2.1. Inactivating and modifying enzymes

Destruction or modification of the structure of antibiotics is one of the most common resistance mechanisms involving enzymes. Depending on the type of reaction they catalyze, the enzymes involved in this resistance mechanism are subdivided into hydrolases, transferases and oxidoreductases (Fig. 3).^9–12

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Fig. 3

The main classes of enzymes modifying antimicrobial drugs.

2.2. Mutation of the antibiotic target site

Since antimicrobial drugs have very specific targets, structural changes to those targets can prevent drug binding, rendering the drug ineffective. Through spontaneous mutations in the gene encoding antibacterial drug targets, bacteria have an evolutionary advantage that permit them to develop resistance to drugs. This mechanism of resistance development is quite common. For example, genetic changes impacting the active site of penicillin binding proteins (PBPs) can inhibit the binding of β-lactam drugs and provide resistance to multiple drugs within this class.¹³ This mechanism is very common among strains of Streptococcus pneumonia, which alter their own PBPs through genetic mechanisms.¹⁴

Similarly, the resistance of many bacterial pathogens to fluoroquinolone antibiotics (such as ciprofloxacin) is mediated by mutations in the ciprofloxacin-targeted DNA gyrase and DNA topoisomerase IV genes.¹⁵

2.3. The target site is changed

Although bacteria such as Streptococcus pneumoniae have mutated antibiotic targets, another similar mechanism for resistance is to obtain additional copies of a gene that encodes a protein that retains activity, but antibiotics cannot bind to it. This is how the pathogen Staphylococcus aureus becomes resistant to most β-lactam antibiotics (such as penicillin). Methicillin-resistant Staphylococcus aureus (MRSA) are resistant to β-lactam antibiotics, which is changed by acquiring additional penicillin binding protein 2 (target of β-lactam) making it resistant to antibiotics. In the presence of β-lactam antibiotics, this additional version called penicillin-binding protein 2a (PBP2a) that cannot function as a target for penicillin binding helping the bacteria to survive.^14,16

Furthermore, a resistance mechanism called target mimicry was discovered, which involves the production of proteins that bind and sequester drugs, thus preventing the drug from binding to its target. For example, Mycobacterium tuberculosis produces a protein that has a regular pentapeptide repeating sequence that appears to mimic the structure of DNA. The protein binds to fluoroquinolones, isolates them, and prevents it from binding to DNA, making Mycobacterium tuberculosis resistant to fluoroquinolones. Proteins that mimic the A site of bacterial ribosomes have also been found to play a role in aminoglycoside resistance.¹⁷

2.4. Overproduction of the target

Bacteria can also overproduce antibiotic targets, which means that the protein targets of antibiotics are excessive compared to the antibiotics themselves. This means that, in the presence of antibiotics, there is enough target protein to continue to function in the cell; this is the mechanism by which E. coli and Haemophilus influenzae are resistant to trimethoprim. Overexpression is sometimes found to combine with mutations that reduce the ability of the antibiotic to bind to its target.¹⁸

4.1. Targeting cysteine biosynthesis

The rationale behind the exploitation of amino acid biosynthesis as a target for antimicrobial adjuvant development is the observation that some pathogens spend part of their life cycle in extremely harsh conditions, such as macrophages or the gastric mucosa, where survival and proliferation require powerful adaptation mechanisms involving metabolic pathways.^38,39 In this case, interference with pathogen adaptation strategies can lead to increased sensitivity to antibiotics. Among potential new drugs targets is an enzyme involved in cysteine biosynthesis.

It has been observed that the importance of cysteine biosynthesis enzymes differs during the life cycle of pathogens: their activity can be dispensable during growth in vitro or acute infections but becomes indispensable during the persistence phase.^40,41 Compared with traditional antibiotics, molecules developed for cysteine biosynthesis and other biosynthetic pathways may have the potential advantage of being more effective against persistence in the host, helping to prevent the development of drug resistance during the clinical incubation period.^42,43

Many studies on the response of microorganisms to environmental stress (such as lack of nutrients, hypoxia, and oxidative stress) have shown that many genes of the cysteine regulator have a positive regulatory effect.⁴⁴

Therefore, the role of cysteine biosynthesis in the development of antibiotic resistance has recently been pointed out.^45,46 The study of mutants lacking the cysteine biosynthesis pathway in Salmonella typhimurium concluded that, due to inhibition of cysteine biosynthesis, unpaired oxidative stress can lead to decrease in the antibiotic resistance, in both vegetative and swarm cell populations. Antibiotic-induced oxidative stress has been widely recognized as a general mechanism of action for many antibacterial drugs,⁴⁷ which may explain the reduction in the rate of resistance observed in bacteria with impaired cysteine biosynthesis. These findings indicate that inhibitors of cysteine biosynthesis may therefore improve the efficacy of antibiotic treatment and reduce the spread of resistance.⁴⁰

Most bacteria and plants carry out cysteine biosynthesis through the reductive sulfate assimilation pathway (RSAP), which is a multi-step sulfate reduction process that culminates in the incorporation of bisulfide into activated form of serine to obtain cysteine⁴⁸ (Fig. 5).

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Fig. 5

Overview of sulfur assimilation and related biochemical pathways.

RSAP starts with the transport of sulfate in the cell and then reducing it to disulfide. This process consumes a lot of energy and is suitable for cellular needs. Bacteria find sulfur in the environment in the form of sulfate and actively transport it across the plasma membrane. After sulfate is reduced to disulfide, the latter is incorporated into cysteine by a member of a large enzyme family called cysteine synthase complex (CSC). The CSC is composed of serine acetyl transferase (SAT, EC 2.3.1.30) and O-acetylserine sulfhydralase (OASS; EC 2.5.1.47), which catalyzes the final step of cysteine biosynthesis.⁴⁹

SAT has a catalytic activity in this complex where the acetyl group of acetyl-CoA is transferred to the hydroxyl group of serine to form O-acetylserine (OAS) and CoA. OAS is unstable and will be spontaneously converted to N-acetyl serine (NAS), which is a natural inducer of cysteine regulation signaling. In fact, high concentrations of bisulfide stabilize cysteine synthase complex (CSC), whereas OAS promotes its dissociation; on the other hand, increasing levels of cysteine can inhibit SAT catalytic activity by feedback control, triggered by binding to its active site.⁵⁰

Depending on the organism and the growth conditions, the last step of cysteine biosynthesis is catalyzed by different thiol lyases which share high homology, but they also show some functions and structures difference. The first identified OASS subtype (OASS-A) was first isolated and identified from Salmonella typhimurium.⁵¹ Subsequently, it was observed how many pathogens have two isoforms of the enzyme: O-acetylserine sulfhydralase (OASS-A encoded by cysK) and O-phosphoserine sulfhydrylase (OASS-B encoded by cysM, EC 2.5.1.65). Which differ in functional and structural properties. In Salmonella typhimurium, the OASS-A and B isomers use O-acetylserine (OAS) as the substrate and S²⁻ as the main source of sulfur. The expression of OASS isozymes depends on environmental conditions (OASS-A under aerobic conditions, OASS-B under anaerobic conditions).⁴⁹ Both are Pyridoxal phosphate PLP-dependent enzymes, belonging to the type II subfamily of the PLP superfamily. They also have the same Bi–Bi ping pong-like reaction mechanism, in which the first half of the reaction is through β-elimination of the β-substituted l-serine external aldimine to generate the intermediate Schiff base of α-amino acrylate. In the second half-reaction, the sulfur source attacks the α-amino acrylate to form l-Cys.⁵² Since its discovery, OASS has been thought to have multiple functions, such as swarming motility in Proteus mirabilis,⁵³ the transcriptional regulation of certain bacteria and nematodes,⁵⁴ and the activation of toxins in Escherichia coli,⁵⁵ and antibiotic resistance in Salmonella typhimurium.^45,46

Based on these facts and considering the need to inhibit both enzymes to prevent the biosynthesis of l-cysteine,⁵⁰ the identification of small molecules that can inhibit these two subtypes of OASS is of great importance for research.

In 2016, Pieroni et al.,^56,57 starting from the evidence that SAT competitively inhibits OASS-A, based on the structural characteristics of the OASS-SAT interaction, a rational design of the first sulfydrylase inhibitor was developed. Taking into account their previous research data,⁵⁸ combined with computational methods^59,60 and spectroscopic methods, such as saturation transfer difference (STD) and nuclear magnetic resonance (NMR), rational design and synthesis a series of 2-phenylcyclopropanecarboxylic acid derivatives targeting two isoforms of St-OASS.⁶¹ In fact, they demonstrated that compounds that bind to the enzyme's active site effectively inhibit the OASS-A and B isoforms by competing with SAT. These findings provide a proof of principle and support the idea that it is possible to develop molecules that can inhibit both OASS-A and B enzymes and pave the way for the development of pharmacological tools to overcome bacterial virulence and resistance (Fig. 6).

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Fig. 6

A schematic diagram of the rational design of the first sulfhydrylase inhibitor based on the structural characteristics of the OASS-SAT interaction.

In 2018, a small series of 1,2-substituted-1H-benzo[d]imidazole derivatives was synthesized, which have a submicromolar range for all forms of leishmaniasis (K_i = 0.15–0.69 μM). The affinity for Leishmania, Mexico CPB2. 8ΔCTE, one of the most promising targets for anti-detox drug design. These compounds confirmed their in vitro activity against the intracellular amoeba of Leishmania infantum. Although they have a certain degree of cytotoxicity (CC₅₀ = 8.0 μM in PMM, CC₅₀ = 32.0 μM in MCR-5), compound 1d (IC₅₀ = 6.8 μM) gave the best results. The molecular coupling and prediction studies of ADME-Tox properties were carried out on the computer to verify the expected target interaction hypothesis and evaluate the drug similarity of these derivatives⁶² (Fig. 7).

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Fig. 7

Structure of 1,2-substituted-1H-benzo[d]imidazole derivatives 1 and compounds 2a,b.

Besides, type I signal peptidase plays an important role in bacterial viability, and it is a promising but rarely developed target for antibacterial drugs. In view of the increasing rate of antimicrobial resistance, a new macrocyclic lipopeptide that combines P2 and P10 with a borate warhead was developed and found to inhibit E. coli type I signal peptidase (EcLepB) and show good antibacterial activity. The structural modification of the macrocycle, peptide sequence and lipophilic tail allowed to obtain fourteen new macrocyclic boronic esters. It can be shown that macrocyclization is well tolerated in terms of EcLepB inhibition and antibacterial activity. Among the synthesized macrocyclic compounds, effective enzyme inhibitors were also identified in the low nanomolar range (for example, compound 2a, EcLepB IC₅₀ = 29 nM), and they also showed good antibacterial activity (for example, compound 2b, Escherichia coli WT MIC = 16 mg mL⁻¹) (Fig. 7). The unique macrocyclic boronic ester described in this article is based on the previously announced EcLepB linear lipopeptide inhibitor, which attempts to solve the problem of cytotoxicity and hemolysis. This article revealed that the structural changes of the macrocycle can affect the cytotoxicity and hemolytic activity, which indicates that the P2 to P1′ linker provides a way to optimize off-target effects. However, for the current compounds, it cannot be distinguished between antibacterial activity and cytotoxicity.⁶³

4.2. Targeting quorum sensing

In many bacterial pathogens, population growth is controlled by quorum sensing (QS), which is an intercellular communication mechanism that controls phenotypic manifestations (such as virulence).⁶⁴ Bacteria use this system to communicate with each other in a given population.⁶⁵ It consists of a signal molecule (called autoinducer) continuously secreted by each type of bacteria, and when the defined concentration of this molecular messenger reaches a threshold, it will activate the QS control process. Several aspects of virulence are affected by QS, so that, the identification of small molecules that can interfere with this cell–cell mechanism is currently a field of great interest.

The most frequently studied QS autoinducers are N-acyl homoserine lactone (AHL) and peptides. AHL is produced by Gram-negative bacteria while peptides are formed by Gram-positive bacteria.⁶⁶ AHL, also known as autoinducer-1 (AI-1), consists of a homoserine lactone ring with additional fatty acid side chains and passively diffuse into and out of Gram-negative bacterial cell.⁶⁷ Although the AHL signal has traditionally been considered as intraspecific communication, studies have shown that AHL can also be used to detect potential environmental competitors.⁶⁸

Peptides (auto-inducible peptides or quorum sensing peptides) are synthesized on the ribosome of Gram-positive bacteria and exhibit post-translational modifications in terms of their stability and functionality.^69,70 When the extracellular concentration of signal peptide accumulates to a trough value, it will be detected by the protein (histidine-sensing kinase).^66,67

Moreover, autoinducer 2 (AI-2) is another type of a signaling molecule that can be found in both Gram-positive and Gram-negative bacteria. AI-2 is widely used for interspecies communication, which is why it is considered as a universal communication signal between different bacterial species.⁷¹ Pseudomonas Quinolone Signal (PQS) autoinducer, also known as quinolone, was identified in Pseudomonas aeruginosa, produced by the protein encoded by pqsABCDH gene, and together with other AHL autoinducers, it controls biofilm formation and the production of virulence factors, Such as lecithin.^72,73 Recently, auto-inducible IQS produced by the protein encoded by ambBCDE has been described in Pseudomonas aeruginosa. This molecule controls the expression of genes related to the production of pyocyanin, rhamnolipid, and elastase.⁷⁴ Thus, QS has been identified in a wide range of Gram-negative and Gram-positive bacteria and is important in many disease-causing species as it has been shown to play a role in biofilm formation.⁷⁵

Fig. 8 shows some autoinducers as AI-1, autoinducing peptide (AIP-1), PQS, γ-butyrolactone and CAI-1 along with their chemical structure. Whereas Fig. 9 illustrates some of the chiral signaling molecules belonging to the acyl-homoserine lactone (AHSLs) group (3–5) used by Gram-negative bacteria, furanosyl borate diester (6) used by Gram-positive and Gram-negative bacteria, and (S)-3-hydroxytridecan-4-one) (7) found in Vibrio species.⁷¹

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Fig. 8

Structures of selected quorum sensing auto-inducers.

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Fig. 9

Some structures of chiral quorum sensing signaling molecules in bacteria.

Acinetobacter baumannii,⁷⁶Pseudomonas aeruginosa^77,78 and Escherichia coli79 are examples of Gram-negative bacteria that have QS systems. QS systems from other Gram-negative bacteria generate signal molecules through LuxI homologous protein synthase. These proteins produce specific AHL for each bacterial species.⁶⁹ The change of AHL depends on the length of the carbon chain.⁷¹Escherichia coli and Salmonella do not have LuxI protein and therefore do not synthesize AHL.⁸⁰ However, both synthesize the SdiA protein that recognizes and binds to AHL produced by other bacteria.⁷⁵Pseudomonas aeruginosa has four known QS systems: las, rhl, PQS and integrated QS (IQS). The first two are mediated by homoserine N-acyl lactone (AHL) autoinducers, the third is mediated by quinolone (PQS signal), and the last is mediated by IQS signal^74,81 (Fig. 10).⁸²

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Fig. 10

A hypothetical model explaining transcriptional regulation of QSI in P. aeruginosa.⁸²

The innate receptors of these auto inducers belong to the LuxR type which act as transcriptional regulators upon activation by their native agonists. In P. aeruginosa, the so-called las and rhl systems have been identified to be the key AHL-based QS systems. The native ligands of the involved cytoplasmic receptors LasR and RhlR are N-(3-oxo-dodecanoyl)-l-homoserine lactone (3-oxo-C₁₂-HSL or OdDHL) and N-butanoyl-l-homoserine lactone (C₄-HSL or BHL), respectively (Fig. 10). Due to these similarities, bacteria can spread between species through this mechanism, which is useful for bacterial coinfection. Generally, in a species, there are multiple communication systems that are interconnected and affect each other.⁸³ In the literature, there are reports describing various quorum sensing systems.^84,85

This mechanism of QS can be found in different bacteria such as Staphylococcus aureus,⁸⁶Bacillus subtilis,⁸⁷Clostridium botulinum,⁸⁸ and S. pneumoniae,⁸⁹ and can regulate different phenotypes.

Mechanisms of QS inhibition in controlling bacterial biofilm formation are shown in Fig. 11: (1) inhibit AIs synthesis; (2) degrade or inactivate AIs by AHL-lactonases, oxidoreductases, antibodies, etc.; (3) interfere with the signal receptors using AI antagonists; (4) interfere with the response regulators thus disturbing signaling cascade; (5) reduce the extracellular AIs accumulation by inhibiting AIs efflux hence inhibited cell-to-cell signaling.^82,90

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Fig. 11

The mechanisms of QS inhibiting agents in controlling bacterial biofilm formation.

Many researchers have focused on the development of LasR antagonists to disrupt the AHL signal in Pseudomonas aeruginosa. The ideal cellular effect of QSI targeting LasR and/or RhlR would be the reduction in the production of virulence factors (such as elastase, hydrogen cyanide, pyocyanin, pyoverdin, rhamnolipid or alkaline protease) as well as the attenuation of biofilm formation.^82–90 The following section provides a detailed but not exhaustive overview of the structures covered by the synthetic QSI that interferes with AHL signals. Generally, compounds targeting LasR and Rh1R can be divided into two categories: structural mimics of AHL and structurally unrelated substances.^84,85 Many synthetic agonists and antagonists of the LuxR-like receptor have structural characteristics that are very similar to natural ligands, or are directly synthesized derivatives thereof (Fig. 12 and ​and13).13). Obviously, the AHL scaffold can be divided into two parts. The head group is composed of a 5-membered homoserine lactone part, while the tail region contains linear N-acyl residue of different lengths. The amide-based linker between the two fragments facilitates a modular approach to direct synthesis and derivatization. Many reports in the literature dealing with synthetic AHL analogs focus on the structural modification of one of these two parts while keeping the other part of the molecule unchanged. Also, unnatural head and tail module combinations are described.^91–103

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Fig. 12

Some reported substituents as head group replacements in AHL to obtain QSI.

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Fig. 13

A diagram of some substituents as tail section replacements to obtain QSI.

The combination of unnatural favorable head and tail groups with each other can produce quite unexpected results. Spring and colleagues have shown that many of these “chimeric” compounds are low or essentially inactive.⁹⁸ Therefore, the conservation of natural 3-oxododecanoyl chains in antagonists with unnatural head groups may be mandatory for strong QS inhibition. Fig. 14 shows some promising AHL-simulated LasR and RhlR antagonists and their effects on Pseudomonas aeruginosa.^{92–94,98–100,103–107}

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Fig. 14

Some LasR and/or RhlR antagonists with reported effects on P. aeruginosa cells.

Various methods of developing quorum sensing inhibitors started by mimicking the chemical structure of quorum sensing signaling molecules. Pseudomonas aeruginosa can be considered as a potential target. The biosynthesis of PQS signal requires the action of a set of enzymes PqsABCDEH and its autocatalytic receptor PqsR (MvfR). Later, many compounds were active in infection models, such as compound 20 which inhibits QS system and biofilm formation^108,109 (Fig. 15).

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Fig. 15

Structure of compounds 20 and 21a–c.

In 2018, Srinivasarao et al.¹¹⁰ designed and synthesized 2-phenylindole-amide-triazole and salicyclic acid-triazole analogues (21a–c). These compounds were screened for in vitro quorum sensing inhibitory (QSI) activity against Pseudomonas aeruginosa. The QSI activity was determined in the LasR expressing P. aeruginosa MH602 reporter strain by measuring green fluorescent protein (GFP) production. 4-(1-heptyl-1H-1,2,3-triazol-4-yl)-N-(2-phenyl-1H-indol-3-yl)butanamide (21a) and 4-(1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)-N-(2-phenyl-1H-indol-3-yl)butanamide (21b) exhibited promising QSI activity with 58.89 and 54.34% at 250 μM, respectively. 1,2,3-Triazole based salicylic acid derivatives exhibited moderate to good activity that 2-hydroxy-4-(1-phenyl-1H-1,2,3- triazol-4-yl)benzoic acid (21c) was the most promising QS inhibitor with 40.28% inhibition at 250 μM (Fig. 15).

In 2018, Onem et al.¹¹¹ studied the quorum sensing effect of synthetic benzimidazole derivatives (N-acyl homoserine lactone analogs). In Pseudomonas aeruginosa PA01, the anti-virulence ability of all synthetic compounds was investigated using different tests as elastase, pyocyanin, and group movement. Thus, 1,3-dihydro-2H-benzimidazol-2-one (22a), 5-methyl-1,3-dihydro-2H-benzimidazol-2-one (22b) and 1,3-diacetyl-1,3-dihydro-2H-benzimidazol-2-one (22c) were found to have anti-QS activity and a significant inhibitory effect on elastase, pyocyanin production and mass movement. In summary, the results indicated that synthetic benzimidazole derivatives can inhibit the spread of bacteria in Pseudomonas aeruginosa, which is essential for infection, and the role of these molecules in other bacteria should be further studied (Fig. 16).

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Fig. 16

Structure of compounds 22a–c and 23a–d.

In 2019, a pyrazolo[3,4-b]pyridine analogs were synthesized, and antibacterial tests were performed on Gram-negative bacteria, Gram-positive bacteria and selected fungi. Compound 23a showed broad spectrum and effective antimicrobial activity. In addition, 23b and 23c showed significant broad-spectrum antibacterial activity. The anti-quorum detection activity of the new members was tested on C. violaceum, where 23c showed high efficacy (pigment inhibition diameter 17 nm), while 23a and 23d revealed moderate efficacy (pigment inhibition diameter 10–15 nm)¹¹² (Fig. 16).

In 2019, four series of l-homoserine lactone analogs were designed, synthesized and their inhibitory activity against Pseudomonas aeruginosa QS was evaluated. The results showed that compounds 24a, 24b of series I and 24c, 24d of series II represent l-homoserine lactone analogs with benzothiazolyl and 4-chlorophenyl substituents, respectively, and have a high inhibitory activity on CV026 and PAO1. Furthermore, most of the compounds of series III with side chains containing phenylurea and dithiocarbamate groups exhibited significant inhibition of QS at CV026 and PAO1. The results of the mechanism of action study confirmed that the analogs 24a, 24b, 24c, 24d, 24f and 24g inhibited the QS system of Pseudomonas aeruginosa by selectively reducing the expression of virulence factors, especially the biofilm formed by PAO1 by 28.6, 38.0, 34.1, 35.3, 36.2 and 40.3%, respectively. The activity of compound 24f is better than all other analogs, and the molecular coupling results showed that compound 24f competes with OdDHL for binding to LasR. This compound inhibited the expression of las system genes, PQS system genes and rhl system related genes, thereby inhibiting the production of virulence factors and the formation of PAO1 biofilms¹¹³ (Fig. 17).

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Fig. 17

Structure of compounds 24a–g and 25a–d.

In 2019, a series of 2-aminobenzimidazoles based on 1,2,3-triazole was synthesized, and the in vitro quorum sensing inhibitory activity against Pseudomonas aeruginosa was evaluated. By measuring the production of green fluorescent protein, the quorum sensing inhibitory activity was determined in the LasR-expressing Pseudomonas aeruginosa reporter strain MH602. The synthesized compound, N-(1H-benzo[d]imidazol-2-yl)-2-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)acetamide (25a) showed a good quorum sensing inhibitory activity of 64.99% at 250 mM. Whereas, N-(1H-benzo[d]imidazol-2-yl)-2-(4-(4-nitrophenyl)-1H-1,2,3-triazol-1-yl)acetamide (25b) was the most promising quorum inhibitor at 250, 125, and 62.5 mM, and the inhibitory rates were 68.23, 67.10, and 63.67%, respectively. Moreover, the trifluoromethyl phenyl analogs 25c and 25d also showed a quorum sensing inhibition of 64.25% and 65.80% at 250 mM, respectively. Compound 25b was the most active quorum sensing inhibitor and revealed low cytotoxicity to normal human embryonic kidney cell lines at the concentrations tested (25, 50 and 100 mM)¹¹⁴ (Fig. 17).

In 2020, Hossain et al.¹¹⁵ designed, synthesized and evaluated some compounds capable of reducing the virulence of Pseudomonas aeruginosa. Compounds 26a, 26b and 26c exhibited the best activity, where compounds 26a and 26b represented the new QSI scaffolds. As anti-virulence agents, these compounds have been shown to reduce the production of all tested PA virulence factors: biofilm, pyocyanin, and rhamnolipids in the range of 40–60%. Furthermore, these compounds have been found to reduce the pro-virulence of ciprofloxacin at sub-inhibitory concentrations (Fig. 18).

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Fig. 18

Structure of compounds 26a–c, 27 and 28.

In 2020, Sabir et al.¹¹⁶ designed and synthesized fourteen anthranilyl-AMP mimics, which contained triazole linkers as potential inhibitors of Pseudomonas quinolone biosynthesis. However, with the exception of deoxycytidine analogs (27), most of these analogs showed no PQS inhibitory activity against the Pseudomonas aeruginosa reporter chain, which inhibited 30% of the PQS activity at 125 μM. Interestingly, the biofilm morphology observed by these compounds under the microscope changes significantly, indicating that they can inhibit PQS or other signaling systems that cause bacterial aggregation. Therefore, future efforts should be focused on modifying these analogs to increase penetration of bacterial cells.

In 2020, twenty-two quorum sensing inhibitors were synthesized, mimicking the structure of autoinducers of Acinetobacter, and 28 of them inhibited biofilms by up to 34%. By inhibiting extracellular polysaccharides and synergistic effects with gentamicin sulfate, the biofilm inhibition effect is further proved¹¹⁷ (Fig. 18).

In 2020, a series of 3-amino-2-oxazolidinone derivatives was designed and synthesized. C. violaceum CV026 was used as the reporter strain to evaluate the inhibitory activity of new oxazolidinone compounds on QS. Thirteen compounds showed good activity (IC₅₀ range 3.69–63.58 mM), where compound 29 inhibition of biofilm formation was the most significant (IC₅₀ = 3.686 ± 0.5790 mM). In vitro, compound 29 significantly inhibited PAO1 biofilm formation (range 42.98–7.67%), virulence factor production (pyocyanin, elastase, rhamnolipid, and protease), and bacterial movement. Furthermore, the combination of compound 29 and antibiotics (meropenem trihydrate) can significantly improve the antibiotic sensitivity of PAO1 cells of the Pseudomonas aeruginosa biofilm. In vivo, compound 29 significantly prolonged the lifespan of wild-type C. elegans N2 infected by Pseudomonas aeruginosa PAO1. In summary, compound 29 can be considered as a drug candidate for drug-resistant Pseudomonas aeruginosa PAO1, providing a tool for finding new antibacterial drug¹¹⁸ (Fig. 19).

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Fig. 19

Structure of compounds 29 and 30a–f.

In 2021, the 1,2,4-triazole derivatives were designed, synthesized and selected for in vitro evaluation of antimicrobial activity and anti-QS activity. Out of fourteen derivatives, compound 30f selectively possessed antibacterial activity against C. violaceum. Further derivatives that possessed an electron withdrawing group and halogen atoms in N-phenylacetamide moiety were moderately active against Chromobacterium violaceum and Xanthomonas campestris pv. Campestris (Xcc). After observing the reduction of violacein production through plate assay, compounds 30a, 30b, 30c, 30d and 30e were subjected to quantification of quorum sensing inhibition. Compounds with the electron-withdrawing group in N-phenylacetamide moiety showed admirable activity with >80% inhibition of violacein. Mainly compound 30b which had no effect on the growth of bacteria was identified as excellent QSI which could be a lead compound for further development¹¹⁹ (Fig. 19).