Fig. 5. IC50 measurements, in vitro engagement, and molecular docking studies support the prioritization of compound D88.
a Dose-dependent inhibition of HHQ, PQS, HQNO, 2-AA, and DHQ production was measured in PA14 cultures at OD600nm = 3.0. The cells grown with the compound’s vehicle were considered the control. b Dose-dependent production of AA in the presence of D88 in PA14 cultures at OD600nm = 3.0. AA is the primary precursor of all the MvfR-regulated small molecules assessed in A. GraphPad Prism 9.2.0 software plotted the IC50 curves against percent inhibition of the HAQs, 2-AA, DHQ, and AA production at each concentration. a, b Data represent the mean value of n = 3 biological replicates. Error bars denote ±SEM. c D88 inhibition of MvfR binding to the pqsA promoter. Overnight grown culture of PA14 expressing MvfR-VSV-G was diluted to OD 600nm 0.01 and grown at 37 °C with and without D88 (50 µM) and/or PQS (38 µM) until OD 600 nm 1.0. Thereafter MvfR-DNA complex were cross-linked and isolated via chromatin immunoprecipitation (ChIP). Coprecipitated DNA was purified and quantified using quantitative real-time polymerase chain reaction qPCR. MvfR binding to the pqsA promoter was calculated using the input method. Data reprsent n = 3 biological replicates, and the error bars denote ±SEM. Statistical analysis was carried out using GraphPad Prism 9.2.0 software. Unpaired t test was applied between the compared group. ∗ indicate significant differences from the control at P < 0.05 (Control vs. PQS = 0.038; PQS vs. PQS + D88 = 0.012). d The representative structure of the MvfR ligand-binding domain (LBD) complex with D88 (magenta sticks) was obtained from the docking analysis using AutoDock Vina. Tyr258 related to pi-interaction and Val170, Leu189, Ile236, and Ile263 related to hydrophobic interaction are shown as blue and green sticks, respectively. e Two-dimensional diagram of MvfR-D88 docking analyzed using PoseView program. A Green dashed line connecting two green dots indicates pi interaction. The solid green line indicates hydrophobic interactions made by hydrophobic residues (Val170, Leu189, Ile236, and Ile263) surrounding D88. AA = anthranilic acid; HHQ = 4-hydroxy-2-heptylquinoline; PQS = 3,4-dihydroxy-2-heptylquinoline; HQNO = ; 2-AA = 2-aminoacetophenone; DHQ = 2,4-dihydroxyquinoline; MvfR-VSV-G = MvfR fused to a vesicular stomatitis virus glycoprotein; Val = ; LeTyr = Tyrosine; Val = Valine; Leu = Leucine; Ile= Isoleucine.