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Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy.

ABSTRACT: BACKGROUND: The nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli. However, relative to some other nitroaromatic prodrugs, nitro-CBI-DEI is a poor substrate for E. coli NfsB. To address this limitation we evaluated other nitroreductase candidates from E. coli and Pseudomonas aeruginosa. FINDINGS: Initial screens of candidate genes in the E. coli reporter strain SOS-R2 identified two additional nitroreductases, E. coli NfsA and P. aeruginosa NfsB, as being more effective activators of nitro-CBI-DEI than E. coli NfsB. In monolayer cytotoxicity assays, human colon carcinoma (HCT-116) cells transfected with P. aeruginosa NfsB were >4.5-fold more sensitive to nitro-CBI-DEI than cells expressing either E. coli enzyme, and 23.5-fold more sensitive than untransfected HCT-116. In three dimensional mixed cell cultures, not only were the P. aeruginosa NfsB expressing cells 540-fold more sensitive to nitro-CBI-DEI than pure cultures of untransfected HCT-116, the activated drug that they generated also displayed an unprecedented local bystander effect. CONCLUSION: We posit that the discrepancy in the fold-sensitivity to nitro-CBI-DEI between the two and three dimensional cytotoxicity assays stems from loss of activated drug into the media in the monolayer cultures. This emphasises the importance of evaluating high-bystander GDEPT prodrugs in three dimensional models. The high cytotoxicity and bystander effect exhibited by the NfsB_Pa/nitro-CBI-DEI combination suggest that further preclinical development of this GDEPT pairing is warranted.

SUBMITTER: Green LK

PROVIDER: S-EPMC3695803 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy.

Green Laura K LK Syddall Sophie P SP Carlin Kendall M KM Bell Glenn D GD Guise Christopher P CP Mowday Alexandra M AM Hay Michael P MP Smaill Jeffrey B JB Patterson Adam V AV Ackerley David F DF

Molecular cancer 20130610

<h4>Background</h4>The nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli. However, relative to some other nitroaromatic prodrugs, nitro-CBI-DEI is a poor substrate for E. coli NfsB. To address this limitation we evaluated other nitroreductase candidates from E. coli ...[more]

PMID: 23758947

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Project description:With the inappropriate use of antibiotics, antibiotic resistance has emerged as a major dilemma for patients infected with Pseudomonas aeruginosa. Elastase B (LasB), a crucial extracellular virulence factor secreted by P. aeruginosa, has been identified as a key target for antivirulence therapy. Quercetin, a natural flavonoid, exhibits promising potential as an antivirulence agent. We aim to evaluate the impact of quercetin on P. aeruginosa LasB and elucidate the underlying mechanism. Molecular docking and molecular dynamics simulation revealed a rather favorable intermolecular interaction between quercetin and LasB. At the sub-MICs of ≤256 μg/ml, quercetin was found to effectively inhibit the production and activity of LasB elastase, as well as downregulate the transcription level of the lasB gene in both PAO1 and clinical strains of P. aeruginosa. Through correlation analysis, significant positive correlations were shown between the virulence gene lasB and the QS system regulatory genes lasI, lasR, rhlI, and rhlR in clinical strains of P. aeruginosa. Then, we found the lasB gene expression and LasB activity were significantly deficient in PAO1 ΔlasI and ΔlasIΔrhlI mutants. In addition, quercetin significantly downregulated the expression levels of regulated genes lasI, lasR, rhlI, rhlR, pqsA, and pqsR as well as effectively attenuated the synthesis of signaling molecules 3-oxo-C12-HSL and C4-HSL in the QS system of PAO1. Quercetin was also able to compete with the natural ligands OdDHL, BHL, and PQS for binding to the receptor proteins LasR, RhlR, and PqsR, respectively, resulting in the formation of more stabilized complexes. Taken together, quercetin exhibits enormous potential in combating LasB production and activity by disrupting the QS system of P. aeruginosa in vitro, thereby offering an alternative approach for the antivirulence therapy of P. aeruginosa infections. KEY POINTS: • Quercetin diminished the content and activity of LasB elastase of P. aeruginosa. • Quercetin inhibited the QS system activity of P. aeruginosa. • Quercetin acted on LasB based on the QS system.

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Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy.

Publications

Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy.

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