Fig 1.
A. Schematic showing the proposed mechanism of oxidative folding in the periplasm of Gram-negative bacteria. DsbA catalyses the formation of a disulfide bond in a protein substrate, then interacts with DsbB to which it transfers electrons so that DsbA is regenerated into its active oxidized state. The electrons are subsequently transferred from DsbB to ubiquinone (UQ) and ultimately to the respiratory complex. B. The binding interface between EcDsbA (black and red) and EcDsbB loop P2 (blue) derived from the crystal structure of the EcDsbAC33A:EcDsbBC130S complex [37]. The EcDsbA hydrophobic groove residues are highlighted in orange shading, the intermolecular disulfide bond is shown as a solid red line and the hydrogen bond with the cisPro loop is shown as a dashed red line. C. The binding interface between PmDsbA (black and red) and the peptide PWATCDS (blue) from the crystal structure of the complex [38]. In this complex there is no disulfide bond as the active site cysteine of PmDsbA was mutated to Ser (S30). The peptide Cys5 residue points away from the binding interface. Residues W2 and P1 of the peptide both interact with the hydrophobic groove (in orange) and these interactions were used as the target for this peptidomimetic design.
Fig 2.
Synthetic route for the tripeptide peptidomimetics.
Fig 3.
Comparison of the docked designed peptidomimetic with the EcDsbA-EcDsbB and PmDsbA-PWATCDS crystal structures.
Calculated electrostatic surfaces of the enzymes are shown, with acidic regions in red, basic regions in blue and non-polar (hydrophobic) regions in white. Electrostatics cut-offs used are +/- 7.5 keV. A. Detail of the EcDsbA complex with EcDsbB from the crystal structure (PDB code 2ZUP [37]) centred on the 97YPSPFATCDFMVR109 sequence of EcDsbB (in light blue) showing Phe101 (F101) binding in the EcDsbA hydrophobic groove (circled). B. Detail of the PmDsbAC30S:PWATCDS crystal structure (PDB code 4OD7) with PWATCDS in magenta. Residue Trp2 (W2) of the peptide binds in the PmDsbA hydrophobic groove (circled). C. Virtual screening identified compound 1 as a potential hit. Three optimal conformations of 1 are shown (in differing shades of green), in their predicted binding mode to the PmDsbAC30S hydrophobic groove. Potential hydrogen bonds between the morpholine moiety and DsbA Pro150, His32 and Asn162 are shown as yellow dashed lines.
Fig 4.
Chemical structures of the 10 peptidomimetic compounds synthesized and tested in this work.
Compound 1 is the hit from the virtual screening from which derivatives 2–10 were designed.
Fig 5.
Compound 10 demonstrated weak inhibitory activity.
A. Differential scanning fluorimetry profile with increasing concentrations of compound 10. Similar to all other compounds tested, there was no significant shift in the unfolding temperature of EcDsbA up to 2 mM of compound 10. B. ITC profile of EcDsbA titration by compound 10, which shows no detectable binding under the conditions used (see methods for details). A similar outcome was found for the other 9 compounds. C. Compound 10 was the only one of the ten tested peptidomimetics that exhibited detectable activity in the DsbA assay, inducing a reduction in DsbA folding activity. D. Plotting the log of the peptidomimetic concentration against the rate of fluorescence increase measured in the enzyme assay allowed fitting of a sigmoidal curve and an estimated IC50 value of ~1 mM for compound 10. The positive control with no compound is shown as a white circle.