Modulation of the Substitution Pattern of 5-Aryl-2-Aminoimidazoles Allows Fine-Tuning of Their Antibiofilm Activity Spectrum and Toxicity

Abstract

We previously synthesized several series of compounds, based on the 5-aryl-2-aminoimidazole scaffold, that showed activity preventing the formation of Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa biofilms. Here, we further studied the activity spectrum of a number of the most active N1- and 2N-substituted 5-aryl-2-aminoimidazoles against a broad panel of biofilms formed by monospecies and mixed species of bacteria and fungi. An N1-substituted compound showed very strong activity against the biofilms formed by Gram-negative and Gram-positive bacteria and the fungus Candida albicans but was previously shown to be toxic against various eukaryotic cell lines. In contrast, 2N-substituted compounds were nontoxic and active against biofilms formed by Gram-negative bacteria and C. albicans but had reduced activity against biofilms formed by Gram-positive bacteria. In an attempt to develop nontoxic compounds with potent activity against biofilms formed by Gram-positive bacteria for application in antibiofilm coatings for medical implants, we synthesized novel compounds with substituents at both the N1 and 2N positions and tested these compounds for antibiofilm activity and toxicity. Interestingly, most of these N1-,2N-disubstituted 5-aryl-2-aminoimidazoles showed very strong activity against biofilms formed by Gram-positive bacteria and C. albicans in various setups with biofilms formed by monospecies and mixed species but lost activity against biofilms formed by Gram-negative bacteria. In light of application of these compounds as anti-infective coatings on orthopedic implants, toxicity against two bone cell lines and the functionality of these cells were tested. The N1-,2N-disubstituted 5-aryl-2-aminoimidazoles in general did not affect the viability of bone cells and even induced calcium deposition. This indicates that modulating the substitution pattern on positions N1 and 2N of the 5-aryl-2-aminoimidazole scaffold allows fine-tuning of both the antibiofilm activity spectrum and toxicity.

Inhibiting bacterial cooperation is an evolutionarily robust anti-biofilm strategy

“…For example, an inhibitor might be given prophylactically to inhibit Salmonella colonization in the gut when the risk of exposure to Salmonella is high, as is currently done with probiotics 72 and vaccines 73 . The potential for clinical application is further supported by our recent reports showing that 2-aminoimidazoles can inhibit biofilm formation in vivo in a subcutaneous model in rats 14 , have low cytotoxicity against different mammalian cell types (tumor cell lines and bone cells 35,74 ) and do not affect the survival of Caenorhabditis elegans, a small nematode that is widely used for toxicity testing and is considered to have high predictive value for toxicity in mammals 75 . However, while clinical potential clearly exists, the assays we report here are most reflective of industrial treatments.…”

“…We next sought to put our anti-biofilm strategy where resistance is counter selected into action. 2-cyclopentenyl-5-(4-chlorophenyl)-2-aminoimidazole, a specific member of the class of 5-aryl-2aminoimidazoles was chosen as EPS inhibitor 34,35 (see below). We have previously reported that 5-aryl-2-aminoimidazoles prevent EPS production of S. Typhimurium by reducing the transcription of csgD and its regulon 36 .…”

“…If fluorescently labelled plasmids pFPV25.1 or pMax were present, 100 µg ml −1 Ampicillin or 50 µg ml −1 kanamycin, respectively, was added to overnight cultures and biofilms. The EPS inhibitor used was 2cyclopentenyl-5-(4-chlorophenyl)-2-aminoimidazole [34][35][36] dissolved in DMSO. The carrier solvent (DMSO) did not have an effect on biofilm formation or planktonic growth at the applied concentration (0.05%).…”

“…We, therefore, sought to design a treatment strategy that inhibited biofilm and, critically, where we can demonstrate that resistant strains are counter selected. Our approach centers upon in-house developed 5-aryl-2-aminoimidazole-based inhibitors 14,[33][34][35][36] of EPS production in Salmonella biofilms. Salmonella species form biofilms outside and inside the host and EPS both helps cells attach and protects against eradication by mechanical cleaning, disinfectants, antibiotics and the host immune system 37 .…”

Candida spp./Bacteria Mixed Biofilms

“…Novel synthetic molecules are under exploration. The activity spectrum of the most active N1and 2N-substituted 5-aryl-2-aminoimidazoles against single-and mixed-biofilms of Gram-positive and Gram-negative bacteria and C. albicans was assessed by Peeters et al [165]. Molecules with substituents at both the N1 and 2N positions had high activity against mixed Gram-positive and yeast biofilms (monospecies and mixed), excluding biofilms formed by Gram-negative bacteria.…”