Discovery and evaluation of 3-(2-isocyanobenzyl)-1H-indole derivatives as potential quorum sensing inhibitors for the control of Pseudomonas aeruginosa infections in vitro

Abstract

Quorum sensing (QS) inhibition stands out as an innovative therapeutic strategy for combating infections caused by drug-resistant pathogens. In this study, we assessed the potential of 3-(2-isocyanobenzyl)-1H-indole derivatives as novel quorum sensing inhibitors (QSIs). Initial screenings of their QS inhibitory activities were conducted against Pseudomonas aeruginosa PAO1 and Chromobacterium violaceum CV026. Notably, six 3-(2-isocyanobenzyl)-1H-indole derivatives (4, 12, 25, 28, 32, and 33) exhibited promising QS, biofilms, and pyocyanin inhibitory activities under minimum inhibitory concentrations (MICs) against P. aeruginosa PAO1. Among them, 3-(2-isocyano-6-methylbenzyl)-1H-indole (IMBI, 32) emerged as the most promising candidate, demonstrating superior biofilm and pyocyanin inhibition. Further comprehensive studies revealed that derivative 32 at 25 μg mL⁻¹ inhibited biofilm formation by 70% against P. aeruginosa PAO1, as confirmed by scanning electron microscopy (SEM). Additionally, derivative 32 substantially increased the susceptibility of mature biofilms, leading to a 57% destruction of biofilm architecture. In terms of interfering with virulence factors in P. aeruginosa PAO1, derivative 32 (25 μg mL⁻¹) displayed remarkable inhibitory effects on pyocyanin, protease, and extracellular polysaccharides (EPS) by 73%, 51%, and 37%, respectively, exceeding the positive control resveratrol (RSV). Derivative 32 at 25 μg mL⁻¹ also exhibited effective inhibition of swimming and swarming motilities. Moreover, it downregulated the expressions of QS-related genes, including lasI, lasR, rhlI, rhlR, pqsR, sdhB, sucD, sodB, and PA5439, by 1.82- to 10.87-fold. Molecular docking, molecular dynamics simulations (MD), and energy calculations further supported the stable binding of 32 to LasR, RhlI, RhlR, EsaL, and PqsR antagonizing the expression of QS-linked traits. Evaluation of the toxicity of derivative 32 on HEK293T cells via CCK-8 assay demonstrated low cytotoxicity. Overall, this study underscores the efficacy of derivative 32 in inhibiting virulence factors in P. aeruginosa. Derivative 32 emerges as a potential QSI for controlling P. aeruginosa PAO1 infections in vitro and an anti-biofilm agent for restoring or enhancing drug sensitivity in drug-resistant pathogens.

This work evaluated 3-(2-isocyanobenzyl)-1H-indole derivatives as quorum sensing inhibitors for the control of Pseudomonas aeruginosa infections in vitro.

Introduction

Multidrug resistance (MDR) poses a substantial and escalating challenge, intensified by the contemporary methods employed to combat infectious diseases. The misuse of antibiotics, prolonged hospital stays during prevalent health crises such as the COVID-19 pandemic, and various epidemics have contributed to the proliferation of MDR infections.¹ This scenario heightens the urgency of identifying novel drugs that can be promptly deployed in critical public health emergencies. The World Health Organization (WHO) has also highlighted antimicrobial resistance as a major threat to human health and called for the urgent development of alternative treatment and infection control strategies. Bacteria utilize quorum sensing (QS) as a mechanism for cell–cell communication, coordinating collective behavioral responses to environmental changes through the generation and sensing of signaling molecules.² Many pathogens causing nosocomial infections rely on QS to regulate the production of virulence factors, with signal secretion and identification being cell density-dependent processes.³ QS-regulated gene expression encompasses critical processes such as virulence factor synthesis, biofilm formation, swarming, and swimming motilities.^4,5 Pathogens produce virulent factors that can harm host cells or evade immune system elimination, leading to illnesses.⁶

Biofilm formation is closely linked to 80% of human bacterial infections and is defined as intricate extracellular polymeric matrices consisting of nucleic acids, proteins, polysaccharides, and lipids, that enable bacterial adhesion to host surfaces and provide protection. Biofilms pose challenges to antibiotic penetration and action, often contributing to antibiotic efflux and the development of chronic infections.⁷ Therefore, an effective strategy for treating bacterial infections involves disrupting cellular communication signals mediated by the QS system, thereby reducing the accumulation of virulence factors and inhibiting biofilm formation.⁸ Unlike traditional antibiotics, quorum sensing inhibitors (QSIs) do not affect bacterial growth or vitality and typically do not interfere with bacterial or host metabolism.⁹ QSIs impose less selective pressure on bacteria, thereby reducing the risk of drug resistance. This underscores the significance of exploring QSIs as a viable and reliable approach to address the challenges posed by bacterial infections.

Pseudomonas aeruginosa, an extensively studied pathogen, is recognized for causing both acute and chronic pulmonary infections, particularly in elderly or immunocompromised individuals such as those with AIDS, cancer, acute leukemia, organ transplantation, or cystic fibrosis (CF).^10,11 This opportunistic Gram-negative bacterium inherently displays resistance to fluoroquinolones.¹² Presently, P. aeruginosa infections present a significant threat not only due to the emergence of multidrug-resistant strains but also because of the widespread formation of persistent biofilms.⁷ In the lungs of CF patients, P. aeruginosa commonly forms biofilms, providing protection against the human defense system and exposure to antibiotics, thereby presenting therapeutic challenges.⁹ The transcriptional regulation of P. aeruginosa QS systems primarily involves LasI/LasR, RhlI/RhlR, and PQS/MvfR, all interconnected and synchronously regulated.¹³ LasI, functioning as a protein synthetase, catalyzes the synthesis of the autoinducer N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) (Fig. 1). This molecule binds to the transcriptional regulator receptor protein LasR, promoting the expression of virulence factor genes, including lasB, apr, and toxA.¹⁴ LasR activation also positively regulates both the RhlI/RhlR and PQS/MvfR gene systems. In the RhlI/RhlR system, N-butanoyl-l-homoserine lactone (C4-HSL) (Fig. 1) is produced by RhlI and detected by the RhlR protein. This system primarily oversees the expression of virulence factors such as rhamnolipid, hydrogen cyanide synthase, swarming motilities, and biofilm formation.¹⁵ In the subsequent distinct QS system of PQS/MvfR, 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and its precursor 2-heptyl-4(1H)-quinolone (HHQ) (Fig. 1) bind to the transcriptional regulator MvfR, leading to the transcription of target genes.¹⁶ Consequently, disrupting the synthesis of these signal molecules or impeding their binding to homologous receptors emerges as a promising strategy to inhibit the QS system of P. aeruginosa, thereby repressing the expression of virulence factors and biofilm formation.

Fig. 1. Chemical structures of signaling molecules in P. aeruginosa (3-oxo-C12-HSL, C4-HSL, HHQ, and PQS) and QSI (IMBI, 32).

In the previous study, we synthesized and validated the efficacy of various 3-(2-isocyanobenzyl)-1H-indole derivatives as QSIs targeting Serratia marcescens.¹⁷ Recognizing the pivotal role of QS in P. aeruginosa infections, we deduced that 3-(2-isocyanobenzyl)-1H-indole derivatives could be a new potential QSI against P. aeruginosa. The investigation aimed to identify a novel class of QSIs against P. aeruginosa through a comprehensive evaluation of indol-isonitrile candidates (parts A and B in Scheme S1†). Building on these promising plate screening results at the beginning, we conducted a functional assessment of these novel derivatives, specifically focusing on biofilm inhibition rates and pyocyanin inhibition rates at the sub-MICs. Following the multi-step screenings for QS inhibition, we finally identified six derivatives (4, 12, 25, 28, 32, and 33) that significantly inhibited QS in P. aeruginosa PAO1. The outcomes revealed that 3-(2-isocyanobenzyl)-1H-indole derivatives notably enhanced inhibitory actions against biofilms of P. aeruginosa PAO1. In the current landscape of widespread antibiotic misuse and the ensuing surge in drug-resistant bacteria, we do believe that using QSI as an innovative therapeutic strategy for combating infections caused by drug-resistant pathogens may be effective in the future.

Results

Initial screening for QS inhibition on solid medium of 3-(2-isocyanobenzyl)-1H-indole derivatives

This study primarily explored the impact of 3-(2-isocyanobenzyl)-1H-indole derivatives 1–35 on the QS inhibitory activities of P. aeruginosa PAO1 and the reporter strain C. violaceum CV026. Our results indicated that derivatives 1, 2, 4, 8, 10, 11, 12, 13, 14, 16, 17, 18, 19, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 exhibited effective QS inhibition against P. aeruginosa PAO1. Specifically, derivatives 1, 2, 4, 8, 12, 13, 23, 24, 25, 26, 27, 28, 31, 32, 33, 34, and 35 demonstrated strong activities in QS inhibitory screenings against P. aeruginosa PAO1 (Fig. S1†). Furthermore, our findings revealed that derivatives 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 17, 18, 23, 24, 25, 28, 29, 31, 32, 33, and 35 were effective QS inhibitors against C. violaceum CV026. Notably, derivatives 2, 4, 5, 6, 10, 12, 13, 14, 17, 18, 23, 25, 31, 32, and 33 exhibited high activities in QS inhibitory screenings against C. violaceum CV026 (Fig. S2†). Subsequently, we determined the minimum inhibitory concentrations (MICs) of all 3-(2-isocyanobenzyl)-1H-indole derivatives (Table S1†) and assessed their biofilm inhibition on P. aeruginosa PAO1 using resveratrol (RSV)¹⁸ as a positive control, a typic and effective QS inhibitor in P. aeruginosa. Our results illustrated a concentration–dependent relationship between the derivatives and biofilm inhibition (Fig. 2). More detail can be found in Table S2.†

Fig. 2. Effects of 3-(2-isocyanobenzyl)-1H-indole derivatives on biofilm inhibition against P. aeruginosa PAO1. (A) Impact of 3-(2-isocyanobenzyl)-1H-indole derivatives on biofilm inhibition at 1/2 MICs. (B) Impact on biofilm inhibition effects by 3-(2-isocyanobenzyl)-1H-indole derivatives at 1/4 MICs. Each experiment was conducted a minimum of three times in triplicate, with at least 400 cells counted for each treatment. DMSO served as the solvent, and the compounds were dissolved in DMSO. Resveratrol (RSV) was utilized as positive control. All data were presented with the standard deviation (SD) from three independent experiments. Statistical differences were assessed by one-way ANOVA test, and the error bars indicate the SD of three biological replicates. p-Values derived from these comparisons are denoted with asterisks: * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the RSV control. “ns” indicates no significance or no label p > 0.05.

Optimization and structure–activity relationship analysis of 3-(2-isocyanobenzyl)-1H-indole rerivatives on biofilm inhibition

Subsequent to the initial screening, we assessed the inhibitory effects of 3-(2-isocyanobenzyl)-1H-indole derivatives on P. aeruginosa PAO1 biofilm development at 1/2 MICs based on reported methods¹⁹ (Fig. 2A) and conducted a comprehensive structure–activity relationship (SAR) analysis, more details can be found in Table 1. Building upon recent reports,²⁰ we postulated that the isocyano group may play a pivotal role in biofilm inhibition activity, which was supported by the derivative 5 (33%), with an amino group instead of an isocyano one, exhibiting weaker inhibitory effects compared to the lead compound 4 (38%), partially confirming the significance of the isocyano group in part A for biofilm inhibition activity. Additionally, for comparison, tryptamine-derived isocyanides, derivatives 1 (19%) and 3 (7%), with alkyl substituents on isocyano groups, displayed much lower inhibition compared to the derivative 4 (38%), which featured a phenyl substituent as one of the preferred factors for maintaining or increasing inhibitory activity. Evaluation of the biofilm inhibition rate at 1/4 MICs further highlighted that derivative 2 (9%) was substantially less effective than derivative 4 (15%), which also confirmed the potential importance of a phenyl substituent on the isocyano group. Analyzing derivatives 7 (16%) and 4 (38%) at 1/2 MICs, which bore different substituents on the indole ring, namely, derivatives substituted by the indole ring (derivative 4) and benzene ring (derivative 7), affirmed that the indole ring should rather be better present in part B. Furthermore, when assessing the biofilm inhibition rate at 1/4 MICs, indole ring derivative 4 (15%) outperformed alkane derivative 6 (12%), reinforcing the recommendation for the indole ring in part B. Consequently, we shifted our focus to the diverse substitutes in the skeleton of 3-(2-isocyanobenzyl)-1H-indole, specifically in parts A or B.

Optimization and structure–activity relationship analysis of 3-(2-isocyanobenzyl)-1H-indole derivatives against biofilm.

Entry	Compd. no.	R	R′	Inhibitory effects
1	4	H	H	38% ± 3.6
2	8	5-MeO	H	21% ± 5.8
3	9	5-F	H	35% ± 1.3
4	10	5-Me	H	17% ± 2.5
5	11	6-Me	H	50% ± 3.5
6	12	6-OMe	H	7% ± 0.5
7	13	6-Br	H	39% ± 0.6
8	14	6-Cl	H	11% ± 2.9
9	15	7-Br	H	21% ± 7.3
10	16	7-Cl	H	26% ± 5.9
11	17	7-Me	H	7% ± 1.9
12	18	8-Me	H	40% ± 5.3
13	19	2-Me	H	13% ± 1.2
14	23	H	6′-Me	27% ± 1.1
15	24	H	6′-Cl	1% ± 1.2
16	25	H	5′-F	19% ± 4.4
17	26	H	5′-Me	1% ± 1.4
18	27	H	4′-Me	24% ± 1.3
19	28	H	4′-F	19% ± 6.0
20	29	H	4′-Br	21% ± 3.7
21	30	H	4′-Cl	56% ± 0.6
22	31	H	3′-F	49% ± 0.5
23	32	H	3′-Me	52% ± 3.8
24	33	H	3′-Cl	4% ± 3.3

Subsequent exploration of parts A and B involved derivatives with diverse electronic property groups on the indole ring, including methyl, methoxy, halogen (–F, –Cl, –Br), phenyl, carbonyl group, and more. In detail, we explored the compounds with different positions and groups, which showed different influences on the biofilm effect. Firstly, we focus on exploring ring A. We analyzed different positions for derivatives 23 (27%), 26 (1%), 28 (19%), and 32 (52%), which were all substituted with the methyl group, but the 3′-position, derivative 32 (52%) which had a significant effect on biofilm compared to other positions, appeared to indicate that 3′ position may hold a dominant position. Combined with the 25 (19%), 28 (19%), and 31 (49%), which were all substituted with the –F group, it also appeared to indicate that 3′-position may hold a dominant position. Therefore, more research work in 3′-position may be valuable in the future. For the different groups: for 6′-position, found that methyl is better than –Cl; for 5′-position, –F is better than –Me; For 4′-position, found that Me- is approximately equal with –F and –Br, except for –Cl; for 3′-position, found that Me- is better than –F and –Cl, but the –F is also higher than the leading compound. In general, maybe electron-donating groups are more suitable than electron-withdrawing groups in ring A, except for the 5′-position. In summary, 3′-position and electron-donating group substitution are more suitable in ring A for biofilm effect. And more importantly, the derivatives 30 (56%), 31 (49%), 32 (52%), are all better than lead compound 4 (38%). This is a significant improvement in finding highly active compounds.

Secondly, we analyzed for different positions substituted in part B (indole part), compounds relationship on biofilm effect, the derivatives 10 (17%), 11 (50%), 17 (7%), and 18 (40%), and 19 (13%), which were all substituted with the methyl group, results indicated, that the derivative 11 (50%) with substitution at 6-position had a significant effect on biofilm compared to other positions, which appeared to indicate that 6-position may hold a dominant position. And later combined with the 13 (39%), 15 (21%), which were all substituted with the –Br group, also appeared to indicate that 6-position may hold a dominant position. So, more substitutions in 6-position may be valuable in the next. And continuedly, we test the compounds with the different substituent groups. Here we analyze and summarize the relationship between compounds with different electrical properties groups on biofilm effect in the indole part. In 5-position, we found that –F better than –Me, –OMe; in 6-position, –Br/–Cl better than –OMe, except the –Me, is very highly effective; in 7-position, we found that –Br/–Cl better than –OMe; in 8-position, we found that –Me is comparable with the –H, lead compound 4 (38%). In general, maybe electron-withdrawing is more suitable than electron-donating groups in ring B for the biofilm effect. On the other hand, it is also indicated that 5-Me, has a very high effect. And derivatives 11 (50%) and 18 (40%) are better than the lead compound. All in all, for ring B in 6-position and the electron-withdrawing group is more suitable. Additionally, indole ring N–H was identified as potentially critical for biofilm activity. Modifications were made to indole N–H, shielded by t-Butyloxy carbonyl group in derivatives 20 (38%) and 21 (47%), as well as alkane substitution in derivative 22 (24%) with N-methyl substituents. Results showed that derivatives 20 and 21, while comparable to the lead derivative 4 (38%), were less effective than the positive compound RSV (48%), but here, based on the SAR, we deduced for indole N–H, maybe indole N–H is protected by t-Butyloxy carbonyl group, or maybe this 2-Ph has a great effect on biofilm. Anyway, this led to the determination that the indole ring N–H free was crucial for biofilm activity, confirming 3-(2-isocyanobenzyl)-1H-indole as a dominating skeleton in this work. And for the linker part, derivatives 34 (34%), and 35 (24%) all show that prolonging the linker for ring A and ring B, just one –CH₂ is suitable, more or less, maybe all is not perfect. Also, adding alkane substitution is not good, all in all, it showed keeping the 3-(2-isocyanobenzyl)-1H-indole skeleton and doing some minor modifications is good for this work.

On the whole, the SAR analysis elucidated the influence of different structural components (A and B) on the biofilm inhibition effects of 3-(2-isocyanobenzyl)-1H-indole derivatives. (i) For part A, the isocyano group emerged as pivotal, with the phenyl substituent on the isocyano group proving suitable. (ii) In part B, the indole ring exhibited superior suitability for biofilm inhibitory effects, particularly with 6-Me-substitution displaying heightened activity. (iii) In part A, benzene activity was enhanced by 4′-Cl-substitution, 3′-F-substitution, and 3′-Me-substitution. Ultimately, for effective biofilm inhibition against P. aeruginosa PAO1, derivative 11 (50%) with 6-methyl in the indole ring in part B, as well as derivatives 30 (56%) and 32 (52%) featuring 4′-Cl-substitution and 3′-Me-substitution in part A, were identified as the most promising structural configurations.

Impact on inhibition of pyocyanin by 3-(2-isocyanobenzyl)-1H-indole derivatives

Pyocyanin, a crucial virulence factor in the QS system, plays a vital role in P. aeruginosa PAO1. We explored the inhibitory effects of 3-(2-isocyanobenzyl)-1H-indole derivatives on pyocyanin production. Our findings revealed a significant decrease in pyocyanin levels upon treatment with 3-(2-isocyanobenzyl)-1H-indole derivatives at 1/2 MICs (Fig. 3). The pyocyanin inhibition exhibited by derivatives 4, 5, 10, 12, 13, 17, 21, 25, 26, 28, 32, and 33 surpassed that of the positive control RSV (38%), with derivatives 13, 26, and 32 displaying notable activities (64%, 60%, and 64%, respectively). Evidently, derivatives 13, 26, and 32 could emerge as promising derivatives for further investigation in the field of QS research based on this study. Comprehensive details of the QS inhibitory effects on pyocyanin inhibition by 3-(2-isocyanobenzyl)-1H-indole derivatives against P. aeruginosa PAO1 are provided in Table S3.†

Fig. 3. Effects of 3-(2-isocyanobenzyl)-1H-indole derivatives on pyocyanin inhibition rates of P. aeruginosa PAO1 at 1/2 MICs. Each experiment was conducted a minimum of three times in triplicate, with at least 400 cells counted for each treatment. DMSO served as the solvent, and the compounds were dissolved in DMSO. Resveratrol (RSV) was utilized as positive control. All data were presented with the standard deviation (SD) from three independent experiments. Statistical differences were assessed by one-way ANOVA test, and the error bars indicate the SD of three biological replicates. p-Values derived from these comparisons are denoted with asterisks: * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the RSV control. “ns” indicates no significance or no label p > 0.05.

Comprehensive evaluation of 3-(2-isocyanobenzyl)-1H-indole derivatives

Continuing our assessment, based on the original plate screening results, we investigated the impact of 3-(2-isocyanobenzyl)-1H-indole derivatives, specifically derivatives 4, 5, 10, 12, 13, 17, 21, 25, 26, 28, 32, and 33, on the QS inhibitory activities of P. aeruginosa PAO1 and C. violaceum CV026. Employing the bacterial QS activity plate screening method and focusing on pyocyanin inhibition, we identified six promising derivatives (4, 12, 25, 28, 32, and 33) out of the above twelve derivatives tested that exhibited robust QS inhibitory effects against P. aeruginosa PAO1 (Fig. S3A†) and C. violaceum CV026 (Fig. S3B†). Subsequently, we assessed the inhibitory effect of rhamnolipid with these six derivatives to identify the most effective ones in terms of QSI. Remarkably, all six derivatives (4, 12, 25, 28, 32, and 33) demonstrated rhamnolipid inhibitory activities against P. aeruginosa PAO1 (Fig. 4B). Among them, derivative 12 exhibited the highest inhibition of P. aeruginosa PAO1, with a reduction of 44% at 1/2 MICs and 30% at 1/4 MICs. However, considering all biofilm inhibitions, pyocyanin inhibitory effects, and screening outcomes against P. aeruginosa PAO1, derivative 32 emerged as particularly intriguing. Consequently, further in-depth research has been initiated with derivative 32 as the primary candidate of focus.

Fig. 4. QS Inhibitory activities, rhamnolipid inhibition effects, and growth profiles of six selected derivatives 4, 12, 25, 28, 32, and 33 against P. aeruginosa PAO1. (A) The chemical structures of the six derivatives (highly active after QS inhibitory testings). (B) Effects of the six derivatives on rhamnolipid inhibition rates of P. aeruginosa PAO1 at 1/2 MICs and 1/4 MICs. Each experiment was conducted at least three times in triplicate, with a minimum of 400 cells counted for each treatment. Compounds were dissolved in DMSO, and the amount of DMSO used as the solvent for the compounds served as negative control. All data were presented with the standard deviation from three independent experiments. Statistical differences were determined by a one-way ANOVA test. The error bars represent the standard deviation of three biological replicates. p-Values derived from these comparisons are highlighted with asterisks, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the DMSO control. “ns” indicates no significance or no label p > 0.05. (C) Growth profiles of P. aeruginosa treated with the six derivatives at 1/2 MICs for 24 h. (D) Growth profiles of P. aeruginosa treated with the six derivatives at 1/4 MICs for 24 h. (E) Growth profiles of P. aeruginosa treated with derivative 32. Growth was evaluated at various concentrations of 32 (6.25, 12.5, and 25 μg mL⁻¹) for 24 h. DMSO was used as the negative control. Error bars represent standard deviations of three measurements. All strains were cultured LB broth (pH 7.0) medium at 37 °C.

MICs and growth profiles

It is noteworthy that P. aeruginosa, characterized as an opportunistic human pathogen, demonstrates multidrug resistance and a propensity for relapse.¹⁰ Moreover, a majority of P. aeruginosa strains exhibit resistance to beta-lactam antibiotics like cephalosporins and penicillins. Additionally, resistance to rifampin and vancomycin has been observed, posing challenges in treatment.¹¹ To assess the susceptibility of P. aeruginosa PAO1 to the optimized 3-(2-isocyanobenzyl)-1H-indole derivatives, relevant MICs were determined using a 2-fold gradient dilution method. The MICs for all derivatives investigated in this study are presented in Table S1.† Specifically, the MICs for the pivotal six derivatives (4, 12, 25, 28, 32, and 33) were 96, 6, 48, 12, 96, and 24 μg mL⁻¹, respectively, with their molecular structures illustrated in Fig. 4A. Subsequently, we explored the growth curves of all six derivatives at 1/2 MICs (Fig. 4C) and 1/4 MICs (Fig. 4D) to discern any significant effects. Notably, derivative 32 exhibited no impact on the growth of P. aeruginosa PAO1 at concentrations ranging from 6.25 to 25 μg mL⁻¹ (Fig. 4E).

Effect on AHLs levels of derivative 32

The levels of AHLs produced by P. aeruginosa PAO1 were quantified to evaluate the putative anti-QS activity of derivative 32. LC-MS/MS analysis confirmed that two major AHLs, that is, C4-HSL and 3-oxo-C12-HSL, were detected in the culture supernatants. Exposure to derivative 32 (6.25, 12.5, and 25 μg mL⁻¹) for 24 h caused a significant decrease in both peaks and areas of C4-HSL and 3-oxo-C12-HSL (more details were presented in the ESI†). Relative quantification analysis demonstrated that derivative 32 treatment at 50, 25, and 12.5 μg mL⁻¹ reduced C4-HSL by approximately 19%, 51%, and 61%, respectively, compared with the control (Fig. S4A†). Additionally, we also detected derivative 32 treatment with 3-oxo-C12-HSL at 50, 25, and 12.5 μg mL⁻¹ significantly reduced 3-oxo-C12-HSL by approximately 41%, 60%, and 72%, respectively, compared with the control (Fig. S4B†). These data demonstrated that derivative 32 possesses anti-QS capacity, which might be caused by interfering with the production of AHLs.

Impact on inhibition of biofilm formation and eradication of mature biofilms of derivative 32

The crystal violet assay was employed to assess the inhibitory impact of derivative 32 on biofilm formation in P. aeruginosa PAO1. At 6.25, 12.5, and 25 μg mL⁻¹, derivative 32 inhibited biofilm formation by 32%, 53%, and 70%, respectively (Fig. 5A). Although the treatment with derivative 32 significantly reduced biofilm formation compared to the DMSO control, it exhibited a higher inhibitory effect than the positive control RSV (55%) at 25 μg mL⁻¹. Subsequently, scanning electron microscopy (SEM) was utilized to evaluate the inhibitory potential of 32 against biofilms (Fig. 5B). SEM images revealed that the biofilm in the DMSO control group exhibited a dense net-like structure connected by fibrous elements. Intriguingly, treatment with 32 at 25 μg mL⁻¹ substantially reduced the biofilm, resulting in a scattered appearance. Moreover, the efficacy of 32 in eradicating mature biofilms was determined using the crystal violet assay. The results demonstrated that derivative 32, at 6.25, 12.5, and 25 μg mL⁻¹, reduced the mature biofilms by 43%, 50%, and 57%, respectively (Fig. 6). This eradicating effect was also higher compared to the positive control RSV (47%) at 25 μg mL⁻¹ and the biofilms were remarkably reduced upon 32 treatments.

Fig. 5. Inhibitory effects of derivative 32 on biofilm formation in P. aeruginosa PAO1. (A) The inhibitory effect of 32 on P. aeruginosa PAO1 biofilm formation. DMSO was used as the negative control, and resveratrol (RSV) served as the positive control. All data are presented with the standard deviation from three independent experiments. Statistical differences were determined by a one-way ANOVA test. The error bars represent the standard deviation of three biological replicates. p-Values derived from these comparisons are highlighted with asterisks, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the DMSO control. “ns” indicates no significance or no label p > 0.05. (B) Scanning electron microscopy (SEM) images of P. aeruginosa PAO1 treated with (a) DMSO, (b) 6.25 μg mL⁻¹, (c) 12.5 μg mL⁻¹, and (d) 25 μg mL⁻¹ of derivative 32.

Fig. 6. Eradication effect of derivative 32 on formed biofilms of P. aeruginosa PAO1. DMSO was used as the negative control, and resveratrol (RSV) served as the positive control. All data are presented with the standard deviation from three independent experiments. Statistical differences were determined by a one-way ANOVA test. The error bars represent the standard deviation of three biological replicates. p-Values derived from these comparisons are highlighted with asterisks, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the DMSO control. “ns” indicates no significance or no label p > 0.05.

Impact on inhibition of virulence factors of derivative 32

At the beginning, we assessed the inhibitory effect of rhamnolipid with these six derivatives to identify the most effective ones in terms of QS. Remarkably, all six derivatives (4, 12, 25, 28, 32, and 33) demonstrated significant rhamnolipid inhibitory activities against P. aeruginosa PAO1 (Fig. 4B). Continuously derivative 32 demonstrated a suppressive effect on virulence factors at 6.25, 12.5, and 25 μg mL⁻¹ without influencing the growth of P. aeruginosa PAO1 even at high dosages (Fig. 4E). Consequently, it is evident that derivative 32 effectively inhibits the virulence factors of P. aeruginosa PAO1 (Fig. 7), and the detailed information is as follows: (I) impact on protease production: protease, a crucial virulence factor of the QS system that can alter host immune responses [30], was significantly suppressed by derivative 32 at 25 μg mL⁻¹, exhibiting a 51% reduction compared to the negative control and a more substantial inhibition than that of the positive control RSV (26%) (Fig. 7A). (II) Impact on pyocyanin synthesis: pyocyanin, a vital virulence factor underlying the QS system in P. aeruginosa PAO1, was effectively inhibited by derivative 32 in a concentration-dependent manner. For example, at 25, 12.5, and 6.25 μg mL⁻¹, derivative 32 suppressed pyocyanin by 73%, 57%, and 41%, respectively, demonstrating superior efficiency compared to RSV (26%) at 25 μg mL⁻¹ (Fig. 7B). (III) Impact on rhamnolipid synthesis: another well-studied virulence factor is rhamnolipid. At 25 μg mL⁻¹, derivative 32 inhibited rhamnolipid by 23%, surpassing the inhibitory effect of RSV (12%) (Fig. 7C). (IV) Impact on EPS production: EPS, an essential element of biofilms with a critical function in providing cell nourishment, maintaining cohesion, and promoting cell proliferation, experienced a significant decrease following treatment with derivative 32. At 25 μg mL⁻¹, EPS decreased by 37%, while the reduction in EPS was only 22% with RSV (Fig. 7D). (V) Swarming and swimming motilities: Investigation of 32 in terms of motilities potential revealed comparable outcomes to pyocyanin in P. aeruginosa PAO1 (Fig. 7E and F).

Fig. 7. Inhibitory effects of derivative 32 on virulence factor production in P. aeruginosa PAO1. The levels of virulence factors were assessed at 6.25, 12.5, and 25 μg mL⁻¹. (A) Protease, (B) pyocyanin, (C) rhamnolipid, (D) extracellular polysaccharides (EPS), (E) swarming motility, and (F) swimming motility. Swimming and swarming motilities were treated with the negative control DMSO, the positive control resveratrol (RSV, 25 μg mL⁻¹), and 32 (6.25, 12.5, and 25 μg mL⁻¹). All data are presented with the standard deviation from three independent experiments. Statistical differences were determined by a one-way ANOVA test. The error bars represent the standard deviation of three biological replicates. p-Values derived from these comparisons are highlighted with asterisks, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the DMSO control. “ns” indicates no significance or no label p > 0.05.

Impact on expression of QS and biofilm-related genes

The influence of derivative 32 on the expression of QS-related genes, namely lasI, lasR, rhlI, rhlR, pqsR, sdhB, sucD, sodB, and PA5439 in P. aeruginosa PAO1, was assessed using RT-qPCR (Fig. 8). The most notable change was observed in PA5439, which exhibited a down-regulation of approximately 10.87-fold following exposure to 25 μg mL⁻¹ of 32. Genes lasI and lasR, associated with QS-related motilities, fimbria production, and adherence, were specifically down-regulated by 5.00- and 4.42-fold, respectively. Genes rhlI and rhlR, implicated in biofilm formation, extracellular polysaccharides, and rhamnolipid biosynthesis, experienced suppression by 7.30- and 9.71-fold, respectively. Additionally, gene pqsR, involved in protease and pyocyanin biosynthesis, underwent a down-regulation of 7.41-fold. Furthermore, genes sdhB, sucD, sodB, and PA5439, which contribute to biofilms or related virulence factor production in P. aeruginosa PAO1, were suppressed by 2.87-, 5.65-, 1.82- and 10.87-fold, respectively. The rplU gene served as an internal control,²¹ and the primers utilized in this study are detailed in Table S4.†

Fig. 8. Downregulation of expression in QS-related genes upon derivative 32 treatment in P. aeruginosa PAO1. The relative fold difference in the expression of QS and biofilm-related genes of P. aeruginosa PAO1 was assessed after treatment with 32 (25 μg mL⁻¹). The results are presented as means ± SD (n = 3). All data are presented with the standard deviation from three independent experiments. Statistical differences were determined by a one-way ANOVA test. The error bars represent the standard deviation of three biological replicates. p-Values derived from these comparisons are highlighted with asterisks, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the DMSO/rplU control. “ns” indicates no significance or no label p > 0.05.

Molecular docking

Molecular docking was utilized to gain a deeper comprehension of the potential anti-virulence and anti-biofilm formation properties of derivative 32. Building on our prior research, five proteins—namely, LasR, RhlI, RhlR, EsaL,²² and PqsR — were identified as transcriptional regulators in QS systems.¹⁷ Particularly, PqsR functions as a transcriptional activator in the QS system of P. aeruginosa PAO1. The crucial isocyano group of 32 established hydrogen bonds with the amino acid residues Ile 195 (3.36 Å) and Met 224 (3.93 Å) of PqsR (Fig. 9A). And the crucial indole-N–H of 32 established hydrogen bonds with the amino acid residues Asp 48 (2.82 Å) of EsaL, one of the possible target proteins in QS system (Fig. 9B). In essence, the blocking of C4-AHL binding sites by derivative 32 is expected to be an effective strategy against transcriptional factors LasR, RhlI, RhlR, EsaL, and PqsR. The findings suggest that the combination of derivative 32 with LasR, RhlI, RhlR, EsaL, and PqsR could impede the expression of QS-related traits.

Fig. 9. Molecular docking profiles. The 3D and 2D schematics illustrating receptor–ligand interactions of derivative 32 with various proteins were generated using the Discovery Studio 4.0 program. Docking was performed with compound 32 and PqsR (A) and Esal (B) in 3D schematics. Docking was performed with compound 32 and PqsR (C) and Esal (D) in 2D schematics.

Molecular dynamic simulations and energy calculations

Molecular dynamics (MD) simulations were employed to explore the binding stability of complexes formed by 32 with various target proteins related to QS and biofilms. Five specific target proteins (LasR, RhlI, RhlR, EsaL, and PqsR) were selected as the initial conformations for MD simulations, building on previous research.¹⁷ The interaction potential energy (E_iap) and the root mean square deviation (RMSD), which encompasses the sum of the Lennards-Jones potential and the Coulombic energy, were calculated over time. The E_iap values between proteins and ligands ranged from −117.7 to −198.4 kcal mol⁻¹ (Fig. 10), with the PqsR-32 complex registering −175.6 ± 8.599 kcal mol⁻¹ (Fig. 10A). In all instances, E_iap reached equilibrium and stabilized after a few picoseconds. The RMSD, indicating the deviation in the position of the ligands' heavy components associated with the proteins, was assessed to verify the reliability of ligand binding to target proteins. Most ligands binding to proteins exhibited an RMSD of less than 0.25 nm (Fig. 10), with the PqsR-32 complex, for example, having an RMSD of 0.09 ± 0.013 kcal mol⁻¹ (Fig. 10C). Following an initial brief equilibrium, the RMSD remained constant in all conditions, indicating stable binding of ligands to target proteins.

Fig. 10. E _iap and RMSD profiles: molecular dynamic simulation (MD) studies were conducted for target proteins bound with derivative 32. (A) Time-dependent plots for E_iap of atomic positions, and (C) time-dependent plots for RMSD of atomic positions, 32 for the H-bonds with target proteins, specifically highlighting PqsR. (B) Time-dependent plots for E_iap of atomic positions, and (D) Time-dependent plots for RMSD of atomic positions, 32 for the H-bonds with target proteins, specifically highlighting Esal.

Cytotoxicity

Motivated by the promising outcomes from in vitro investigations, we delved into assessing the cytotoxic effects of derivative 32 using the CCK-8 assay as per the manufacturer's instructions. Specifically, the cell viability was gauged through the CCK-8 test, and the 50% inhibitory concentration (IC₅₀) of 32 was determined. The determined IC₅₀ concentration for human embryonic kidney (HEK) 293 T cells was 59 μM, showcasing that derivative 32 exhibited minimal toxicity for this cell type, making it a suitable candidate as a QSI against P. aeruginosa infection (Fig. S5†). Additionally, cytotoxicity assays were conducted on mouse red blood cells and fibroblast cells to evaluate the safety profile of the investigated derivatives, affirming their low hazard to cells, with all inhibition rates remaining lower (Fig. S6†).

Discussion

In the present decade, the abuse/misuse of antibiotics has led to a concerning rise in MDR, posing a significant risk to both the global economy and healthcare.²³ Traditional antibiotics typically operate by disrupting processes like bacterial cell wall formation, DNA replication, protein synthesis, and the efflux pump mechanism.^24,25 To effectively tackle bacterial infections, it is crucial to identify new targets. The bacterial communication system, referred to as the QS system, plays a vital role in regulating the production of virulence factors, biofilm formation, and bacterial resistance.^26,27 Biofilms serve as protective barriers employed by bacteria to resist the host immune system and antibiotics. Biofilms are formed as a result of various disorders that substantially increase bacterial resistance.²⁸ By hindering the formation of biofilms, QSIs can contribute to reducing resistance to antibiotics.²⁴ Additionally, biofilms serve as protective shields adopted by bacteria to evade the host immune system and antibiotics. These biofilms consist of exopolysaccharide protein polymers secreted by bacteria, attaching to surfaces, whether abiotic or biotic. The extracellular polymers encompass exopolysaccharides such as alginate, lipids, Psl and Pel polysaccharides, proteins, and extracellular DNA (eDNA). Consequently, inhibiting QS emerges as a distinct therapeutic strategy with the potential to address antibiotic resistance. P. aeruginosa produces various virulence factors associated with host infection, including adhesins, proteolytic enzymes, pyocyanin, siderophores, and rhamnolipids, as part of its strategy to combat the host immune system and sustain bacterial persistence.²⁹ Additionally, these virulent factors are non-essential for bacterial growth and survival. As a result, a centralized treatment approach will not hinder bacterial growth and prevent the development of selective pressure. This method demonstrates effectiveness in reducing the emergence of bacterial resistance.³⁰ Progress in QS research holds the potential to generate new antibiotic derivatives with unique modes of action, presenting a hopeful strategy in the ongoing fight against antibiotic resistance.

In this study, the inhibitory effect of QS on P. aeruginosa PAO1 was initially assessed by examining the expression levels of vital virulence factors, such as pyocyanin and biofilms. The design of indol-isonitrile derivatives targeting the specific inhibition of the virulence factor pyocyanin in P. aeruginosa PAO1 was also addressed. This blue-green phenazine pigment possesses redox properties and exerts pro-inflammatory and immunosuppressive activities by inducing oxidative stress in host cells.^31,32 Therefore, the study included an evaluation of the inhibition rates of pyocyanin by indol-isonitrile derivatives against P. aeruginosa PAO1. Based on the bioactivity chasing for all derivatives, the functional assessment of the novel derivatives in biofilm inhibition rate and pyocyanin inhibition rate assays demonstrated that incorporation in parts A and B, particularly within the indol-isonitrile scaffold, resulted in highly potent QS activity, displaying increased efficacy compared to the lead compound (4). Subsequently, six derivatives (4, 12, 25, 28, 32, and 33) exhibiting the most promising QS inhibitory activities were identified. Upon observing the heightened QS inhibitory activities of six derivatives (Fig. S3A and S3B†), prompting consideration for further enhancement, an investigation into another virulence factor, rhamnolipids, was conducted to validate the results. Rhamnolipids are amphiphilic glycolipids with surfactant properties that can intercalate into biofilms, inducing cell lysis.³³ In the inflammatory response, the destruction of phagocytes releases eDNA, facilitating biofilm formation.³⁴ Additionally, rhamnolipids promote lipopolysaccharide aggregation to the outer membrane, enhancing bacterial adhesion to host surfaces.³⁵ Moreover, they regulate bacterial swarming motility, a crucial aspect of bacterial biofilm maturation. Swarming motility involves the coordinated translocation of bacterial flora on semisolid surfaces and is vital for pathogenicity and antibiotic resistance, primarily governed by QS-related genes such as lasB and pvdQ.^36,37 Consequently, a study on rhamnolipid inhibition was conducted on the six derivatives (4, 12, 25, 28, 32, and 33). Intriguingly, the results revealed well-defined rhamnolipid inhibitory effects against P. aeruginosa PAO1 for all six derivatives (4, 12, 25, 28, 32, and 33) (Fig. 4B).

In our ongoing investigation into QSIs,^19,38,39 we identified 3-(2-isocyanobenzyl)-1H-indole derivatives as a novel class with potential QS inhibitory effects against P. aeruginosa. To enhance QS inhibitory activity against P. aeruginosa, we employed screening strategies that included assessing anti-biofilm formation activities. Biofilm inhibition rates were measured at 1/2 MICs and 1/4 MICs to evaluate the impact on biofilm inhibition (Fig. 2). At 1/2 MICs, the majority of the 3-(2-isocyanobenzyl)-1H-indole derivatives exhibited inhibition of biofilm formation against P. aeruginosa PAO1, although there was a decline in inhibition rates at 1/4 MICs. Interestingly, derivative 32 demonstrated excellent efficacy against P. aeruginosa even at 1/4 MICs. Furthermore, the design of indol-isonitrile derivatives aimed at inhibiting pyocyanin was conducted. Pyocyanin inhibition rates were employed to assess the QSI and anti-biofilm activities of the indol-isonitrile derivatives against P. aeruginosa. Our results indicated that derivative 32 significantly inhibited pyocyanin production, suggesting potent QS inhibitory activity.

Additionally, the SAR analysis indicated that derivatives featuring 3-(2-isocyanobenzyl)-1H-indole as a backbone generally exhibit more potential than those with a benzene ring or alkane core in part A. Furthermore, in part B, the phenyl substituent proves more effective than the alkyl group in the isocyano group. Therefore, 3-(2-isocyanobenzyl)-1H-indole emerges as a dominant structure in biofilm inhibition against P. aeruginosa. This structural insight underscores the significance of 1H-indole and 3-2-isocyanobenzyl as two crucial chemical factors. Among all 35 3-(2-isocyanobenzyl)-1H-indole derivatives, derivative 32 demonstrated outstanding QSI and anti-biofilm activities against P. aeruginosa. Notably, owing to its substantial role in inhibiting pyocyanin production at sub-MICs, derivative 32 exhibited the most promising potency in QS inhibition against P. aeruginosa (Fig. 3). Ultimately, when integrating the inhibitory effects of biofilms, rhamnolipids, pyocyanin, and the initial bacterial QS inhibitory activity screenings on plates of P. aeruginosa PAO1 and C. violaceum CV026, along with other similar effects of 3-(2-isocyanobenzyl)-1H-indole derivatives, derivative 32 emerged as the most promising candidate and warranted in-depth investigation.

The potential QSI activities of 32 were further validated by the suppression of QS-related virulence factors in P. aeruginosa, including protease, pyocyanin, rhamnolipid, EPS, swarming motility, swimming motility, and more (Fig. 7). This study further evidenced the potential QSI activities of 32 through the downregulation of QS-related genes such as lasI, lasR, rhlI, rhlR, pqsR, sdhB, sucD, and PA5439, which govern biofilm formation in P. aeruginosa PAO1 (Fig. 8). Two critical virulence factors, rhamnolipids and pyocyanin were significantly suppressed by 32, aligning with the downregulation of the related genes rhlI and pqsR. Derivative 32 also inhibited extracellular proteases produced by P. aeruginosa, which are pivotal for cytolytic activities and host infections.⁴⁰ Swarming, mediated by flagella and dominated by lasI and lasR, plays a key role in pathogen colonization, including initial attachment and biofilm formation.⁴¹ Moreover, the gene rhlI regulates fimbriae subunits, essential for adhesion and colonization by P. aeruginosa.

Exposure to derivative 32 and the concurrent downregulation of relevant genes aligned with the inhibition of biofilm formation, attachment capacity, and swarming motility (Fig. 8). Biofilms, formed on biotic or abiotic surfaces, are highly structured by bacterial populations attached to a surface or embedded in a scaffold of self-produced extracellular matrix.⁴² As a crucial component of biofilms, EPS⁴³ was significantly suppressed by exposure to 32, up to 51% at 25 μg mL⁻¹ (Fig. 7D). Additionally, molecular docking of 32 with the target proteins (Fig. 9) provided a closer insight into the relationship between anti-virulence, anti-biofilm activities, and the inhibition of 32 on the QS systems.

The utilization of QSIs represents an alternative strategy for mitigating P. aeruginosa pathogenicity and lowering the likelihood of developing resistant strains. Bacteria undergo phenotypic changes in response to their surroundings, transitioning from free-swimming cells to biofilm communities. These biofilms enhance cellular cooperativity and resistance to stressors and antibiotics. Biofilms act as the first barrier against antibiotics, significantly contributing to microbial drug resistance.⁴⁴ Once P. aeruginosa biofilms are established, drug resistance becomes inevitable and more serious.^45,46 In this study, derivative 32 exhibited a 70% inhibition of biofilm formation (Fig. 5) and 57% eradication of mature biofilms (Fig. 6) against P. aeruginosa at 25 μg mL⁻¹. Notably, derivative 32 also demonstrated remarkable inhibitory activities against other strains, such as C. violaceum CV026. The biofilm inhibition effect of 32 was further confirmed through SEM images (Fig. 5B), illustrating substantial inhibitory and eradication effects at various concentration levels. Derivative 32 emerges as a promising choice for addressing drug resistance through a novel antibiotic strategy targeting QS.

Conclusion

In summary, this study evaluated the impact of 3-(2-isocyanobenzyl)-1H-indole derivatives on the suppression of QS and QS-related virulence factors in P. aeruginosa. Notably, six derivatives (4, 12, 25, 28, 32, and 33) demonstrated pronounced inhibition effects on pyocyanin and rhamnolipids in P. aeruginosa PAO1, with derivative 32 exhibiting potent QS inhibitory and anti-biofilm activities. Furthermore, some key virulence factors were effectively inhibited by derivative 32 at sub-MICs, surpassing the efficacy of the positive control (RSV). Derivative 32 not only inhibited the development of biofilms but also eradicated mature biofilms of P. aeruginosa. The potential mechanisms underlying its QS inhibitory activity were further elucidated through docking analysis and RT-qPCR analysis. In conclusion, our findings suggest that derivative 32 holds promise for development as an effective QSI and anti-biofilm agent against P. aeruginosa and can be anticipated to offer a valuable solution for combating drug-resistant bacterial infections.

Experimental

Synthesis of 3-(2-isocyanobenzyl)-1H-indole derivatives

3-(2-Isocyanobenzyl)-1H-indole derivatives used in this study were synthesized as compounds targeting QS for antibiofilm drug development based on our recently published study,¹⁷ and all the 3-(2-isocyanobenzyl)-1H-indole derivative structures for QS inhibiting evaluation against P. aeruginosa PAO1 in this study were presented. (Details can be seen in the ESI†).

Bacterial strains

The bacterial strains used in this study included the reporter strains C. violaceum CV026 and wild type P. aeruginosa PAO1. Specifically, C. violaceum CV026 was received from the Guangdong Provincial Center for Microbial Strains (Guangzhou, China). The wild type P. aeruginosa PAO1 was kindly provided by Q. Gong (Ocean University of China). All the strains were cultured at 37 °C with Luria Bertani (LB) broth (pH 7.0) medium unless otherwise specified.

Initial screening for QS inhibition on solid medium

Since the recombinant bioassay method for AHL QS system responds to a broad spectrum of signal molecules, it could also respond to a broad spectrum of QSIs.⁴⁷ In the initial color screening for QSI activity, we employed the biomonitoring strain CV026 for AHL inhibitor detection. And also, used the wild type P. aeruginosa PAO1 for directly detecting the QSI at the same time.⁴⁸ CV026 would produce violacein only with the exogenous addition of C4-HSL. CV026 was incubated in LB broth plus 50 μg ml⁻¹ kanamycin with shaking overnight, and the assay was conducted with 20 μM C4-HSL added to the LB agar. An aliquot of 15 μl of reporter strain overnight culture was added to the melting 15 ml of LB agar medium (37 °C), and tested bacteria were inoculated to the plates with a sterile toothpick. The concentration of agarose used in CV026 plates was 1.0% (w/v). Plates were incubated overnight at 37 °C. QS Inhibition from both CV026 assay plates and PAO1 bioassay plates was observed by a ring of colorless but viable cells around the test growing colony.

QS Inhibitory screenings of the 3-(2-isocyanobenzyl)-1H-indole derivatives

The 3-(2-isocyanobenzyl)-1H-indole derivative structures (listed in Scheme S2†) were used for testing QS inhibitory activity against P. aeruginosa PAO1. The antibiotics used in this study were purchased from Sangon Biotech Co., Ltd (Shanghai, China). A total of 35 3-(2-isocyanobenzyl)-1H-indole derivatives were investigated for their QS inhibitory activities against P. aeruginosa PAO1 according to published methods.⁴⁹C. violaceum CV026 was also used as a reporter strain for the screening of QSI.⁴⁰

Determination of MICs and growth curves

After confirming the QS inhibitory activities of 3-(2-isocyanobenzyl)-1H-indole derivatives, MICs of derivatives against P. aeruginosa PAO1 were determined using the 2-fold serially diluted method.⁴¹ Specifically, stock solutions were prepared by dissolving derivatives in DMSO. The Clinical and Laboratory Standards Institute (CLSI, 2015) was adopted for the determination of MICs for derivatives with an inoculum of 1–5 × 10⁵ CFU mL⁻¹. The derivatives were serially diluted twofold in Mueller–Hinton broth. For growth measurement, P. aeruginosa PAO1 was grown overnight in LB medium at 37 °C. Then the culture was diluted to OD₆₀₀ = 0.05 and subcultured to mid-log phase before being diluted to OD₆₀₀ = 0.05. Then the culture (3 mL) was aliquoted with an appropriate amount of test compound. Cell growth was tested following 1, 3, 5, 7, 9, 11, and 24 h of incubation at 37 °C. Finally, the 150 μL of the resulting culture was transferred to a clear 96-well microtiter plate and OD₆₂₀ was measured.

Determination of AHLs levels

The putative anti-QS capacity of derivative 32 was assessed by quantitating C4-HSL and 3-oxo-C12-HSL levels secreted by P. aeruginosa PAO1.^50,51 Briefly, 0.1% overnight cultures of P. aeruginosa PAO1 were inoculated into 50 mL of LB in the presence or absence of derivative 32 (6.25, 12.5, and 25 μg mL⁻¹) and cultured at 37 °C for 24 h. The same amount of DMSO was added as the negative control. After cultivation, cells were removed by 15 min centrifugation (4 °C). The supernatant was extracted three times using acidified ethyl acetate (1 : 1, v/v). The solvent was evaporated under reduced pressure, and residues were dissolved in methanol. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was adopted for AHLs quantification.³⁹ Briefly, peaks corresponding to C4-HSL and 3-oxo-C12-HSL were detected based on their MS/MS fragmentations and the retention time of AHLs standards. We selected the ion m/z 102 for quantification on account of its specificity and better signal-to-noise ratio. The peak area calculation was performed by the extracted ion chromatograms. Results were normalized to the DMSO control for relative quantification.

Biofilm formation inhibition assay

Biofilms of P. aeruginosa PAO1 were cultivated in LB broth supplemented with or without derivatives in 24-well plates.⁵² After 24 h of static incubation, cultures and planktonic cells were removed, and sessile cells were stained with 0.05% crystal violet, and rinsed using distilled water. After dissolution with 95% ethanol, biofilm biomass was measured at OD₅₇₀. After cultivation, coverslips were washed with PBS, fixed with 2.5% glutaraldehyde, and dehydrated with ethanol. Samples were then freeze-dried, gold-coated, and detected with SEM (JSM6360, JEOL, Tokyo, Japan).

Mature biofilm eradication assay

For biofilm eradication assay,⁵³ briefly, biofilms were cultivated in LB broth in 24-well plates at 37 °C overnight. Then, the biofilms were washed with PBS, and then added to fresh LB broth and derivatives. After culture overnight again, the biofilm was washed with PBS, then fixed with methanol, stained with crystal violet, dissolved with ethanol, and eventually quantified by OD₅₇₀.

Virulence factor assays

Firstly, overnight P. aeruginosa PAO1 cultures were added to LB broth (1 : 100, v/v) provided with derivatives in the concentrations (6.25–25 μg mL⁻¹) cultivated at 37 °C and 180 rpm. DMSO and RSV (25 μg mL⁻¹) were served as the controls. After incubation, 1 mL of culture was centrifuged for 5 min at 4 °C. The supernatants were stored at −20 °C for further testing.

Pyocyanin content was determined according to the previously reported method with minor modifications.³⁷P. aeruginosa PAO1 was grown overnight in LB medium at 37 °C. The culture was diluted to OD₆₀₀ = 0.05 and subcultured for 5–7 h to reach the mid-log phase before being diluted to OD₆₀₀ = 0.05. Then test tubes containing 5 mL aliquot of culture and test compounds were grown for 18 h at 37 °C (200 rpm). To extract pyocyanin, the culture was centrifuged at 10000 rpm for 5 min, and the supernatant was collected and extracted with 3 mL of chloroform. Then, 1 mL of 0.2 M HCl was mixed with the chloroform layer, and the red upper aqueous phase was collected by centrifugation at 5000 rpm for 10 min. The absorbance of these solutions was measured at 520 nm. The percentage of pyocyanin production⁵⁴ was calculated as percentage of pyocyanin = (Abs_sample − Abs_blank)/(Abs_negative − Abs_blank) × 100%.

Rhamnolipids were assessed using the orcinol method.¹⁶ Briefly, 300 μL of culture supernatant was extracted twice with 600 μL of ethyl ether. The ether layer was evaporated at 35 °C under reduced pressure, and residuals were dissolved in 100 μL of deionized water. A total of 900 μL of orcinol solution (0.19% orcinol in 53% [v/v] H₂SO₄, Sigma-Aldrich, USA) was mixed with 100 μL of each sample. After 30 min of heating at 80 °C, the cooled samples were then determined at 421 nm.

For protease determination.⁵⁵ Briefly, 75 μL of culture supernatant was mixed with 125 μL of 2% azocasein buffered substrate in 1 M Tris–HCl (pH 8.0), and incubated at 37 °C for 15 min. After incubation, 600 μL of 10% trichloroacetic acid was added to each reaction tube, and after 30 min, the mixture was centrifuged. An aliquot of 700 μL of 1 M NaOH was added to the supernatant to terminate the reaction, and then determined by OD₄₄₀.

To measure EPS,¹²P. aeruginosa PAO1 culture was incubated at 37 °C, and 180 rpm for 24 h in 24-well plates. Then, biofilms were washed with distilled water, followed by the addition of 500 μL of 0.9% NaCl, 5% phenol, and 2.5 mL of 0.2% hydrazine sulfate. Then incubation for 1 h in the dark, then determined by OD₄₉₀.

For motility inhibition assays, swimming and swarming motilities were performed as previously described⁵⁶ with minor modifications. Briefly, 2 μL of overnight P. aeruginosa PAO1 culture (OD₆₂₀ = 0.5) was inoculated with derivatives at the center of the swimming agar (1% tryptone, 0.5% NaCl, 0.3% agar, pH 7.2) and swarming agar medium (1% tryptone, 0.5% NaCl, 0.5% glucose, 0.3% agar, pH 7.2), respectively. RSV and DMSO were used as controls. Plates were cultivated at 37 °C overnight, and migration was then recorded.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Briefly, 40 μL P. aeruginosa PAO1 of 24 h culture was added to 4 mL of fresh LB broth supplemented with DMSO or 25 μg mL⁻¹ of 32. Overnight, cells were harvested by centrifugation. Total RNA was isolated from the P. aeruginosa PAO1 pellet cells with an RNA extraction kit (Sangon Biotech, Shanghai, China). Isolated RNA was reversely transcribed into cDNA using RT6 cDNA synthesis kit (Sangon Biotech, Shanghai, China). The RT-qPCR reactions were accomplished using the LineGene 9600 Plus (Sangon Biotech, Shanghai, China). The rplU gene was used as the internal control.²¹ More details for the primers are shown in ESI.†

Statistical analysis

All assays were performed at least three times, and data were expressed as means ± standard deviation (SD). Graphs were constructed using Origin 8.6 software (OriginLab, Northampton, MA, USA). One-way analysis of variance (ANOVA) was performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) for comparing differences between groups, followed by the Tukey–Kramer test. A P value ≤ 0.05 was considered statistically significant.

Data availability

All the data can be found in the MS and ESI part, thank you! The protein datasets generated during the current study are available in the PDB database, were retrieved from the https://www.rcsb.org/. All data generated or analyzed during this study are included in this manuscript.

Author contributions

J-W and JY-Y conceived and designed research. JY-Y, WH-W and J-W conducted experiments. J-W and N-S contributed new reagents or analytical tools. P-D, N-S, J-W, and JY-Y drafted and prepared the manuscript. L-Y, AQ-J and B-W applied for funding and supervised research. All authors read and approved the manuscript.

Conflicts of interest

The authors declare no competing financial interest.