Hit Identification of New Potent PqsR Antagonists as Inhibitors of Quorum Sensing in Planktonic and Biofilm Grown Pseudomonas aeruginosa

Abstract

Current treatments for <i>Pseudomonas aeruginosa</i> infections are becoming less effective because of the increasing rates of multi-antibiotic resistance. Pharmacological targeting of virulence through inhibition of quorum sensing (QS) dependent virulence gene regulation has considerable therapeutic potential. In <i>P. aeruginosa</i>, the <i>pqs</i> QS system regulates the production of multiple virulence factors as well as biofilm maturation and is a promising approach for developing antimicrobial adjuvants for combatting drug resistance. In this work, we report the hit optimisation for a series of potent novel inhibitors of PqsR, a key regulator of the <i>pqs</i> system, bearing a 2-((5-methyl-5<i>H</i>-[1,2,4]triazino[5,6-<i>b</i>]indol-3-yl)thio) acetamide scaffold. The initial hit compound <b>7</b> (PAO1-L IC₅₀ 0.98 ± 0.02 μM, PA14 inactive at 10 μM) was obtained through a virtual screening campaign performed on the PqsR ligand binding domain using the University of Nottingham Managed Chemical Compound Collection. Hit optimisation gave compounds with enhanced potency against strains PAO1-L and PA14, evaluated using <i>P. aeruginosa pqs</i>-based QS bioreporter assays. Compound <b>40</b> (PAO1-L IC₅₀ 0.25 ± 0.12 μM, PA14 IC₅₀ 0.34 ± 0.03 μM) is one of the most potent PqsR antagonists reported showing significant inhibition of <i>P. aeruginosa</i> pyocyanin production and <i>pqs</i> system signaling in both planktonic cultures and biofilms. The co-crystal structure of <b>40</b> with the PqsR ligand binding domain revealed the specific binding interactions occurring between inhibitor and this key regulatory protein.