Identification of Inhibitors of PvdQ, an Enzyme Involved in the Synthesis of the Siderophore Pyoverdine.

The growing prevalence of drug-resistant bacterial pathogens is of significant concern in the United States and worldwide. Of particular concern are the multidrug resistant Gram-negative bacteria including Klebsiella pneumoniae, Acinetobacter species, Enterobacter species, and Pseudomonas aeruginosa.(1,2) Gram-negative human pathogens such as P. aeruginosa typically require intracellular iron levels in the micromolar range for growth and infectivity.3,4 The low abundance of iron in a typical host environment has provided a selective pressure for P. aeruginosa to develop a mechanism to extract iron from the extracellular milieu. Targeting siderophore biosynthesis as a strategy to reduce virulence5 has received much attention recently. Salicyl-AMS (5′-O-(N-salicylsulfamoyl)adenosine), a nM inhibitor of the myocobactin biosynthetic enzyme MbtA,6−8 reduces the growth of M. tuberculosis in mouse lungs.9 Importantly, this work validates the approach that preventing pathogen access to essential nutrients and demonstrates the bioavailability of the Salicyl-AMS inhibitor and the primary importance of mycobactin over other iron-acquisition pathways. P. aeruginosa produces pyoverdine, a peptide siderophore that scavenges extracellular iron.10 Secreted pyoverdine binds to Fe3+ with high affinity (Kf ∼ 1024 M–1 at pH 7.0) and the resulting complex is taken into the bacterial cell through a specific receptor.11 Pyoverdine also plays a role in the regulation of other P. aeruginosa virulence factors12−14 and biofilm formation.15−17 It has been shown that pyoverdine-deficient mutant strains are not infectious in the mouse lung,18 plant,19 and C. elegans.20 Pyoverdine is biosynthesized by four nonribosomal peptide synthetases (NRPSs) and 10 additional modifying enzymes.10,21 The modular NRPS enzymes contain multiple catalytic domains joined in a single protein that catalyze peptide production in an assembly line fashion. During synthesis, the nascent peptide is covalently bound to an integrated peptidyl carrier protein domain and delivered to the neighboring catalytic domains. In this modular architecture, each NRPS module catalyzes the incorporation of a single substrate into the final peptide product.22 Pyoverdine is composed of a conserved dihydroquinoline-type chromophore and a peptide tail that varies among different Pseudomonas species (Figure ​(Figure11A).11,21 Additionally, most strains produce variable pyoverdine isoforms with N-terminal succinate, succinamide, or glutamate moieties bound to the chromophore. PvdL, the first NRPS protein of the pyoverdine pathway, is shared among all sequenced pseudomonads and generates the peptide backbone that is converted into this chromophore.21 Interestingly, PvdL contains a N-terminal module with homology to fatty acyl-CoA ligases.23 We recently24 showed that this unusual NRPS architecture incorporates a myristate molecule, subsequently identified as either myristic or myristoleic acid,25 at the N-terminus of an intermediate in pyoverdine biosynthesis. Additionally, we demonstrated that the incorporated fatty acid, which is not present on mature pyoverdine, is removed by PvdQ,24 one of the 10 auxiliary proteins necessary for pyoverdine synthesis (Figure ​(Figure11B).10 PvdQ belongs to a family of N-terminal nucleophile (Ntn) hydrolases that catalyze the cleavage of amide bonds via an acylated enzyme intermediate.26 PvdQ exhibits promiscuity in activity and also cleaves acyl-homoserine lactones that are involved in quorum signaling.27,28 Figure 1 Structure of pyoverdine and the role of PvdQ in biosynthesis. (A) The mature pyoverdine siderophore is an undecapeptide containing an N-terminal sidearm, the chromophore, and a species-specific peptide chain. (B) PvdQ catalyzes the removal of the myristoyl ... To examine the role of PvdQ in pyoverdine maturation, we developed a high-throughput biochemical assay to find inhibitors of the PvdQ acylase activity.24 The assay monitored the hydrolysis of p-nitrophenyl myristate (Figure ​(Figure2A)2A) and showed good reproducibility and signal-to-noise parameters, with Z′ scores of 0.7–0.9 within one plate and 0.6 overall. In this proof-of-concept study, we screened 1280 compounds, identifying aryl bromides 1 and 2 (Figure ​(Figure2B),2B), which exhibit IC50 values of 130 μM and 65 μM and bind in the fatty acid binding pocket.24 This success with a small library suggested that a more thorough effort might lead to compounds with higher affinity. We therefore conducted a high-throughput screen with a larger chemical library and 4-methylumbelliferyl laurate (4-MU laurate), a fluorogenic substrate with improved signal-to-noise properties, to identify more potent scaffolds for PvdQ inhibition that can serve as tool compounds for understanding pyoverdine maturation and therapeutic leads for P. aeruginosa infection. Figure 2 High-throughput screen for PvdQ inhibitors. (A) PvdQ hydrolysis of either p-nitrophenyl myristate or 4-MU-laurate substrates result in production of compounds that can be detected through absorbance or fluorescence. (B) Comparison of biochemical PvdQ ...