Inhibition of biofilm formation, quorum sensing and virulence factor production in Pseudomonas aeruginosa PAO1 by selected LasR inhibitors / Inhibición de la formación de biopelículas, detección de quórum y producción de factores de virulencia en Pseudomonas aeruginosa PAO1 mediante inhibidores de LasR seleccionados

The quorum sensing network of Pseudomonas aeruginosa mediates the regulation of genes controlling biofilm formation and virulence factors. The rise of drug resistance to Pseudomonas aeruginosa infections has made quorum sensing–regulated biofilm formation in clinical settings a major issue. In the present study, LasR inhibitors identified in our previous study were evaluated for their antibiofilm and antiquorum sensing activities against P. aeruginosa PAO1. The compounds selected were (3-[2-(3,4-dimethoxyphenyl)-2-(1H-indol-3-yl)ethyl]-1-(2-fluorophenyl)urea) (C1), (3-(4-fluorophenyl)-2-[(3-methylquinoxalin-2-yl)methylsulfanyl]quinazolin-4-one) (C2) and (2-({4-[4-(2-methoxyphenyl)piperazin-1-yl]pyrimidin-2-yl}sulfanyl)-N-(2,4,6-trimethylphenyl)acetamide) (C3). The minimum inhibitory concentrations of C1 and C2 were 1000 μM, whereas that of C3 was 500 μM. At sub-MICs, the compounds showed potent antibiofilm activity without affecting the growth of P. aeruginosa PAO1. Electron microscopy confirmed the disruption of biofilm by the selected compounds. The antiquorum sensing activity of the compounds was revealed by the inhibition of violacein in Chromobacterium violaceum and the inhibition of swimming and swarming motilities in P. aeruginosa PAO1. Furthermore, the compounds also attenuated the production of quorum sensing–mediated virulence factors. The qRT-PCR revealed the downregulation of quorum sensing regulatory genes, namely lasI, lasR, rhlI, rhlR, lasB, pqsA and pqsR. The selected compounds also exhibited lower cytotoxicity against peripheral blood lymphocytes. Thus, this study could pave a way to explore these compounds for the development of therapeutic agent against Pseudomonas aeruginosa biofilm–related infections.(AU)

Inhibition of biofilm formation, quorum sensing and virulence factor production in Pseudomonas aeruginosa PAO1 by selected LasR inhibitors.

The quorum sensing network of Pseudomonas aeruginosa mediates the regulation of genes controlling biofilm formation and virulence factors. The rise of drug resistance to Pseudomonas aeruginosa infections has made quorum sensing-regulated biofilm formation in clinical settings a major issue. In the present study, LasR inhibitors identified in our previous study were evaluated for their antibiofilm and antiquorum sensing activities against P. aeruginosa PAO1. The compounds selected were (3-[2-(3,4-dimethoxyphenyl)-2-(1H-indol-3-yl)ethyl]-1-(2-fluorophenyl)urea) (C1), (3-(4-fluorophenyl)-2-[(3-methylquinoxalin-2-yl)methylsulfanyl]quinazolin-4-one) (C2) and (2-({4-[4-(2-methoxyphenyl)piperazin-1-yl]pyrimidin-2-yl}sulfanyl)-N-(2,4,6-trimethylphenyl)acetamide) (C3). The minimum inhibitory concentrations of C1 and C2 were 1000 µM, whereas that of C3 was 500 µM. At sub-MICs, the compounds showed potent antibiofilm activity without affecting the growth of P. aeruginosa PAO1. Electron microscopy confirmed the disruption of biofilm by the selected compounds. The antiquorum sensing activity of the compounds was revealed by the inhibition of violacein in Chromobacterium violaceum and the inhibition of swimming and swarming motilities in P. aeruginosa PAO1. Furthermore, the compounds also attenuated the production of quorum sensing-mediated virulence factors. The qRT-PCR revealed the downregulation of quorum sensing regulatory genes, namely lasI, lasR, rhlI, rhlR, lasB, pqsA and pqsR. The selected compounds also exhibited lower cytotoxicity against peripheral blood lymphocytes. Thus, this study could pave a way to explore these compounds for the development of therapeutic agent against Pseudomonas aeruginosa biofilm-related infections.

Combined machine learning and pharmacophore based virtual screening approaches to screen for antibiofilm inhibitors targeting LasR of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a virulent pathogen affects patients with cystic fibrosis and nosocomial infections. Quorum sensing (QS) mechanism plays a crucial role in causing these ailments by mediating biofilm formation and expressing virulent genes. A novel approach to circumvent this bacterial infection is by hindering its QS network. Targeting LasR of las system serves beneficial as it holds the top position in QS system cascade. Here, we have integrated machine learning, pharmacophore based virtual screening, molecular docking and simulation studies to look for new leads as inhibitors for LasR. Support vector machine (SVM) learning algorithm was used to generate QSAR models from 66 antagonist dataset. The top three models resulted in correlation coefficient (R2) values of 0.67, 0.86, and 0.91, respectively. The correlation coefficient (R2test) values on external test set were found to be 0.62, 0.57, and 0.55, respectively. A four-point pharmacophore model was developed. The pharmacophore hypothesis AAAD_1 was used to screen for potential leads against MolPort database in ZincPharmer. The leads which showed predicted pIC50 value of >8.00 by SVM models were subjected to docking analysis that reranked the compounds based on docking scores. Four top leads namely ZINC3851967 N-[3,5-bis(trifluoromethyl)phenyl]-5-tert-butyl-6-chloropyrazine-2-carboxamide, ZINC4024175 4-Amino-1-[(2R,3S,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-oxopyrimidine-5-carbonitrile, ZINC2125703 N-[(5-Methoxy-4,7-dimethyl-2-oxo-2H-chromen-3-yl)acetyl]-beta-alanine, and ZINC3851966 N-[3,5-Bis(trifluoromethyl)phenyl]5-tert-butylpyrazine-2-carboxamide were selected. These compounds were checked for its stability by performing a molecular dynamics simulation for a period of 100 ns. The ADME properties of the leads were also determined. Hence, the compounds identified in this study can be used as possible leads for developing a novel inhibitor for LasR.Communicated by Ramaswamy H. Sarma.

High-Throughput Virtual Screening for a New Class of Antagonist Targeting LasR of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes fatal effects in patients with cystic fibrosis and immunocompromised individuals and leads to around 1000 deaths annually. The quorum sensing mechanism of P. aeruginosa plays a major role in promoting biofilm formation and expression of virulent genes. Hence, quorum sensing inhibition is a promising novel approach to treat these bacterial infections as these organisms show a wide range of antibiotic resistance. Among the interconnected quorum sensing network of P. aeruginosa, targeting the las system is of increased interest as its principal receptor protein LasR is the earliest activated gene. It is also shown to be involved in the regulation of other virulence-associated genes. In this study, we have applied high-throughput virtual screening, an in silico computational method to identify a new class of LasR inhibitors that could serve as potent antagonists to treat P. aeruginosa-associated infections. Three-tire structure-based virtual screening was performed on the Schrödinger small molecule database, which resulted in 12 top hit compounds with docking scores lesser than -11.0 kcal/mol. Three of these best-scored compounds CACPD2011a-0001928786 (C1), CACPD2011a-0001927437 (C2), and CACPD2011a-0000896051 (C3) were further analyzed. The binding free energies of these compounds in complex with the target protein LasR (3IX4) were evaluated, and the pharmacokinetic properties were determined. The stability of the docked complexes was assessed by running a molecular dynamics simulation for 100 ns. Molecular dynamics simulation analysis revealed that all three compounds were found to be in stable contact with the protein over the entire simulation period. The antagonistic effect of these compounds was validated using the LasR reporter gene assay in the presence of acyl homoserine lactone. Significant reduction in the ß-galactosidase enzyme activity was achieved at 100 nM concentration for all three compounds pursued. Hence, the present study provides strong evidence that these three compounds could serve as quorum sensing inhibitors of P. aeruginosa LasR protein and can be a probable candidate to treat Pseudomonas-associated infections.