Activation and inhibition of PqsR in <i>P. aeruginosa</i> by the 2-alkyl-4(1<i>H</i>)-quinolones.

<p>*EC₅₀ determined in a <i>P. aeruginosa</i> Δ<i>pqsA</i> CTX::<i>pqsA'-lux</i> strain; **EC₅₀ determined in a <i>P. aeruginosa</i> Δ<i>pqsAH</i> CTX::<i>pqsA'</i>-lux strain; – no activity.</p

3-NH₂-7Cl-C9-QZN inhibits AQ-dependent <i>P. aeruginosa</i> virulence factor production and biofilm development.

<p>(<b>A</b>) expression of <i>lecA</i> in <i>P. aeruginosa</i> PAO1 is inhibited by 3-NH₂-7Cl-C9-QZN as reflected by a <i>lecA'-lux</i> chromosomal reporter fusion; (<b>B</b>) production of pyocyanin by <i>P. aeruginosa</i> PAO1 grown in the presence of 0, 50 or 100 µM 3-NH₂-7Cl-C9-QZN and in a corresponding Δ<i>pqsA</i> mutant. The inhibition of pyocyanin production by 3-NH₂-7Cl-C9-QZN is observed as an absence of green pigmentation in culture supernatants (inset; numbers correspond to columns); (<b>C</b>) Semi-quantitative analysis by LC-MS/MS of HHQ, NHQ, NQNO and PQS production by <i>P. aeruginosa</i> PAO1 grown in the absence or presence of 200 µM 3-NH₂-7Cl-C9-QZN. The Δ<i>pqsA</i> mutant was used as a negative control; (<b>D</b>) Biofilm development is reduced in <i>P. aeruginosa</i> by a Δ<i>pqsA</i> mutation and following treatment of the wild type PAO1 strain with 3-NH₂-7Cl-C9-QZN.</p

Crystallographic data collection and refinement statistics.

*<p>Values in parentheses are for highest-resolution shell.</p>a<p>R_merge = Σh Σi|<i>I</i>i(h)−<<i>I</i>(h)>/|Σh Σi <i>I</i>i(h), where <i>I</i> is the observed intensity and <<i>I</i>> is the average intensity of multiple observations from symmetry-related reflections calculated with XDS.</p>b<p>Correlation co-efficient value calculated using XDS to determine the resolution cutoff.</p>c<p>All values calculated using REFMAC. R_work = Σh||Fo|h−|Fc|h|/Σh|Fo|h, where Fo and Fc are the observed and calculated structure factors, respectively. R_free computed as in R_work, but only for (5%) randomly selected reflections, which were omitted in refinement.</p

Response of PqsR^CBD ligand binding site mutants to AQs.

<p>(<b>A</b>) PqsR-6His is functional in <i>P. aeruginosa</i>. The <i>pqsR</i> gene with or without a 6His tag on pME6032 was introduced into a <i>P. aeruginosa pqsR</i> deletion mutant (Δ<i>pqsR</i>) containing a chromosomal miniCTX::<i>pqsA'-lux</i> reporter gene fusion. Relative PqsR activity was determined as a % of the maximum bioluminescence produced by the miniCTX::<i>pqsA'-lux</i> reporter fusion in a wild type <i>P. aeruginosa</i> PAO1 background carrying the empty pME6032 vector; (<b>B</b>) Light output from the <i>P. aeruginosa</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain transformed with one of each of the 13 site-specific mutations introduced into the PqsR^CBD ligand-binding pocket. Bioluminescence is presented as % of <i>pqsA</i> promoter activity with respect to PAO1 Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> expressing the PqsR-6His protein (WT); (<b>C</b>) Western blot analysis confirming expression of each of the PqsR-6His mutant proteins; (<b>D</b>) Light output from the <i>P. aeruginosa</i> Δ<i>pqsA</i> Δ<i>pqsH</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain transformed with either the gene coding for PqsR-6His or one of the 13 site-specific mutants and supplemented with either HHQ or PQS (40 µM). Bioluminescence is presented as % of <i>pqsA</i> promoter activity with respect to the <i>P. aeruginosa</i> Δ<i>pqsA</i> Δ<i>pqsH</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain expressing PqsR-6His.</p

Activation and inhibition of PqsR in <i>P. aeruginosa</i> by the 2-alkyl-4(3<i>H</i>)-quinazolinones.

<p>*EC₅₀ determined in a <i>P. aeruginosa</i> Δ<i>pqsA</i> miniCTX::<i>pqsA'-lux</i> strain; **IC₅₀ determined in a <i>P. aeruginosa</i> wild type strain incorporating a miniCTX::<i>pqsA'-lux</i> fusion; – no activity; <b>^‡</b>compounds exhibited growth inhibition; X compounds did not dissolve in MeOH at a workable concentration; 3-NH₂-7Cl-PhOBn-QZN (46) is barely soluble.</p

3-NH₂-7Cl-C9-QZN is a competitive inhibitor of PqsR.

<p>(<b>A</b>) PqsR activity as reflected by the maximum bioluminescence produced by a miniCTX::<i>pqsA'-lux</i> fusion in a Δ<i>pqsA</i> mutant in the presence of 100 µM QZN (inset) and increasing concentrations of PQS; (<b>B</b>) Topology diagram of the PqsR^CBD ligand-binding site occupied by the QZN shown in stick (orange), with hydrogen bonds coloured purple; (<b>C</b>) Orientation of 4-quinolone ring (right panel) and 7Cl-substituted QZN ring (left panel) within the PqsR ligand-binding pocket. The 7Cl is accommodated within a crevice forming a hydrogen bond (dotted line) with Thr265 (left panel); (<b>D</b>) Superposed PqsR^CBD-3-NH₂-7Cl-C9-QZN and PqsR^CBD-NHQ structures with residues shown as stick (orange and yellow respectively).</p