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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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Structure of PvdQ bound to HTS hit compound 3. (A)Electron density is shown, calculated with coefficients of the formFo-Fc generated prior to building the ligand in the active site. Densityis contoured at 3σ (gray) and 8σ (red). (B) Active siteof the enzyme is shown of the PvdQ bound to 3. Superposedon the structure is the fatty acid chain from covalently acylatedstructure from PDB 3L94.
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fig4: Structure of PvdQ bound to HTS hit compound 3. (A)Electron density is shown, calculated with coefficients of the formFo-Fc generated prior to building the ligand in the active site. Densityis contoured at 3σ (gray) and 8σ (red). (B) Active siteof the enzyme is shown of the PvdQ bound to 3. Superposedon the structure is the fatty acid chain from covalently acylatedstructure from PDB 3L94.

Mentions: We examined the bindingof the new compounds with X-ray crystallography. The initial HTS lead 3 and the fluorinated analogue 4 were selectedfor structural studies. The binding mode of biaryl nitriles to PvdQwas first studied by solving the structure of PvdQ bound to 3 at a resolution of 2.0 Å (Figure 4). Hit compound 3 sits in the acyl-binding site withthe 4-chlorophenyl directed into the pocket away from the catalyticnucleophile (Figure 4). Hydrophobic residuesLeu169, Leu269, Val374, Leu375, Trp378, and Pro401, as well as themethyl group of Thr166 surround the 4-chlorophenyl ring. The 2-chloropyridinelies in a hydrophobic pocket created by Met245, Phe248, Phe240, andHis284. Leu266 fits between the two aromatic rings of and contributesto the hydrophobic cavities for both rings. Hydrophobic interactionsdominate both spaces that accommodate the aromatic rings of the biarylnitrilescaffold.


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 HTS hit compound 3. (A)Electron density is shown, calculated with coefficients of the formFo-Fc generated prior to building the ligand in the active site. Densityis contoured at 3σ (gray) and 8σ (red). (B) Active siteof the enzyme is shown of the PvdQ bound to 3. Superposedon the structure is the fatty acid chain from covalently acylatedstructure from PDB 3L94.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Structure of PvdQ bound to HTS hit compound 3. (A)Electron density is shown, calculated with coefficients of the formFo-Fc generated prior to building the ligand in the active site. Densityis contoured at 3σ (gray) and 8σ (red). (B) Active siteof the enzyme is shown of the PvdQ bound to 3. Superposedon the structure is the fatty acid chain from covalently acylatedstructure from PDB 3L94.
Mentions: We examined the bindingof the new compounds with X-ray crystallography. The initial HTS lead 3 and the fluorinated analogue 4 were selectedfor structural studies. The binding mode of biaryl nitriles to PvdQwas first studied by solving the structure of PvdQ bound to 3 at a resolution of 2.0 Å (Figure 4). Hit compound 3 sits in the acyl-binding site withthe 4-chlorophenyl directed into the pocket away from the catalyticnucleophile (Figure 4). Hydrophobic residuesLeu169, Leu269, Val374, Leu375, Trp378, and Pro401, as well as themethyl group of Thr166 surround the 4-chlorophenyl ring. The 2-chloropyridinelies in a hydrophobic pocket created by Met245, Phe248, Phe240, andHis284. Leu266 fits between the two aromatic rings of and contributesto the hydrophobic cavities for both rings. Hydrophobic interactionsdominate both spaces that accommodate the aromatic rings of the biarylnitrilescaffold.

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