Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target

Abstract

Elastase B (lasB) is a multifunctional metalloenzyme secreted by the gram-negative pathogen <i>Pseudomonas aeruginosa</i>, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)₂]ClO₄ (Ag-phendione) and [Cu(phendione)₃](ClO₄)₂.4H₂O (Cu-phendione) had anti-<i>P. aeruginosa</i> action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using <i>in silico</i> approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) <i>lasB</i> gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of <i>Galleria mellonella</i> larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag⁺ and Cu²⁺ complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the <i>in vitro</i> cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-<i>p</i>-nitrobenzylamide, with Cu-phendione having the best inhibitory action (K <i>_<i>i</i> </i> = 90 nM). Treating living bacteria with a sub-inhibitory concentration (½ × MIC value) of the test compounds caused a significant reduction in the expression of the <i>lasB</i> gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the <i>in vivo</i> model, increasing the survival time of <i>G. mellonella</i> larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against <i>P. aeruginosa</i>.