Antibiotics are widely used around the world for the treatment of infectious diseases. The hysterical use of antibiotics led to the emergence of resistant strains of almost all pathogenic bacteria [1]. The discovery of virulence-arresting drugs (VADs) targeting bacterial virulence, represents one of the advanced approachs for combating antimicrobial resistance (AMR) and bacterial infections. This highlights the urgent need for novel therapeutic strategies depending on neutralizing virulence factors rather than killing bacteria, thereby reducing selective pressure for resistance development [2].

Pseudomonas aeruginosa (P. aeruginosa or PA) is a frequent opportunistic Gram-negative bacteria in clinical settings that can cause a variety of lengthy-treat infections. The case is much more compilicated in patients with impaired immune systems, such as those with cancer, cystic fibrosis, HIV, burn victims, etc. [3]. WHO listed P. aeruginosa as the second most resistant bacteria after Acinetobacter baumannii which poses a considerable risk with about 81.7 % resistance rate. Every year, antimicrobial-resistant infections cause 700,000 to over 16 million fatalities around the world [4]. Quorum sensing (QS) inhibition is a promising strategy for reducing bacterial resistance and eliminating the expression of virulence factor genes linked to bacterial population density [5]. Quorum-sensing regulates various cellular processes such as pathogenic gene expression, toxin production, formation of biofilms, and the production of many virulence factors such as pyocyanin, elastase, and protease [6]. It controls genes expression by self-producing extracellular signaling molecules known as autoinducers (AIs) [7]. There are multiple approaches to block QS such as inhibiting the synthesis, inactivation or degradation of the AIs [8], or competing with the AIs for binding to their receptor [9]. Accordingly, it is likely that application of such strategy might contribute to treating P. aeruginosa infections.

P. aeruginosa has three quorum sensing systems; lasI/lasR, rhlI/rhlR, and Pseudomonas quinolone signal (Pqs) [10]. The lasI/lasR system relies on N-acyl-l-homoserine lactone (AHL) and N-3-oxododecanoyl-l-homoserine lactone (3-OC12-HSL) [11], while the rhlI/rhlR system develops N-butyryl-l-homoserine lactone (C4-HSL) as autoinducers [12]. The third system, (Pqs), employs several types of 2-alkyl quinolones (AQs), however, 2-nonyl-4-quinolone (NHQ) and 2-heptyl-4-quinolone (HHQ) are the most prevalent signal molecules [13]. The biosynthesis of NHQ and HHQ signal molecules requires the transcriptional regulator (PqsR), also known as multiple virulence factor regulator (MvfR). PqsR plays a vital role in Pqs and regulates the expression of rhlI and consequently controls many virulence functions such as hydrogen cyanide and pyocyanin production, bacterial motility, and biofilm formation. This signifies PqsR as an interesting target for developing novel quorum sensing inhibitors (QSIs) [5]. Fig. 1 illustrates the known PqsR agonists (NHQ and HHQ) and representative examples of the reported antagonists (I – VIII).

The proposed design strategy for development of small molecule antivirulence agents against P. aeruginosa quorum sensing is based primarily on analysis of the structural features of the known PqsR agonists (NHQ and HHQ) and antagonists (I – VIII), listed in Fig. 1.

The PqsR anatagonists (I – V) were developed based on isosteric replacement of the quinolone nucleus as well as modulation of the side chain of the native autoinducers NHQ and HHQ. Compound I is a member of the first series of quinazolinone derivatives PqsR competitive antagonists (IC50 = 5 μM), showed potent antibiofilm and antivirulence activity. This compound involves replacement of the quinolone nucleus of the autoinducer by quinazolinone, but retaining the alkyl side chain. the observed antagonist effect is attributed to stronger binding with PqsR co-inducer binding domain (PqsRCBD) than the native agonist NHQ [14]. Further modification of compound I by replacing the alkyl side chain with an ether linker to a substituted aryl moiety resulted in the hit antagonist II, which significantly inhibited the expression of P. aeruginosa PqsA promoter at IC50 = 3.2 μM. It also demonstrates decreased levels of pyocyanin, Pqs, and HHQ in PAO1-L, PA14 strains and PAK6085 clinical isolate. Additionally, this compound potentiated the effect of ciprofloxacin in the early stages of biofilm treatment [15].

A second approach involves isosteric replacement of the quinazolinone ring by benzimidazole, while keeping the hydroxyether or thioaminde linker to the aryl substituent. Compound III inhibited PqsR-controlled PqsA-lux transcriptional reporter fusion in P. aeruginosa at IC50 = 0.07 μM. Besides, it exhibited better efficacy against P. aeruginosa cystic fibrosis isolates with significant inhibition of virulence factor pyocyanin and 2-alkyl-4(1H)-quinolones [16]. Compound IV (M64) is the most effective PqsR inhibitor among the benzimidazole-based series [17]. It effectively reduces the level of pyocyanin at IC50 = 300 nM, suppresses acute and persistent/relapsing infections in mice, and was significantly effective against MDR isolates of P. aeruginosa [17,18]. Alternatively, a series of triazino-indole derivatives were synthesized and evaluated for their antiquorum sensing inhibitory activity. Compound V showed a potent inhibitory effect against both PAO1-L and PA14 strains, at IC50 = 0.25 ± 0.12 μM and IC50 = 0.34 ± 0.03 μM, respectively. It also markedly suppressed pyocyanin synthesis and Pqs signaling in both planktonic cultures and biofilms [19].

An alternative QS inhibition has been reported, based on structural modulation of the acyl homoserine lactone (AHL) autoinducer family. Several reviews reported numerous natural and synthetic furan and bromofuranon-based QSIs [20]. Notably, novel analogs of (5-oxo-2,5-dihydrofuran-3-yl)methyl alkanoates, e.g. compound VI, were found to efficiently inhibit Pseudomonal QS signaling and biofilm formation, achieving inhibition levels ranging from 80 % to 90 % [21]. Compound VII, a bromofuranone, showed activity against both planktonic and biofilm-forming cells [22]. Structure-activity relationship analysis of bromofuranons turned out that the presence of bromine is necessary for activity against persister cells and increases the susceptibility of PAO1 persister cells to ciprofloxacin at sub-minimal inhibitory doses (sub-MIC) [23]. Moreover, the bicyclic bromofuranone (VIII) was reported to inhibit the virulence factor elastase in P. aeruginosa [24]. This compound also demonstrated ability to suppress biofilm formation in both Escherichia coli and P. aeruginosa [25].

Considering these findings, we strive to design and synthesis series of hybrid molecules involving a benzofuran skeleton linked to an alkyl or aryl moiety through a thiosemicarbazides spacer, (Fig. 2), as potential QSIs targeting P. aeruginosa PqsR. The three structural components matched to the previously cited pharmacophoric features of PqsR antagonists. Firstly, the benzofyran or its 5-bromo congener considered as an isosteric alternate for the quinolone moiety in PqsR (NHQ and HHQ) quorum sensing system of P. aeruginosa. Secondly, the terminal alkyl/aryl moiety affords a bulky substituent favouring antagonistic rather than agonist effect. Finally, The thiosemicarbazide linker is a substitute for the aminoalkylether or the thioamide linkers in the reported antagonists (II – V).

Molecular modeling have become a leading strategy in drug discovery and development tactics [26]. Consequently, the proposed strategy is computationally confirmed through a molecular docking study, into the binding site of PqsR ligand-binding domain (PqsRLBD), as well as flexible alignment of the targetd benzofuran-based derivatives and the benzimidazole derivative IV (M64) as a validated anatagonist (Fig. 3). The declared target consists of two subdomains: pocket A (outer/superficial) and pocket B (deep pocket) connected by an antiparallel β-sheet forming a curved channel [14]. Fig. 3a shows the crystal structure of PqsRLBD co-crystalized with the antagonist IV (M64). Analysis of the interactions disclose fitting of the benzimidazole nucleus of M64 in pocket B, which is the same site of the quinolone ring of the native autoinducers, NHQ or HHQ. Additionally, The phenoxy group of M64 intercalates in pocket A through a hydrophobic interaction with Tyr258, aligning the side chain of NHQ. While the amidic carbonyl group of the linker binds through H-bonding with Gln194. [18]. The results provide an outstanding basis showing that the antagonistic activity is attributed to fitting into the curved channel and the active location of pockets A and B.

The flexible alignment study results demonstrate an alignment score S = −107.48 kcal/mol, indicating adepuate alignment both in terms of internal strain and overlap of molecular features. Fig. 3b shows the alignment of M64 with 2-(4-chlorophenylthiosemicarbazide)benzofuran, and allylthiosemicarbazide analogue of 5-bromobenzofuran series, as representative examples of the designed compounds. Further evidence supporting the postulated design is illustrated by the overlay of the mentioned compounds and M64 in the binding site of PqsR ligand-binding domain (PqsRLBD) as shown in Fig. 3c. The benzofuran nucleus aligned with the phenoxy group of M64 occupying pocket A. Meanwhile, the attached alkyl or aryl substituent on the thiosemicarbazide linker aligned with the nitrobenzimidazole moiety of M64 and accordingly bind to pocket B. The thiosemicarbazide linker, is a multiple hetero atom chain, which will attain H-bonding interactions to the respective amino acids in the antiparallel β-sheet curved channel of the binding domain.

Remarkably, the presence of a bromo substituent located at C-5 of the benzofuran ring, in the second series, enhances the affinity to the hydrophobic pocket A and attains extra binding interactions. Obviously, bromofuranones (VII and VIII) were reported as antiquorum sensing relevant to the autoinducers of the acyl homoserine lactones. Ultimately, the deliberate strategy reveals a unique and potent scaffold, that represents the designed compounds, (Fig. 2). The synthesized compounds will be primarily, evaluated for their antibacterial activity against P. aeruginosa, to assess the respective minimal inhibitory concentrations (MIC). The most active candidates were tested for their QS inhibitory ability, inhibition of biofilm formation, and reducing the levels of the virulence factors. Moreover, compounds that might exhibit promising in vitro activity potential will be tested in vivo for the ability to suppress the severity of P. aeruginosa infection.