Design, Synthesis, and Biochemical Characterization of Non-Native Antagonists of the Pseudomonas aeruginosa Quorum Sensing Receptor LasR with Nanomolar IC₅₀ Values

Abstract

Quorum sensing (QS), a bacterial cell-to-cell communication system mediated by small molecules and peptides, has received significant interest as a potential target to block infection. The common pathogen Pseudomonas aeruginosa uses QS to regulate many of its virulence phenotypes at high cell densities, and the LasR QS receptor plays a critical role in this process. Small molecule tools that inhibit LasR activity would serve to illuminate its role in P. aeruginosa virulence, but we currently lack highly potent and selective LasR antagonists, despite considerable research in this area. V-06-018, an abiotic small molecule discovered in a high-throughput screen, represents one of the most potent known LasR antagonists, but has seen little study since its initial report. Herein, we report a systematic study of the structure-activity relationships (SARs) that govern LasR antagonism by V-06-018. We synthesized a focused library of V-06-018 derivatives and evaluated the library for bioactivity using a variety of cell-based LasR reporter systems. The SAR trends revealed by these experiments allowed us to design probes with 10-fold greater potency than V-06-018 and 100-fold greater potency than other commonly used N-acyl L-homoserine lactone (AHL)-based LasR antagonists, along with high selectivities for LasR. Biochemical experiments to probe the mechanism of antagonism by V-06-018 and its analogs support these compounds interacting with the native ligand-binding site in LasR and, at least in part, stabilizing an inactive form of the protein. The compounds described herein are the most potent and efficacious antagonists of LasR known, and represent robust probes both for characterizing the mechanisms of LuxR-type QS and for chemical biology research in general in the growing QS field.

Antipathogenic Compounds That Are Effective at Very Low Concentrations and Have Both Antibiofilm and Antivirulence Effects against Pseudomonas aeruginosa

“…Our two MHYs showed similar effects at concentrations 10 6 or 10 8 times lower than these. The anti-QS effect of these two MHYs was also maximized at around 100 pM, which is much lower than the concentration of the previously screened anti-QS substances that were usually effective at concentrations higher than 0.1 μM ( 15 , 16 , 27 , 28 ). At first glance, MHY1383 and MHY1387 appear to have weak anti-QS effects, but our results show that they can actually reduce the toxicity of P. aeruginosa virulence factors in a small-animal model ( Fig.…”

Heterocyclic Chemistry Applied to the Design of N-Acyl Homoserine Lactone Analogues as Bacterial Quorum Sensing Signals Mimics

“…The substituted amino indoles were obtained from a two-steps procedure, including the nitrosation and the reduction of the nitroso intermediate with hydrazine in presence of palladium as catalyst (Scheme 5). Blackwell and colleagues [45] reported also a series of AHL analogues in which the lactone is replaced by an aromatic moiety, some of them exhibiting very potent antagonistic activity against LasR reporter systems. On the basis of the phenyl derivative 34 discovered by a high-throughput screen of small chemical molecule libraries structural variations on the ring, notably using heterocyclic scaffolds, were performed for searching derivatives with improved potencies.…”

Non-antibiotic methods against Pseudomonas aeruginosa include QS inhibitors: a narrative review

“…QS is negotiated by a solid, interconnected system consisting of the following 4 public systems: 2 LuxR/LuxI-type (LasR/ LasI and RhlR/RhlI), together with an orphan LuxR-type receptor (QscR), and the quinolone system PqsR. LasI catalyzes the production of the diffusible QS signal N-3oxododecanoyl homoserine lactone (3OC12-HSL), 3OC12-HSL connects to its analogous receptor, the transcription factor LasR, which can then activate the appearance of multiple genes, including lasI, rhlI and rhlR, analogously, RhlI catalyzes the generation of the diffusible beacon butyryl-HSL (C4-HSL), which connects to the transcription factor RhlR, signal-bound RhlR stimulates the expression of RhlI and some genes, some of which continue to activate the LasR regulon (14)(15)(16)(17)(18). PqsR is unrelated to LuxR-type receptors, which is negotiated by the signal 2-heptyl-3hydroxy-4-quinolone (Pseudomonas quinolone signal, PQS) and its biosynthetic precursor 2-heptyl-4-quinolone (HHQ) (19,20).…”

“…For example, LasR's activation can cause a cascade of reactions to turn on several PA virulence factors by activating the RhlIR and PQS systems (14,15). LasR and RhlR are suppressed by QscR, which is stimulated through innate sign 3OC12-HSL (18,21). PqsR system and AHL systems interact with each other (22,23).…”