Application of Dual Inhibition Concept within Looped Autoregulatory Systems toward Antivirulence Agents against <i>Pseudomonas aeruginosa</i> Infections

<i>Pseudomonas aeruginosa</i> quorum-sensing (QS) is a sophisticated network of genome-wide regulation triggered in response to population density. A major component is the self-inducing pseudomonas quinolone signal (PQS) QS system that regulates the production of several nonvital virulence- and biofilm-related determinants. Hence, QS circuitry is an attractive target for antivirulence agents with lowered resistance development potential and a good model to study the concept of polypharmacology in autoloop-regulated systems <i>per se</i>. Based on the finding that a combination of PqsR antagonist and PqsD inhibitor synergistically lowers pyocyanin, we have developed a dual-inhibitor compound of low molecular weight and high solubility that targets PQS transcriptional regulator (PqsR) and PqsD, a key enzyme in the biosynthesis of PQS-QS signal molecules (HHQ and PQS). <i>In vitro</i>, this compound markedly reduced virulence factor production and biofilm formation accompanied by a diminished content of extracellular DNA (eDNA). Additionally, coadministration with ciprofloxacin increased susceptibility of PA14 to antibiotic treatment under biofilm conditions. Finally, disruption of pathogenicity mechanisms was also assessed <i>in vivo</i>, with significantly increased survival of challenged larvae in a <i>Galleria mellonella</i> infection model. Favorable physicochemical properties and effects on virulence/biofilm establish a promising starting point for further optimization. In particular, the ability to address two targets of the PQS autoinduction cycle at the same time with a single compound holds great promise in achieving enhanced synergistic cellular effects while potentially lowering rates of resistance development

Structure of the hit compound 1 and two newly synthesized derivatives.

<p>Structure of the hit compound 1 and two newly synthesized derivatives.</p

Biological activities of compounds 47 and 48 possessing a changed relative position of the thiophene sulfur compared to their regioisomers 21 and 29.

a<p>See footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087851#pone-0087851-t002" target="_blank">Table 2</a>.</p

Metabolic stability of compounds 29, 30 and 48 and reference compounds against rat liver microsomes^a.

a<p>0.225 mg/mL protein, NADP⁺-regenerating system, [inhibitor]: 0.5 µM, incubation at 37°C, samples taken at 0, 15, 30, and 60, 90 min, determination of parent compound by MS.</p>b<p>Cl_blood: estimated blood clearance in rats as calculated based on <i>in vitro</i> intrinsic clearance. The values are representative for two independent experiments that essentially gave the same results.</p

Influence of the Dyrk inhibitors 29 and 48 at three different concentrations (A–C) on the cell growth of V79 hamster lung fibroblasts in comparison to harmine.

<p>Compound <b>11</b> is of the same chemotype as <b>29</b> and <b>48</b> but was inactive with respect to Dyrk/Clk inhibition. Intracellular ATP levels were quantified as a measure for cell viability and proliferation rates. Results of one out of three independent experiments are presented, which all showed the same tendency. The standard deviation is indicated as y-error bars. Asterisks indicate statistical significance (p value <0.05).</p

Biological activity of 5-(thiophen-2-yl)pyrimidine derivatives with diversification at the 4-position of the thiophene core.

a<p>See footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087851#pone-0087851-t002" target="_blank">Table 2</a>.</p

The Dyrk1B inhibitor 29 triggers the production of reactive oxygen species in U2OS cells.

<p>Incubation of U2OS cells with <b>29</b> caused an increased intracellular conversion of didydroethidium to the stronger fluorescent ethidium, indicating raised levels of reactive oxygen species compared with the ethanol control. Data shown are representative of two independent experiments. Asterisks indicate statistical significance (p value <0.05).</p

Synthesis of Compound 48<i>^a</i>.

<p><i>^a</i>Reagents and conditions: a) nBuLi, diethyl ether, −78°C, 60 min; trimethylsilylchloride, −78°C, 60 min; b) nBuLi, THF, −78°C, 35 min; (<i>i</i>PrO)₃B, −78°C, 90 min; pinacol, RT, 10 min; c) Dibromothiophene, Na₂CO₃, Pd(PPh₃)₄, Dioxan/Water, 90°C; d) 3-Pyridylboronic acid, Cs₂CO₃, Pd(dppf)Cl₂, DMF, Reflux.</p

Potency of the dual Dyrk/Clk inhibitors 29, 30, 33, 41 and 48 against the target kinases.

a<p>Given are mean values of at least two independent experiments, S.D.<10%.</p

Synthesis of target compounds 33 to 46<i>^a</i>.

<p><i>^a</i>Reagents and conditions: a) Tosylmethylisocyanate, Na₂CO₃, Methanol, Reflux, 4 hours; b) Cs₂CO₃, Pd(dppf)Cl₂, DMF/Water; Reflux, 15 hours; c) Na₂CO₃, Pd(PPh₃)₄, Dioxane/Water, 90°C, 48 hours; d) Cs₂CO₃, Pd(dppf)Cl₂, DMF/Water; Reflux, 15 hours.</p