Identification of Small-Molecule Antagonists of the Pseudomonas aeruginosa Transcriptional Regulator PqsR: Biophysically Guided Hit Discovery and Optimization

Abstract

The Gram-negative pathogen Pseudomonas aeruginosa produces an intercellular alkyl quinolone signaling molecule, the Pseudomonas quinolone signal. The pqs quorum sensing communication system that is characteristic for P. aeruginosa regulates the production of virulence factors. Therefore, we consider the pqs system a novel target to limit P. aeruginosa pathogenicity. Here, we present small molecules targeting a key player of the pqs system, PqsR. A rational design strategy in combination with surface plasmon resonance biosensor analysis led to the identification of PqsR binders. Determination of thermodynamic binding signatures and functional characterization in E. coli guided the hit optimization, resulting in the potent hydroxamic acid derived PqsR antagonist 11 (IC(50) = 12.5 μM). Remarkably it displayed a comparable potency in P. aeruginosa (IC(50) = 23.6 μM) and reduced the production of the virulence factor pyocyanin. Beyond this, site-directed mutagenesis together with thermodynamic analysis provided insights into the energetic characteristics of protein-ligand interactions. Thus the identified PqsR antagonists are promising scaffolds for further drug design efforts against this important pathogen.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Although the potency of compound 11 was less than that of HHQ analogues 19 and 20, compound 11 has a lower molecular weight and is therefore more amenable to further optimization for use in vivo. This second report also helped define structural features of PqsR ligands that determine agonism versus antagonism (229), making rational design of potent PqsR antagonists more feasible moving forward.…”

“…Surpris- ingly, however, elastase production and rhamnolipid production were not inhibited even at higher concentrations of the compound, but no explanation for these results was provided (127). In a follow-up report by the same group, rational design strategy and SPR biosensor analysis were used to identify additional PqsR binders (229). The -opioid receptor agonist (Ϯ)-trans-U50488, which was previously shown to activate quinolone signaling in P. aeruginosa (230), was used as a starting point for fragment-based design of novel inhibitors.…”

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.…”

New Approaches to Control Infections: Anti-biofilm Strategies against Gram-negative Bacteria

“…8) were studied and interesting agonistic or antagonistic activities were observed, depending on the substitution pattern. [18] In Vibrio spp., a second quorum sensing system was discovered and termed autoinducer-2 (AI-2). This system is believed to allow interspecies communication.…”

“…Promising results, especially from interference with quorum sensing and adhesins, must now be further translated into early clinical phases to prevent the aforementioned scenarios of untreatable infections with Gram-negative bacteria. (17)(18)(19) and PapG (20,21), and P. aeruginosa LecA (23)(24)(25) and LecB (26)(27)(28)(29).…”