1. Introduction
The discovery of antibiotics during the twentieth century is considered one of the most important achievements in the history of medicine, having saved humans from a large number of life-threatening and debilitating diseases [
1,
2]. Although antibiotics have proven to be powerful drugs for the control of infectious diseases, their extensive and unrestricted use over the last century has imposed selective pressure upon bacteria, leading to the development of resistance. According to the World Health Organization (WHO), antimicrobial resistance is a worldwide problem which is considered a major threat to the treatment of infectious diseases [
3]. In addition to this serious problem, the premise that bacteria in biofilms are highly resistant to antimicrobials (100–1000 times more) when compared to their planktonic counterparts presents another obstacle towards the treatment of bacterial infections [
4,
5]. For some bacteria, working together as a group provides a means to build a defense that is impossible to achieve by planktonic cells [
2]. Hence, these observations highlight a strong need to develop therapies that can provide sustainable and long-term effectiveness against bacterial biofilms [
2,
6].
Typically, therapies with antibiotics are meant to affect bacterial viability, placing a strong selective pressure on bacteria to develop resistance mechanisms. For that reason, much research has been conducted in order to break out of this vicious cycle by interfering with bacterial systems responsible for pathogenicity/virulence [
7]. In this context, attention has been focused on strategies for interfering with the quorum sensing (QS) systems of pathogenic bacteria, in order to target their pathogenicity/virulence and to develop new anti-infective therapies. QS is an intercellular communication system mediated by diffusible chemical signal molecules termed autoinducers (AIs) that control gene expression patterns and therefore allows bacteria to synchronize their behavior [
8]. In many cases, the responses prompted by QS signals contribute directly to pathogenesis through the production of virulence determinants, such as toxins and proteases. Additionally, QS can contribute to behaviors such as biofilm development that enable bacteria to acquire resistance against antimicrobial compounds [
2]. If these efforts to coordinate bacteria behavior are blocked, it is possible that bacterial adaptability will be reduced, facilitating the host immune system to combat the infection and thus reducing the strong selective pressure imposed by conventional antibiotics [
9]. Moreover, bacteria will be less able to form organized microbial communities that promote pathogenesis and resistance, such as biofilms [
2]. QS inhibitors (QSI) can also improve therapy with antibiotics increasing their effectiveness, favoring the use of lower doses and avoiding the indiscriminate use of broad-spectrum antibiotics [
10].
The relationships between QS, virulence regulation and biofilm formation have been most extensively studied in
Pseudomonas aeruginosa. Therefore, it is not surprising that most of the research on QSI has been centered on this bacterium as a model system [
9].
P. aeruginosa is a Gram-negative opportunistic pathogen associated with biofilm-related nosocomial infections such as ventilator-associated pneumonia and chronic lung infection in cystic fibrosis patients [
11]. Furthermore, it is associated with a high incidence of antibiotic resistance and biofilm formation [
12].
P. aeruginosa employs at least four different QS circuits to regulate the production of virulence factors and promote biofilm development/maturation, namely the LasRI and RhlRI (LuxRI-type systems), the
Pseudomonas quinolone signal (Pqs), and the Integrated Quorum Sensing Signal (IQS) [
11]. QS genes function in a hierarchical manner with the prominent LasRI system controlling the activity of RhlRI circuit and subsequently the Pqs. The IQS is also strongly controlled by LasRI under rich medium conditions [
13]. LasRI and RhlRI systems comprises a transcriptional regulator (LasR and Rh1R, respectively) and its cognate
N-acyl homoserine lactone (AHL) signal (
N-(3-oxododecanoyl)-
l-homoserine lactone (3-oxo-C12-HSL) and
N-butyryl-
l-homoserine lactone (C4-HSL), respectively), which is synthesized by the AHL synthase (LasI or RhlI, respectively) [
14,
15]. These systems are responsible for the regulation of virulence factors production, such as protease, exotoxin A, siderophores and pyocyanin, among others. Additionally, the AHL-based QS system triggers a third
P. aeruginosa quinolone signaling system by producing the AI 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS). This system regulates the expression of virulence factors, biofilm formation, and bacterial motility [
12]. As such, new chemical compounds that can disrupt
P. aeruginosa QS signaling pathways and related mechanisms are welcome for the treatment of important infectious diseases.
The thiazole ring is one of the most important scaffolds in medicinal chemistry [
16]. In our previous work, a hydrazonyl-thiazole-based compound, (
E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)-4-(p-tolyl)thiazole (
HL,
Scheme 1), was synthesized in order to test its activity against cancer cells. Due to poor solubility in culture medium, the activity of
HL could not be tested, so its cobalt(III) complex ([Co(
HL)
2]BF
4,
Scheme 1) was prepared. Our results revealed that Co(
HL)
2 showed stronger activity on human mammary adenocarcinoma (MCF-7) cancer cells than cisplatin [
17]. Additionally, the antibacterial activity of Co(
HL)
2 was tested by the disc diffusion method on several Gram-positive (
Staphylococcus aureus,
Clostridium sporogenes,
Bacillus subtilis, and
Kocuria rhizophila) and Gram-negative (
Proteus hauseri,
P. aeruginosa,
Escherichia coli, and
Salmonella enterica) strains. The results obtained showed that Co(
HL)
2 possesses antibacterial activity comparable to antibiotic amikacin [
18]. The latter result encouraged us to test the activity of Co(
HL)
2 on QS inhibition and QS-dependent phenotypes, namely biofilm development and virulence factors production.
In the present work, Co(HL)2 was investigated as a P. aeruginosa LasI/LasR QS/biofilm formation inhibitor and virulence attenuator (pyocyanin and pyoverdine inhibition), as well as its interactions with a possible target, the transcriptional activator protein LasR, by molecular docking simulations. To the best of our knowledge, this work reports the first investigation of the activity of a metal complex on P. aeruginosa signaling pathways and its biofilms. Interestingly, the results show that Co(HL)2 can inhibit this type of cell-to-cell communications system (LasI/LasR) and underlying phenotypes and that this complex aids the HL to have a stronger interaction with the target protein than the known inhibitors (e.g., furvina).