We report silyl-lipid derivatives of N-acyl l-homoserine lactones (AHLs) that have nanomolar activities in LuxR-type quorum sensing receptors in Gram-negative bacterial pathogens. A collection of silyl-lipid AHLs were designed and synthesized to represent three general structural classes based on native AHL signals and synthetic LuxR-type receptor modulators. The synthetic routes feature straightforward hydrosilylation and aryl silylation reactions to access silyl-lipid groups that are not readily accessible in analogous all-carbon chemistry. Of the 17 compounds evaluated, eight silyl-lipid AHLs were identified with either nanomolar agonistic or submicromolar antagonistic activities in the LasR receptor from the common pathogen Pseudomonas aeruginosa using E. coli reporter gene assays. Several silyl-lipid AHL agonists retained high activities in LasR in a native P. aeruginosa reporter system and also were active in another related LuxR-type receptor, EsaR from Pantoea stewartii. Light scattering and computational experiments indicate that the silyl-lipid group can alter the aggregation capabilities and lipophilicities of AHLs relative to native all-carbon tails, engendering larger aggregate formation in water and higher lipophilicities on average. These properties, along with their strong activity profiles in LuxR-type receptors, suggest silyl-lipid AHLs could provide value as chemical probes to study the mechanisms of quorum sensing in Gram-negative bacteria and the roles of signal lipophilicity in this chemical communication process.

The opportunistic pathogen Pseudomonas aeruginosa controls almost 10% of its genome, including myriad virulence genes, via a cell-to-cell chemical communication system called quorum sensing (QS). Small molecules that either inhibit or activate QS in P. aeruginosa represent useful research tools to study the role of this signaling pathway in infection and interrogate its viability as an antivirulence target. However, despite active research in this area over the past 20+ years, there are relatively few synthetic compounds known to strongly inhibit or activate QS in P. aeruginosa. Most reported QS modulators in this pathogen are of low potency or have structural liabilities that limit their application in biologically relevant environments such as mimics of the native N-acyl l-homoserine lactone (AHL) signals. Here, we report the results of a high-throughput screen for abiotic small molecules that target LasR, a key QS regulator in P. aeruginosa. We screened a 25,000-compound library and discovered four new structural classes of abiotic LasR modulators. These compounds include antagonists that surpass the potency of all known AHL-type compounds and mimetics thereof, along with an agonist with potency approaching that of LasR's native ligand. The novel structures of this compound set, along with their anticipated robust physicochemical profiles, underscore their potential value as probe molecules to interrogate the roles of QS in this formidable pathogen.

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N-acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde, and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also cause mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR-specific inhibitory activities over a range of concentrations below that causing toxicity. ortho-Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho-vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho-vanillin as a small molecule QS modulator against RhlR. IMPORTANCE:Quorum sensing (QS) regulates many aspects of bacterial pathogenesis and has attracted much interest as a target for anti-virulence therapies over the past 30 years, for example, antagonists of the LasR and RhlR QS receptors in Pseudomonas aeruginosa. Potent and selective QS inhibitors remain relatively scarce. However, natural products have provided a bounty of chemical scaffolds with anti-QS activities, but their molecular mechanisms are poorly characterized. The current study serves to fill this void by examining the activity of an important and wide-spread class of natural product QS modulators, benzaldehydes, and related derivatives, in LasR and RhlR. We demonstrate that ortho-vanillin can act as a competitive inhibitor of RhlR, a receptor that has emerged and may supplant LasR in certain settings as a target for P. aeruginosa QS control. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities.

Cell-to-cell signaling, or quorum sensing (QS), in Gram-negative bacteria is governed by small molecule signals (N-acyl L-homoserine lactones, AHLs) and their cognate intracellular receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Quantitative assays to characterize the direct binding of ligands to these receptors are largely non-existent. We report herein a Förster Resonance Energy Transfer (FRET) assay that leverages (i) conserved endogenous tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore-AHL conjugates, and (ii) isolation/stabilization of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor—either in vitro or in cells—and was shown to be compatible with six LuxR-type receptors. These methods will advance fundamental investigations of the mechanisms of LuxR-type proteins and the development of small molecule modulators of QS.

Quorum sensing (QS) allows bacteria to assess their local cell density using chemical signals and plays a prominent role in the ability of common pathogens to infect a host. Non-native molecules capable of attenuating bacterial QS represent useful tools to explore the role of this pathway in virulence. As individual bacterial species can have multiple QS systems and/or reside in mixed communities with other bacteria capable of QS, chemical tools that are either selective for one QS system or "pan-active" and target all QS pathways are of significant interest. Herein we outline the analysis of a set of compounds reported to target one QS system in Pseudomonas aeruginosa for their activity in two other QS circuits in this pathogen and the discovery of molecules with novel activity profiles, including subsets that agonize all three QS systems, agonize one but antagonize the other two, or strongly antagonize just one.