Microbial infections are one of the leading causes of death globally. The risks of such infections have become particularly pronounced with the emergence of multidrug-resistant, pathogenic strains of common bacteria [1]. Pseudomonas aeruginosa, an aerobic Gram-negative bacterium, is the most common pathogen in hospital wards globally [2]. Infection with antibiotic-resistant strains of P. aeruginosa can lead to chronic infection in the lungs of cystic fibrosis (CF) patients, the destruction of the lung epithelium, and death [3]. The virulence factors of P. aeruginosa include effector proteins, extracellular proteases, small molecule toxins, adhesins, extracellular polysaccharides, and lipopolysaccharides. In recent decades, P. aeruginosa has become resistant to a large number of antibiotics (e.g., fluoroquinolones, aminoglycosides, carbapenems) and even third-generation cephalosporins [4]. Alarmingly, the number of pan-resistant specimens cannot be treated with any existing anti-P. aeruginosa antibiotic in the clinic is increasing.

Quorum sensing is a bacterial intraspecies and interspecies signaling mechanism that regulates bacterial lifecycle activities by transmitting signals through synthesizing, releasing, and receiving autoinducers (AIs). Bacteria sense the density of surrounding populations through AIs and regulate gene expression through the QS systems [5]. Depending on the signaling molecules in use, QS systems can be classified into several types: N-acyl-homoserine lactones (AHLs), autoinducer peptides (AIPs), and autoinducer factor-2 (AI-2) [6]. The P. aeruginosa QS systems encompass three signaling mechanisms: las, rhl, and pqs. The las and rhl systems employ signaling molecules with high serine endolipids. The las system consists of the LasI protease, which produces the signaling molecule N-3-oxo-dodecanoyl-l-homoserine lactone (3-oxo-C12-HSL/OdDHL), and the LasR protease, which acts as a receptor protein [7]. The rhl system consists of the RhlI protease, the RhlR protein receptor, and the signaling molecule N-butyryl-l-homoserine lactone (C4-HSL/BHL) [8]. The pqs system employs 2-heptyl-3-hydroxy-4-quinolone as a signaling molecule. Mutual regulation between these signaling systems is known, with studies demonstrating interactions between the pqs and the las and rhl systems [9]. As such, these three interconnected signaling systems of P. aeruginosa modulate each other's activities, controlling the production of virulence factors, biofilm formation, and bacterial motility [5]. This ability to regulate virulence factors and biofilm formation makes the QS systems a relevant target in designing new compounds to address P. aeruginosa antibiotic resistance [10]. Indeed, prior studies have demonstrated that small molecules targeting the QS systems can reduce the virulence of P. aeruginosa [11,12].

Pathogenic, antibiotic-resistant bacteria have been observed with increasing frequency over the past few decades and represent an increasing threat to human health. In addition to enhancing the ability of bacteria to acquire nutrients and construct more comfortable ecological niches, QS systems also enhance bacterial defenses against eukaryotic hosts, competing bacteria, and environmental stresses. Several molecules that act as QSIs have been isolated or synthesized. These compounds effectively inhibit bacterial growth, virulence mechanisms, and biofilm formation across various environments [13,14]. Therefore, QS inhibitors have received considerable attention in physiological and clinical studies as potential leads for developing antibacterial therapies [15,16]. Furthermore, with the development of new technologies such as proteomics and genomics, it is now possible to elucidate the genotypes and phenotypes associated with QS and thus enhance our understanding of the mechanisms by which QS pathways are activated or repressed.

Amide bonds are widely found in nature and have essential applications across science owing to their unique chemical and biological properties. For example, amide bonds are the critical building blocks that establish the primary structure of proteins and also feature among many biologically active molecules as marketed drugs or clinical candidates (Fig. 1). Drugs containing amide structures serve many different therapeutic areas, including antibacterial, antioxidant, antidiabetic, and anticancer indications [[16], [17], [18], [19]]. QS signal is a transduction molecule containing an amide bond ubiquitous in the P. aeruginosa QS systems. It controls the expression of several virulence factors and biofilm formation in P. aeruginosa. To date, most of the reported QS inhibitors mimic QS signal molecules. These compounds are usually obtained by modifying the head group and tail group of QS signal molecules. Sanshools are N-alkyl-substituted amides extracted from the spice-producing Zanthoxylum species. Sanshools are widely used in food, medicine, and cosmetics. Their various biological properties, which include antitumor, anesthetic, and antibacterial activities, have drawn the attention of multiple researchers [[20], [21], [22]]. Therefore, we designed and synthesized a series of amide bond-containing derivatives inspired by the structures of QS signal and sanshool molecules. Various saturated and unsaturated fatty acids were condensed with amines derived from the sanshool skeleton, and the resultant derivatives were evaluated for inhibitory activity against the P. aeruginosa QS systems (Fig. 2). Unnatural QS signal derivatives comprise the most extensively studied class of synthetic QSIs. However, new classes of compounds structurally distinct from natural QS signal derivatives need to be explored to map structure-activity relationships more comprehensively and provide optimal candidate compounds. In this paper, we report a series of novel amides synthesized as potential QSIs from various amines and assay their activity via an improved screening methodology. These compounds were found to suppress the expression of the lasB, rhlA, and pqsA genes based on the expression level of reporter compounds of the QS systems in P. aeruginosa PAO1. To our knowledge, QSIs based on this structural motif have never been reported for P. aeruginosa. To identify potential QSI candidates, we measured the effect of the most promising compounds on the expression of QS-related virulence phenotypes in PAO1, including swarming motility and elastase, rhamnolipid, pyocyanin, and pyoverdine secretion. The compounds’ mechanisms of action were evaluated using RT-qPCR, SPR and molecular docking. In addition, we performed infection experiments using a Galleria mellonella larval assay to assess the biological activities of the compounds in vivo. These results will enable a greater understanding of the mechanism of action of novel QS inhibitors (QSIs) and the role of the QS system and its signaling molecules in bacterial pathogenicity.