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Identification of inhibitors of PvdQ, an enzyme involved in the synthesis of the siderophore pyoverdine.

Wurst JM, Drake EJ, Theriault JR, Jewett IT, VerPlank L, Perez JR, Dandapani S, Palmer M, Moskowitz SM, Schreiber SL, Munoz B, Gulick AM - ACS Chem. Biol. (2014)

Bottom Line: Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase.ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318.Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

View Article: PubMed Central - PubMed

Affiliation: The Broad Institute , Cambridge, Massachusetts 02142, United States.

ABSTRACT
Pseudomonas aeruginosa produces the peptide siderophore pyoverdine, which is used to acquire essential Fe(3+) ions from the environment. PvdQ, an Ntn hydrolase, is required for the biosynthesis of pyoverdine. PvdQ knockout strains are not infectious in model systems, suggesting that disruption of siderophore production via PvdQ inhibition could be exploited as a target for novel antibacterial agents, by preventing cells from acquiring iron in the low iron environments of most biological settings. We have previously described a high-throughput screen to identify inhibitors of PvdQ that identified inhibitors with IC50 values of ∼100 μM. Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

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High-throughput screenfor PvdQ inhibitors. (A) PvdQ hydrolysisof either p-nitrophenyl myristate or 4-MU-lauratesubstrates result in production of compounds that can be detectedthrough absorbance or fluorescence. (B) Comparison of biochemicalPvdQ inhibition activities of prior art compounds (1, 2) to the high-throughput screening lead 3 and the optimizedprobe 4.
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fig2: High-throughput screenfor PvdQ inhibitors. (A) PvdQ hydrolysisof either p-nitrophenyl myristate or 4-MU-lauratesubstrates result in production of compounds that can be detectedthrough absorbance or fluorescence. (B) Comparison of biochemicalPvdQ inhibition activities of prior art compounds (1, 2) to the high-throughput screening lead 3 and the optimizedprobe 4.

Mentions: To examine the role of PvdQ in pyoverdine maturation, wedevelopeda high-throughput biochemical assay to find inhibitors of the PvdQacylase activity.24 The assay monitoredthe hydrolysis of p-nitrophenyl myristate (Figure 2A) and showed good reproducibility and signal-to-noiseparameters, with Z′ scores of 0.7–0.9 within one plateand 0.6 overall. In this proof-of-concept study, we screened 1280compounds, identifying aryl bromides 1 and 2 (Figure 2B), which exhibit IC50 values of 130 μM and 65 μM and bind in the fatty acidbinding pocket.24 This success with a smalllibrary suggested that a more thorough effort might lead to compoundswith higher affinity. We therefore conducted a high-throughput screenwith a larger chemical library and 4-methylumbelliferyl laurate (4-MUlaurate), a fluorogenic substrate with improved signal-to-noise properties,to identify more potent scaffolds for PvdQ inhibition that can serveas tool compounds for understanding pyoverdine maturation and therapeuticleads for P. aeruginosa infection.


Identification of inhibitors of PvdQ, an enzyme involved in the synthesis of the siderophore pyoverdine.

Wurst JM, Drake EJ, Theriault JR, Jewett IT, VerPlank L, Perez JR, Dandapani S, Palmer M, Moskowitz SM, Schreiber SL, Munoz B, Gulick AM - ACS Chem. Biol. (2014)

High-throughput screenfor PvdQ inhibitors. (A) PvdQ hydrolysisof either p-nitrophenyl myristate or 4-MU-lauratesubstrates result in production of compounds that can be detectedthrough absorbance or fluorescence. (B) Comparison of biochemicalPvdQ inhibition activities of prior art compounds (1, 2) to the high-throughput screening lead 3 and the optimizedprobe 4.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215858&req=5

fig2: High-throughput screenfor PvdQ inhibitors. (A) PvdQ hydrolysisof either p-nitrophenyl myristate or 4-MU-lauratesubstrates result in production of compounds that can be detectedthrough absorbance or fluorescence. (B) Comparison of biochemicalPvdQ inhibition activities of prior art compounds (1, 2) to the high-throughput screening lead 3 and the optimizedprobe 4.
Mentions: To examine the role of PvdQ in pyoverdine maturation, wedevelopeda high-throughput biochemical assay to find inhibitors of the PvdQacylase activity.24 The assay monitoredthe hydrolysis of p-nitrophenyl myristate (Figure 2A) and showed good reproducibility and signal-to-noiseparameters, with Z′ scores of 0.7–0.9 within one plateand 0.6 overall. In this proof-of-concept study, we screened 1280compounds, identifying aryl bromides 1 and 2 (Figure 2B), which exhibit IC50 values of 130 μM and 65 μM and bind in the fatty acidbinding pocket.24 This success with a smalllibrary suggested that a more thorough effort might lead to compoundswith higher affinity. We therefore conducted a high-throughput screenwith a larger chemical library and 4-methylumbelliferyl laurate (4-MUlaurate), a fluorogenic substrate with improved signal-to-noise properties,to identify more potent scaffolds for PvdQ inhibition that can serveas tool compounds for understanding pyoverdine maturation and therapeuticleads for P. aeruginosa infection.

Bottom Line: Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase.ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318.Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

View Article: PubMed Central - PubMed

Affiliation: The Broad Institute , Cambridge, Massachusetts 02142, United States.

ABSTRACT
Pseudomonas aeruginosa produces the peptide siderophore pyoverdine, which is used to acquire essential Fe(3+) ions from the environment. PvdQ, an Ntn hydrolase, is required for the biosynthesis of pyoverdine. PvdQ knockout strains are not infectious in model systems, suggesting that disruption of siderophore production via PvdQ inhibition could be exploited as a target for novel antibacterial agents, by preventing cells from acquiring iron in the low iron environments of most biological settings. We have previously described a high-throughput screen to identify inhibitors of PvdQ that identified inhibitors with IC50 values of ∼100 μM. Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

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Related in: MedlinePlus