Limits...
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.

Show MeSH

Related in: MedlinePlus

Structure of PvdQ bound to 4. (A)Final probe compoundML318 is shown with electron density, calculated with coefficientsof the form Fo-Fc generated prior to building the ligand in the activesite, also included. Density is contoured at 3σ (gray) and 8σ(red). (B) Active site of the enzyme is shown of the PvdQ bound to 4. As in Figure 4, the fatty acid chainfrom covalently acylated structure from PDB 3L94 is also shown.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215858&req=5

fig5: Structure of PvdQ bound to 4. (A)Final probe compoundML318 is shown with electron density, calculated with coefficientsof the form Fo-Fc generated prior to building the ligand in the activesite, also included. Density is contoured at 3σ (gray) and 8σ(red). (B) Active site of the enzyme is shown of the PvdQ bound to 4. As in Figure 4, the fatty acid chainfrom covalently acylated structure from PDB 3L94 is also shown.

Mentions: Co-crystallization of PvdQwith the more potent biaryl nitrile 4 offered additionalinsights (Figure 5). Crystals diffracted at2.3 Å and the density of 4 was positioned in thePvdQ acyl-binding pocket analogously to 3, allowing facileplacement of the ligand. Residues Thr166,Leu266, Leu269, Val374, and Trp378 make contacts to 4-fluorophenylas with 4-chlorophenyl of 3. Again, the nitrile was surroundedby Pro401, Trp402, and Val403 residues, which provided key side chainand π-stacking interactions. Parallel-displaced and T-shapedπ-stacking to Phe240 and Trp378, respectively, also enforcedstrong binding of the aromatic rings within the active site. Interestingly,the replacement of 2-chloro substituent on pyridine with trifluoromethylprovided the largest structural change between the binding of ligands 3 and 4. Whereas the only residue within 4 Åof the pyridyl chlorine atom of 3 is His284, each fluorineatom from the CF3 interacts with hydrophobic functionalityon Phe240, Ile274, Trp378, Trp402, and Val403 side chains. These hydrophobicinteractions are along the same vector observed for the natural myristateligand and these interactions could account for the increase in potencyobserved for 4.


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)

Structure of PvdQ bound to 4. (A)Final probe compoundML318 is shown with electron density, calculated with coefficientsof the form Fo-Fc generated prior to building the ligand in the activesite, also included. Density is contoured at 3σ (gray) and 8σ(red). (B) Active site of the enzyme is shown of the PvdQ bound to 4. As in Figure 4, the fatty acid chainfrom covalently acylated structure from PDB 3L94 is also shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Structure of PvdQ bound to 4. (A)Final probe compoundML318 is shown with electron density, calculated with coefficientsof the form Fo-Fc generated prior to building the ligand in the activesite, also included. Density is contoured at 3σ (gray) and 8σ(red). (B) Active site of the enzyme is shown of the PvdQ bound to 4. As in Figure 4, the fatty acid chainfrom covalently acylated structure from PDB 3L94 is also shown.
Mentions: Co-crystallization of PvdQwith the more potent biaryl nitrile 4 offered additionalinsights (Figure 5). Crystals diffracted at2.3 Å and the density of 4 was positioned in thePvdQ acyl-binding pocket analogously to 3, allowing facileplacement of the ligand. Residues Thr166,Leu266, Leu269, Val374, and Trp378 make contacts to 4-fluorophenylas with 4-chlorophenyl of 3. Again, the nitrile was surroundedby Pro401, Trp402, and Val403 residues, which provided key side chainand π-stacking interactions. Parallel-displaced and T-shapedπ-stacking to Phe240 and Trp378, respectively, also enforcedstrong binding of the aromatic rings within the active site. Interestingly,the replacement of 2-chloro substituent on pyridine with trifluoromethylprovided the largest structural change between the binding of ligands 3 and 4. Whereas the only residue within 4 Åof the pyridyl chlorine atom of 3 is His284, each fluorineatom from the CF3 interacts with hydrophobic functionalityon Phe240, Ile274, Trp378, Trp402, and Val403 side chains. These hydrophobicinteractions are along the same vector observed for the natural myristateligand and these interactions could account for the increase in potencyobserved for 4.

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.

Show MeSH
Related in: MedlinePlus