Open in a separate window produces the peptide siderophore pyoverdine, which

Open in a separate window produces the peptide siderophore pyoverdine, which is used to acquire essential Fe3+ ions from the environment. pathogens is usually of significant concern in the United States and worldwide. Of particular concern are the multidrug resistant Gram-negative bacteria including species, species, and 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 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 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. produces pyoverdine, a peptide siderophore that scavenges extracellular iron.10 Secreted pyoverdine binds to Fe3+ with high affinity (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 herb,19 and species (Determine ?(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 usually shared among all sequenced pseudomonads and generates the peptide backbone that is converted into this chromophore.21 Interestingly, PvdL contains Lopinavir (ABT-378) 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 Lopinavir (ABT-378) of an intermediate in pyoverdine biosynthesis. Additionally, we exhibited that the incorporated fatty acid, which is not present on mature pyoverdine, is usually removed by PvdQ,24 one of the 10 auxiliary proteins necessary for pyoverdine synthesis (Physique ?(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 Open in a separate window Determine 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 group from the pyoverdine precursor. 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 infection. Open in a separate window Physique 2 High-throughput screen for PvdQ inhibitors. (A) PvdQ hydrolysis of either (PAO1 strain) in the presence of metal chelator ethylenediamine-had two readouts; absorption at 600 Lopinavir (ABT-378) nm was measured as a reporter of growth inhibition, while absorption at 405 nm was Lopinavir (ABT-378) measured as a reporter of pyoverdine production.30 Biarylnitrile 3 was Rabbit Polyclonal to Cortactin (phospho-Tyr466) selected for further development on the basis of activity against PvdQ, activity in the whole cell assay, specificity as determined by lack of activity in other PubChem Bioassays, lack of toxicity with HeLa cells, and chemical tractability for generation of compound analogues. The hit compound 3 exhibited an IC50 of 40 nM against PvdQ acylase in biochemical assays (Physique ?(Figure2).2). In preliminary growth assays, the hit compound displayed an IC50 of 59 M against PAO1.29 In addition, 3 was not active in any other assay submitted to PubChem at the time of analysis, including toxicity studies with other bacteria, including and position (8,11) led to at least 7-fold decreased potency. By comparison, analogues with an electron-withdrawing group at the position (9, 10, 12, 13) performed better. Table 1 SAR Analysis of Eastern Analogues Open in a separate windows analogRM34-Cl0.04 0.017H0.30 0.0282-OCH30.40 0.0894-F0.07 0.02104-CF30.39 0.10112-OCH31.7 0.6122-Cl, 4-Cl0.25 0.04132-F, 4-Cl0.06 0.02 Open in a separate window aAverage of at least three replicates standard deviation. With regards to the western half of the scaffold, synthetic efforts focused on removing the potentially labile 2-chloro substituent around the pyridine ring without sacrificing potency (Table 2). Removal of the 2-chloro (24) resulted in a 7-fold increase in IC50. The 3-chloro analogue (14) showed reduced potency relative to Lopinavir (ABT-378) lead 3. Meanwhile, the 5-chloro analogue 15 increased potency.