Autoinducer-fluorophore conjugates enable FRET in LuxR proteins in vitro and in cells.

Cell-to-cell signaling, or quorum sensing (QS), in many Gram-negative bacteria is governed by small molecule signals (N-acyl-L-homoserine lactones, AHLs) and their cognate 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. Reports of quantitative direct-binding experiments on LuxR-type proteins are scarce, and robust and generalizable methods that provide such data are largely nonexistent. We report herein a Förster resonance energy transfer (FRET) assay that leverages (1) conserved tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore-AHL conjugates, and (2) isolation 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 proteins. These methods will advance fundamental investigations of LuxR-type protein mechanism and the development of small molecule QS modulators.

An Integrated Approach toward NanoBRET Tracers for Analysis of GPCR Ligand Engagement.

Gaining insight into the pharmacology of ligand engagement with G-protein coupled receptors (GPCRs) under biologically relevant conditions is vital to both drug discovery and basic research. NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a robust and sensitive method to quantify ligand engagement with specific GPCRs genetically fused to NanoLuc luciferase or the luminogenic HiBiT peptide. However, development of fluorescent tracers is often challenging and remains the principal bottleneck for this approach. One way to alleviate the burden of developing a specific tracer for each receptor is using promiscuous tracers, which is made possible by the intrinsic specificity of BRET. Here, we devised an integrated tracer discovery workflow that couples machine learning-guided in silico screening for scaffolds displaying promiscuous binding to GPCRs with a blend of synthetic strategies to rapidly generate multiple tracer candidates. Subsequently, these candidates were evaluated for binding in a NanoBRET ligand-engagement screen across a library of HiBiT-tagged GPCRs. Employing this workflow, we generated several promiscuous fluorescent tracers that can effectively engage multiple GPCRs, demonstrating the efficiency of this approach. We believe that this workflow has the potential to accelerate discovery of NanoBRET fluorescent tracers for GPCRs and other target classes.

Liquid Crystal Emulsions That Intercept and Report on Bacterial Quorum Sensing.

We report aqueous emulsions of thermotropic liquid crystals (LCs) that can intercept and report on the presence of N-acyl-l-homoserine lactones (AHLs), a class of amphiphiles used by pathogenic bacteria to regulate quorum sensing (QS), monitor population densities, and initiate group activities, including biofilm formation and virulence factor production. The concentration of AHL required to promote "bipolar" to "radial" transitions in micrometer-scale droplets of the nematic LC 4'-pentyl-cyanobiphenyl (5CB) decreases with increasing carbon number in the acyl tail, reaching a threshold concentration of 7.1 µM for 3-oxo-C12-AHL, a native QS signal in the pathogen Pseudomonas aeruginosa. The LC droplets in these emulsions also respond to biologically relevant concentrations of the biosurfactant rhamnolipid, a virulence factor produced by communities of P. aeruginosa under the control of QS. Systematic studies using bacterial mutants support the conclusion that these emulsions respond selectively to the production of rhamnolipid and AHLs and not to other products produced by bacteria at lower (subquorate) population densities. Finally, these emulsions remain configurationally stable in growth media, enabling them to be deployed either in bacterial supernatants or in situ in bacterial cultures to eavesdrop on QS and report on changes in bacterial group behavior that can be detected in real time using polarized light. Our results provide new tools to detect and report on bacterial QS and virulence and a materials platform for the rapid and in situ monitoring of bacterial communication and resulting group behaviors in bacterial communities.

Bacterial Quorum Sensing Signals Self-Assemble in Aqueous Media to Form Micelles and Vesicles: An Integrated Experimental and Molecular Dynamics Study.

Many species of common bacteria communicate and coordinate group behaviors, including toxin production and surface fouling, through a process known as quorum sensing (QS). In Gram-negative bacteria, QS is regulated by N-acyl-l-homoserine lactones (AHLs) that possess a polar homoserine lactone headgroup and a nonpolar aliphatic tail. Past studies demonstrate that AHLs can aggregate in water or adsorb at interfaces, suggesting that molecular self-assembly could play a role in processes that govern bacterial communication. We used a combination of biophysical characterization and atomistic molecular dynamics (MD) simulations to characterize the self-assembly behaviors of 12 structurally related AHLs. We used static light scattering and measurements of surface tension to characterize the assembly of four naturally occurring AHLs (3-oxo-C8-AHL, 3-oxo-C12-AHL, C12-AHL, and C16-AHL) in aqueous media and determine their critical aggregation concentrations (CACs). MD simulations and alchemical free energy calculations were used to predict thermodynamically preferred aggregate structures for each AHL. Those calculations predicted that AHLs with 10 or 12 tail carbon atoms should form spherical micelles and that AHLs with 14 or 16 tail carbon atoms should form vesicles in solution. Characterization of solutions of AHLs using negative stain transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed aggregates with sizes consistent with spherical micelles or small unilamellar vesicles for 3-oxo-C12-AHL and C12-AHL and the formation of large vesicles (∼250 nm) in solutions of C16-AHL. These experimental findings are in general agreement with our simulation predictions. Overall, our results provide insight into processes of self-assembly that can occur in this class of bacterial amphiphiles and, more broadly, provide a potential basis for understanding how AHL structure could influence processes that bacteria use to drive important group behaviors.

The luminescent HiBiT peptide enables selective quantitation of G protein-coupled receptor ligand engagement and internalization in living cells.

G protein-coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the ß-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

Equilibrium and Kinetic Measurements of Ligand Binding to HiBiT-tagged GPCRs on the Surface of Living Cells.

G-protein coupled receptors (GPCRs) remain at the forefront of drug discovery efforts. Detailed assessment of features contributing to GPCR ligand engagement in a physiologically relevant environment is imperative to the development of new therapeutics with improved efficacy. Traditionally, binding properties such as affinity and kinetics were obtained using biochemical radioligand binding assays. More recently, the high specificity of resonance energy transfer has been leveraged toward the development of homogeneous cell-based proximity assays with capacity for real-time kinetic measurements. This suite of ligand binding protocols couples the specificity of bioluminescent resonance energy transfer (BRET) with the sensitivity afforded by the luminescent HiBiT peptide. The BRET format is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent Tracers. At the same time, high affinity complementation of HiBiT with the cell impermeable LgBiT limits the bright bioluminescence donor signal to the cell surface and eliminates luminescence background from unoccupied receptors present in intracellular compartments.

N-Acyl Homoserine Lactone Analog Modulators of the Pseudomonas aeruginosa Rhll Quorum Sensing Signal Synthase.

Virulence in the Gram-negative pathogen Pseudomonas aeruginosa relies in part on the efficient functioning of two LuxI/R dependent quorum sensing (QS) cascades, namely, the LasI/R and RhlI/R systems that generate and respond to N-(3-oxo)-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone, respectively. The two acyl homoserine lactone (AHL) synthases, LasI and RhlI, use 3-oxododecanoyl-ACP and butyryl-ACP, respectively, as the acyl-substrates to generate the corresponding autoinducer signals for the bacterium. Although AHL synthases represent excellent targets for developing QS modulators in P. aeruginosa, and in other related bacteria, the identification of potent and signal synthase specific inhibitors has represented a significant technical challenge. In the current study, we sought to test the utility of AHL analogs as potential modulators of an AHL synthase and selected RhlI in P. aeruginosa as an initial target. We systematically varied the chemical functionalities of the AHL headgroup, acyl chain tail, and head-to-tail linkage to construct a small library of signal analogs and evaluated them for RhlI modulatory activity. Although the native N-butyryl-l-homoserine lactone did not inhibit RhlI, we discovered that several of our long-chain, unsubstituted acyl-d-homoserine lactones and acyl-d-homocysteine thiolactones inhibited while a few of the 3-oxoacyl-chain counterparts activated the enzyme. Additional mechanistic investigations with acyl-substrate analogs and docking experiments with AHL analogs revealed two distinct inhibitor and activator binding pockets in the enzyme. This study provides the first evidence of the yet untapped potential of AHL analogs as signal synthase modulators of QS pathways.

N-Acyl l-Homocysteine Thiolactones Are Potent and Stable Synthetic Modulators of the RhlR Quorum Sensing Receptor in Pseudomonas aeruginosa.

The RhlR quorum sensing (QS) receptor in the pathogen Pseudomonas aeruginosa plays a prominent role in infection, and both antagonism and agonism of RhlR have been shown to negatively regulate important virulence phenotypes. Non-native lactone ligands are known to modulate RhlR activity, but their utility as chemical probes is relatively limited due to hydrolytic instability. Herein, we report our design and biological evaluation of a suite of hybrid AHL analogs with structures merging (1) features of reported lead RhlR ligands and (2) head groups with improved hydrolytic stabilities. The most promising compounds identified were N-acyl l-homocysteine thiolactones, which displayed enhanced stabilities relative to lactones. Moreover, they were highly selective for RhlR over another key QS receptor in P. aeruginosa, LasR. These compounds are among the most potent RhlR modulators known and represent robust chemical tools to dissect the complex roles of RhlR in the P. aeruginosa QS circuitry.

Structure-Function Analyses of the N-Butanoyl l-Homoserine Lactone Quorum-Sensing Signal Define Features Critical to Activity in RhlR.

Pseudomonas aeruginosa is an opportunistic pathogen that coordinates the production of many virulence phenotypes at high population density via quorum sensing (QS). The LuxR-type receptor RhlR plays an important role in the P. aeruginosa QS process, and there is considerable interest in the development of chemical approaches to modulate the activity of this protein. RhlR is activated by a simple, low molecular weight N-acyl l-homoserine lactone signal, N-butanoyll-homoserine lactone (BHL). Despite the emerging prominence of RhlR in QS pathways, there has been limited exploration of the chemical features of the BHL scaffold that are critical to its function. In the current study, we sought to systematically delineate the structure-activity relationships (SARs) driving BHL activity for the first time. A focused library of BHL analogues was designed, synthesized, and evaluated in cell-based reporter gene assays for RhlR agonism and antagonism. These investigations allowed us to define a series of SARs for BHL-type ligands and identify structural motifs critical for both activation and inhibition of the RhlR receptor. Notably, we identified agonists that have ∼10-fold higher potencies in RhlR relative to BHL, are highly selective for RhlR agonism over LasR, and are active in the P. aeruginosa background. These compounds and the SARs reported herein should pave a route toward new chemical strategies to study RhlR in P. aeruginosa.

A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence.

Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities.

Mechanism of agonism and antagonism of the Pseudomonas aeruginosa quorum sensing regulator QscR with non-native ligands.

Pseudomonas aeruginosa is an opportunistic pathogen that uses the process of quorum sensing (QS) to coordinate the expression of many virulence genes. During quorum sensing, N-acyl-homoserine lactone (AHL) signaling molecules regulate the activity of three LuxR-type transcription factors, LasR, RhlR and QscR. To better understand P. aeruginosa QS signal reception, we examined the mechanism underlying the response of QscR to synthetic agonists and antagonists using biophysical and structural approaches. The structure of QscR bound to a synthetic agonist reveals a novel mode of ligand binding supporting a general mechanism for agonist activity. In turn, antagonists of QscR with partial agonist activity were found to destabilize and greatly impair QscR dimerization and DNA binding. These results highlight the diversity of LuxR-type receptor responses to small molecule agonists and antagonists and demonstrate the potential for chemical strategies for the selective targeting of individual QS systems.

Chemical Interrogation of LuxR-type Quorum Sensing Receptors Reveals New Insights into Receptor Selectivity and the Potential for Interspecies Bacterial Signaling.

Cell-cell signaling between bacteria, including quorum-sensing (QS) communication systems, may play a role in the establishment and maintenance of polymicrobial communities. To better understand and model these interactions, we must uncover the degree to which neighboring species recognize each another's signals. In the current study, we tested the likelihood of whether the QS systems of two opportunistic pathogens (Acinetobacter baumannii and Pseudomonas aeruginosa) that frequently arise in polymicrobial infections would be affected by the QS signals of neighboring species. Through the synthesis and screening of a library of native and non-native N-acyl l-homoserine lactones (AHLs), we found that the AbaR LuxR-type receptor protein of A. baumannii is highly selective for its native AHL signal. However, a homologous LuxR-type receptor in P. aeruginosa, LasR, is far more promiscuously activated by AHLs relative to AbaR, suggesting that LasR-regulated QS could be more susceptible to activation by neighboring species. To explain the observed difference in signal selectivity between AbaR and LasR, we developed a model based on (i) the activity profiles of these proteins and (ii) previously reported structural data and activity profiles for related LuxR-type receptors. This model may facilitate the study of signal selectivities for hundreds of LuxR-type QS receptors from bacteria, many of which grow in polymicrobial communities and may sense each other's signals. In addition, we discovered a set of AHLs that could be used to selectively activate LasR and selectively inhibit AbaR in polymicrobial experiments.