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Identification and validation of a novel anti-virulent that binds to pyoverdine and inhibits its function
Xu Wang1, Quinn Kleerekoper2, Alexey V Revtovich1
1Department of BioSciences, Rice University , Houston, TX, USA.
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 high-throughput 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. 1H-15N and 1H-13C 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. Abbreviations: NMR: nuclear magnetic resonance; SAR: structure-activity relationship; MD: molecular dynamics; RMSF: root-mean-square fluctuation; HSQC: heteronuclear single quantum correlation; DMSO: dimethyl sulfoxide; Δδavg: average amide chemical shift change; DSS: 2,2-dimethyl-2-silapentane-5-sulfonate; RMSD: root-mean-square deviation; LJ-SR: Lennard-Jones short-range; Coul-SR: Coulombic short-range; FRET: fluorescence resonance energy transfer.
Insights
Researchers screened compounds to inhibit pyoverdine, a virulence factor in Pseudomonas aeruginosa infections. A compound, PQ3c, effectively quenched pyoverdine fluorescence and improved survival in C. elegans models, offering a new therapeutic target.
Area of Science:
- Microbiology
- Drug Discovery
- Biochemistry
Background:
- Pseudomonas aeruginosa causes severe infections, particularly in immunocompromised patients.
- Rising antimicrobial resistance necessitates novel treatment strategies.
- Pyoverdine, a P. aeruginosa siderophore, is crucial for virulence.
Purpose of the Study:
- To identify compounds that inhibit pyoverdine's function by quenching its fluorescence.
- To evaluate the therapeutic potential of identified compounds against P. aeruginosa infections.
- To elucidate the binding mechanism and site of the most effective inhibitor.
Main Methods:
- High-throughput screening of compounds for pyoverdine fluorescence quenching.
- In vivo efficacy testing using Caenorhabditis elegans infection models.
- Biophysical characterization using fluorescence spectroscopy and nuclear magnetic resonance (NMR).
- Molecular docking and molecular dynamics simulations to model the inhibitor-pyoverdine complex.
Main Results:
- Several compounds effectively quenched pyoverdine fluorescence.
- Two compounds significantly improved C. elegans survival against P. aeruginosa.
- PQ3c demonstrated potent inhibition and its binding site on pyoverdine was identified.
- Molecular simulations revealed electrostatic interactions and pi-orbital stacking stabilize PQ3c binding.
Conclusions:
- PQ3c represents a promising scaffold for developing new pyoverdine inhibitors.
- The identified binding site offers a novel target for therapeutic intervention.
- This work provides a new direction for combating P. aeruginosa infections.
