that in JP5023 and JP4856, except for the expression of icaR and codY genes which was upregulated, the expression of icaA, icaD, icaR, fnbB, eno, fib, ebps, saeR, psmα, psmβ, and agrA genes was downregulated to varying degrees with the treatment of SYG-180-2-2 (Figure 10). These results were consistent with the adhesion of bacteria and the detection of PIA.

In order to study the effect of SYG-180-2-2 on human cytotoxicity, we used BEAS-2B in our experiments to evaluate the cytotoxicity of SYG-180-2-2 with the CCK-8 assay. There was no effect on the cytotoxicity when SYG-180-2-2 was used (Figure 11A). When the cells were seeded at 3,000 cells/well, the cell morphology was not abnormal under the microscope (Figure 11B). Obviously, SYG-180-2-2 is not cytotoxic at a subinhibitory concentration.