Structural Basis for Native Agonist and Synthetic Inhibitor Recognition by the Pseudomonas aeruginosa Quorum Sensing Regulator PqsR (MvfR)

Abstract

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.

Discovery of scmR as a global regulator of secondary metabolism and virulence in Burkholderia thailandensis E264

“…Interference with the function of MvfR results in lowered virulence by P. aeruginosa (52,53). Consequently, MvfR has advanced to a target for the development of strategies to combat P. aeruginosa infections.…”

Exploiting Interkingdom Interactions for Development of Small-Molecule Inhibitors of Candida albicans Biofilm Formation

“…However, both HHQ and PQS are coinducers of the virulence-associated LysR-type transcriptional regulator PqsR (41). The structural moieties that underpin the interaction between HHQ/PQS and PqsR remain to be fully characterized, although recent studies have reported diverse classes of PqsR antagonists (53)(54)(55) and implicated the hydrophobic pocket situated within the PqsR protein (56). Therefore, in order to assess whether the lead compounds could elicit a virulence response from P. aeruginosa, phenazine production and pqsA promoter activity (57) were monitored in a pqsA mutant where the capacity to produce native HHQ and PQS had been lost.…”

Pharmacological Inhibition of the Pseudomonas aeruginosa MvfR Quorum-Sensing System Interferes with Biofilm Formation and Potentiates Antibiotic-Mediated Biofilm Disruption

“…We recently showed that another HAQ, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), promotes biofilm formation and biofilm tolerance to antibiotics by inducing eDNA release that occurs as a result of autolysis due to self-poisoning of the respiratory chain by this molecule (14). A few studies have reported that inhibitors of PqsD or MvfR interfere with biofilm formation, but their antibiofilm potency was limited to the high micromolar range (11,(15)(16)(17). We recently described a benzamide-benzimidazole (BB) series of highly potent and cell-permeable inhibitors of MvfR (18).…”