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Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.

Duprez W, Bachu P, Stoermer MJ, Tay S, McMahon RM, Fairlie DP, Martin JL - PLoS ONE (2015)

Bottom Line: By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized.Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors.The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.

ABSTRACT
Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

No MeSH data available.


Related in: MedlinePlus

Synthetic route for the tripeptide peptidomimetics.
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pone.0133805.g002: Synthetic route for the tripeptide peptidomimetics.

Mentions: All purchased chemicals were used without further purification. All solvents were HPLC grade. Boc-Trp-OH (1.00 mmol) was dissolved in 10 mL of dioxane under nitrogen. Carbonyldiimidazole (1.02 mmol) was then added in a stepwise manner and the resulting mixture stirred for 3 h at room temperature and then heated to 50°C for 30 min. The desired amine (1.03 mmol) was added to the reaction mixture at room temperature and stirring was continued for 48 h (Fig 2). The solvent was removed under reduced pressure and crude product was extracted with ethyl acetate (3 x 20ml). The organic extracts were washed with 1M hydrochloric acid (15 mL), saturated sodium bicarbonate solution (15 mL) and water (15 mL), then dried over anhydrous MgSO4 and the solvent was removed under reduced pressure. To this crude compound in water (1 mL) was added anisole (2.00 mmol). The solution was then cooled to 0°C prior to addition of trifluoroacetic acid (30.00 mmol) in 1 mL of water. After stirring for 1 h at 0°C, the reaction mixture was allowed to warm to room temperature and stirring was continued for 12 h. The reaction mixture was then diluted with ethyl acetate (15 mL) and washed with saturated sodium bicarbonate solution (15 mL), water (15 mL) and brine solution (15 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure to afford the crude free amine which was directly used in the next step without further purification. To a solution of the above crude amine in dichloromethane (5 mL) was added carbonyldiimidazole (1.2 mmol) under nitrogen at room temperature. After 3 h stirring, morpholine or 1-Boc-piperazine (1.55 mmol) was added to the reaction mixture and stirred for another 12 h at room temperature. The solvent was removed under reduced pressure, diluted with ethyl acetate (15 mL) and washed with 1M hydrochloric acid (15 mL), saturated sodium bicarbonate solution (15 mL), brine solution (15 mL) and dried over anhydrous MgSO4. The crude product was then purified by preparative HPLC (Gradient 0 to 100% of 95/5 acetonitrile/water solution over 25 min) and freeze-dried. NMR spectral data of all synthesized compounds 1–10 are included in the supporting information (S1–S10 Figs). NMR spectra were recorded on Bruker Avance DRX-600 and Varian 400 MHz spectrometers at 298 K with TMS as internal standard. High-resolution mass spectrometry (HRMS) was performed on a Bruker micro-TOF by direct infusion in acetonitrile/H2O 70:30 at 3 μL/min using sodium formate clusters as an internal calibrant. Semi-preparative RP-HPLC purification of the compounds was performed using a Waters Delta 600 chromatography system fitted with a Waters 486 tuneable absorbance detector with detection at 214 nm. Purification was performed by eluting with solvents A (0.1% TFA in water) and B (9:1 CH3CN:H2O, 0.1% TFA) on a Vydac C18 250 x 22 mm (300 Å) steel jacketed column at 20 mL/min. NH peak of Indole is not observed in some of the compounds in CDCl3 due to peak broadness.


Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.

Duprez W, Bachu P, Stoermer MJ, Tay S, McMahon RM, Fairlie DP, Martin JL - PLoS ONE (2015)

Synthetic route for the tripeptide peptidomimetics.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520593&req=5

pone.0133805.g002: Synthetic route for the tripeptide peptidomimetics.
Mentions: All purchased chemicals were used without further purification. All solvents were HPLC grade. Boc-Trp-OH (1.00 mmol) was dissolved in 10 mL of dioxane under nitrogen. Carbonyldiimidazole (1.02 mmol) was then added in a stepwise manner and the resulting mixture stirred for 3 h at room temperature and then heated to 50°C for 30 min. The desired amine (1.03 mmol) was added to the reaction mixture at room temperature and stirring was continued for 48 h (Fig 2). The solvent was removed under reduced pressure and crude product was extracted with ethyl acetate (3 x 20ml). The organic extracts were washed with 1M hydrochloric acid (15 mL), saturated sodium bicarbonate solution (15 mL) and water (15 mL), then dried over anhydrous MgSO4 and the solvent was removed under reduced pressure. To this crude compound in water (1 mL) was added anisole (2.00 mmol). The solution was then cooled to 0°C prior to addition of trifluoroacetic acid (30.00 mmol) in 1 mL of water. After stirring for 1 h at 0°C, the reaction mixture was allowed to warm to room temperature and stirring was continued for 12 h. The reaction mixture was then diluted with ethyl acetate (15 mL) and washed with saturated sodium bicarbonate solution (15 mL), water (15 mL) and brine solution (15 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure to afford the crude free amine which was directly used in the next step without further purification. To a solution of the above crude amine in dichloromethane (5 mL) was added carbonyldiimidazole (1.2 mmol) under nitrogen at room temperature. After 3 h stirring, morpholine or 1-Boc-piperazine (1.55 mmol) was added to the reaction mixture and stirred for another 12 h at room temperature. The solvent was removed under reduced pressure, diluted with ethyl acetate (15 mL) and washed with 1M hydrochloric acid (15 mL), saturated sodium bicarbonate solution (15 mL), brine solution (15 mL) and dried over anhydrous MgSO4. The crude product was then purified by preparative HPLC (Gradient 0 to 100% of 95/5 acetonitrile/water solution over 25 min) and freeze-dried. NMR spectral data of all synthesized compounds 1–10 are included in the supporting information (S1–S10 Figs). NMR spectra were recorded on Bruker Avance DRX-600 and Varian 400 MHz spectrometers at 298 K with TMS as internal standard. High-resolution mass spectrometry (HRMS) was performed on a Bruker micro-TOF by direct infusion in acetonitrile/H2O 70:30 at 3 μL/min using sodium formate clusters as an internal calibrant. Semi-preparative RP-HPLC purification of the compounds was performed using a Waters Delta 600 chromatography system fitted with a Waters 486 tuneable absorbance detector with detection at 214 nm. Purification was performed by eluting with solvents A (0.1% TFA in water) and B (9:1 CH3CN:H2O, 0.1% TFA) on a Vydac C18 250 x 22 mm (300 Å) steel jacketed column at 20 mL/min. NH peak of Indole is not observed in some of the compounds in CDCl3 due to peak broadness.

Bottom Line: By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized.Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors.The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.

ABSTRACT
Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

No MeSH data available.


Related in: MedlinePlus