New bicyclic brominated furanones as potent autoinducer-2 quorum-sensing inhibitors against bacterial biofilm formation

Highlights

• New bicyclic brominated furanones were synthesized as potent QS inhibitors.

• Inhibitors showed high inhibitory effects on the periodontopathogen biofilm formation.

• Active compounds significantly inhibited the F. nucleatum AI-2 activity.

• The inhibitors showed neither cytotoxicity nor induction of inflammatory response.

Abstract

Bacterial behaviors such as virulence factor secretion and biofilm formation are critical for survival, and are effectively regulated through quorum sensing, a mechanism of intra- and interspecies communication in response to changes in cell density and species complexity. Many bacterial species colonize host tissues and form a defensive structure called a biofilm, which can be the basis of inflammatory diseases. Periodontitis, a chronic inflammatory disease affecting the periodontium, is caused by subgingival biofilms related to periodontopathogens. In particular, Fusobacterium nucleatum is a major co-aggregation bridge organism in the formation and growth of subgingival biofilms, linking the early and late colonizers in periodontal biofilms. According to our previous study, the intergeneric quorum-sensing signal molecule autoinducer-2 (AI-2) of F. nucleatum plays a key role in intra- and interspecies interactions of periodontopathogens, and may be a good target for periodontal biofilm inhibition. Recently, brominated furanones produced by the macroalga Delisea pulchra were shown to inhibit biofilm formation via AI-2, and have been investigated toward the goal of increasing the inhibition effect. In this study, we describe the synthesis of new bromofuranone analogs, i.e., 3-(dibromomethylene)isobenzofuran-1(3H)-one derivatives, and demonstrate their inhibitory activities against biofilm formation by periodontopathogens, including F. nucleatum, Porphyromonas gingivalis, and Tannerella forsythia.

Introduction

Quorum sensing (QS) is the process of cell-to-cell communication in microorganisms mediated by small signaling molecules referred to as autoinducers, which are secreted by bacteria and fungi [1], [2], [3]. Bacteria can effectively regulate numerous phenotypes, including virulence factor expression, bioluminescence, and biofilm formation, in response to their changes in population density through recognition of the threshold reached by autoinducers, the so-called quorum, via intracellular or intercellular communication [4]. In particular, formation of a biofilm can not only enhance the viability of bacteria in response to antibiotics but can also be the basis of the development of chronic inflammatory diseases, including endocarditis, cystic fibrosis, osteomyelitis, chronic urinary tract infections, chronic prostatitis, and periodontal diseases [5].

QS molecules can be classified into three major types: oligopeptides, acyl-homoserine lactones (AHLs, autoinducer-1), and autoinducer-2 (AI-2). Oligopeptides and AHLs are used by gram-negative and gram-positive bacteria, respectively, in intraspecies communication [6]. AI-2 is a universal QS molecule secreted by both gram-negative and gram-positive bacteria [7], [8], [9], and plays a critical role in the virulence of pathogenic bacteria and in biofilm formation [10]. AI-2 mediates both intra- and interspecies communication, and can thus be a good target for the regulation of bacterial infection. Accordingly, AI-2 inhibitors are ideal compounds for bacterial biofilm inhibition in multispecies bacterial communities.

More than 700 species of bacteria have been found in the human oral cavity, which establish mixed-species communities [11]. Among these oral bacteria, periodontopathogens, including Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Tannerella forsythia, and spirochetes, form subgingival biofilms and cause periodontitis, which is a chronic inflammatory disease. Since AI-2 is an interspecies QS molecule that controls intergeneric signaling, it may induce oral biofilm formation and increase the bacterial virulence of periodontopathogens [12], [13], [14]. Furthermore, Kolenbrander et al. [15] suggested that, compared to commensal bacteria, periodontopathogens produce much higher levels of AI-2; such high concentrations would enhance pathogen biofilm maturation, leading to periodontitis. In particular, F. nucleatum is known as a major co-aggregation bridge organism that connects late pathogenic colonizers and early commensal colonizers in a periodontal biofilm [15], [16], [17], [18], [19]. We previously reported that the level of F. nucleatum AI-2 was reduced by QS inhibitors (QSIs), including (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone and d-ribose [20]. Because of the diversity of oral bacteria, it is hard to selectively eliminate periodontopathogens without disturbing oral commensals. Therefore, oral biofilm formation regulation using QSIs is one of the most hopeful protective means for preserving oral health [21].

Brominated furanones produced from the macroalga Delisea pulchra are known to prevent microbial colonization [22], [23]. In addition, these compounds have been shown to inactivate LuxS, which is required for AI-2 synthesis, and to inhibit the QS activity of various bacterial species [4], [24], [25], [26], [27]. Recently, Yang et al. [28] reported a bicyclic version of brominated furanones that could potentially reduce their toxicity while retaining their biofilm inhibitory activities. However, compared with reported monocyclic brominated furanones, the inhibitory activities of the bicyclic furanones were relatively low [28]. These results suggest that modification of the exocyclic vinyl position, which is an essential structural element for the inhibition of LuxS [29], is not an appropriate strategy for the development of effective QSI's. Therefore, discovery of new potent and safer brominated furanone candidates is still desired. In this study, to improve the QS inhibitory activity and biofilm inhibition efficacy of periodontal bacteria, we report the synthesis and evaluation of 3-(dibromomethylene)isobenzofuran-1(3H)-one derivatives, which have different ring sites from the existing bicyclic compound. In particular, we investigated the structure-activity relationships of the biofilm inhibition effects of various new furanone derivatives prepared with new ring structures and possible side chains on the new ring.