Identification and validation of a novel anti-virulent that binds to pyoverdine and inhibits its function

Abstract

Pseudomonas aeruginosa: causes serious infections in patients with compromised immune systems and exhibits resistance to multiple antibiotics. The rising threat of antimicrobial resistance means that new methods are necessary for treating microbial infections. We conducted a highthroughput screen for compounds that can quench the innate fluorescence of the chromophore region of the P. aeruginosa siderophore pyoverdine, a key virulence factor. Several hits were identified that effectively quench pyoverdine fluorescence, and two compounds considerably improved the survival of Caenorhabditis elegans when worms were challenged with P. aeruginosa. Commercially available analogs of the best hit, PQ3, were tested for their ability to rescue C. elegans from P. aeruginosa and to interact with pyoverdine via fluorescence and solution NMR spectroscopy. 1 H-15 N and 1 H-13 C HSQC NMR were used to identify the binding site of PQ3c. The structure model of pyoverdine in complex with PQ3c was obtained using molecular docking and molecular dynamics simulations. PQ3c occupied a shallow groove on pyoverdine formed by the chromophore and N-terminal residues of the peptide chain. Electrostatic interactions and π-orbital stacking drove stabilization of this binding. PQ3c may serve as a scaffold for the development of pyoverdine inhibitors with higher potency and specificity. The discovery of a small-molecule binding site on apo-pyoverdine with functional significance provides a new direction in the search of therapeutically effective reagent to treat P. aeruginosa infections.

An In Vitro Cell Culture Model for Pyoverdine-Mediated Virulence

“…For instance, Imperi and colleagues identified 5-fluorocytosine from a screen for pyoverdine biosynthetic inhibitors and validated its therapeutic properties in a murine lung infection model [ 32 ]. Our lab has identified several compounds that inhibit pyoverdine function and rescue C. elegans hosts during P. aeruginosa pathogenesis [ 43 ]. Inhibitors of the type III secretion system, elastase LasB, and quorum-sensing have also been identified and validated using these model pathosystems [ 52 , 53 , 54 , 55 ].…”

“…For this reason, gallium (III) rescues G. mellonella or C. elegans from P. aeruginosa pathogenesis [ 31 , 42 ]. We also recently reported several molecules that block pyoverdine function by interacting with the chromophore region of the siderophore [ 43 , 44 ]. These inhibitors decreased the expression of PvdS-regulated genes and rescued C. elegans from P. aeruginosa pathogenesis.…”

Novel Immune Modulators Enhance Caenorhabditis elegans Resistance to Multiple Pathogens

“…These drugs have MIC/EC ratios that range from 0.57 to 2.7 ( 18 ). In contrast, compounds that prevent pyoverdine biosynthesis or function, such as 5-fluorocytosine, LK11, LK31a, and PQ3c, exhibit MIC/EC ratios that range from 15 to >35 ( 14 , 18 , 19 ). For compounds with an MIC/EC ratio of >10, indicating a nonantimicrobial mechanism, the expression of 115 genes involved in C. elegans host defense pathways was evaluated using a previously designed custom nanoString codeset ( 18 ).…”

“…Using a well-developed C. elegans - P. aeruginosa pathosystem, we have previously carried out a moderately sized, phenotype-based screen of several fragment-based small molecule libraries. Hits from this screen appeared to fall into several broad categories, including conventional antimicrobials (i.e., those that prevent bacterial growth) ( 14 , 69 ), drugs that interfere with bacterial factors required for virulence (e.g., by preventing the production or function of pyoverdine) ( 18 , 19 ), and those that appear to stimulate host immunity. Here, we characterized five molecules—LK32, LK34, LK35, LK38, and LK56—that fall into this last category.…”

“…We previously carried out a high-throughput, high-content, fragment-based phenotypic screen for small molecules capable of extending C. elegans survival during exposure to P. aeruginosa in liquid ( 14 ). In the process, ∼70 novel small molecules were identified, some of which possessed antibacterial or anti-virulence properties ( 14 , 18 , 19 ). However, a number of hits had no apparent effect on bacterial growth (suggesting that they are not antimicrobials) and also did not prevent the production or the function of pyoverdine (the most important virulence determinant in the assay used for the screen).…”