Compound 10 demonstrated weak inhibitory activity.

<p><b>A.</b> Differential scanning fluorimetry profile with increasing concentrations of compound <b>10</b>. Similar to all other compounds tested, there was no significant shift in the unfolding temperature of EcDsbA up to 2 mM of compound <b>10</b>. <b>B.</b> ITC profile of EcDsbA titration by compound <b>10</b>, which shows no detectable binding under the conditions used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133805#sec002" target="_blank">methods</a> for details). A similar outcome was found for the other 9 compounds. <b>C.</b> Compound <b>10</b> was the only one of the ten tested peptidomimetics that exhibited detectable activity in the DsbA assay, inducing a reduction in DsbA folding activity. <b>D.</b> 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 <b>10</b>. The positive control with no compound is shown as a white circle.</p

Synthetic route for the tripeptide peptidomimetics.

<p>Synthetic route for the tripeptide peptidomimetics.</p

Comparison of the docked designed peptidomimetic with the EcDsbA-EcDsbB and PmDsbA-PWATCDS crystal structures.

<p>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. <b>A.</b> Detail of the EcDsbA complex with EcDsbB from the crystal structure (PDB code 2ZUP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133805#pone.0133805.ref037" target="_blank">37</a>]) centred on the ⁹⁷YPSPFATCDFMVR¹⁰⁹ sequence of EcDsbB (in light blue) showing Phe101 (F101) binding in the EcDsbA hydrophobic groove (circled). <b>B.</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). <b>C.</b> Virtual screening identified compound <b>1</b> as a potential hit. Three optimal conformations of <b>1</b> 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.</p

The DsbA-DsbB interaction.

<p><b>A.</b> 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>B.</b> The binding interface between EcDsbA (black and red) and EcDsbB loop P2 (blue) derived from the crystal structure of the EcDsbAC33A:EcDsbBC130S complex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133805#pone.0133805.ref037" target="_blank">37</a>]. 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 <i>cis</i>Pro loop is shown as a dashed red line. <b>C.</b> The binding interface between PmDsbA (black and red) and the peptide PWATCDS (blue) from the crystal structure of the complex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133805#pone.0133805.ref038" target="_blank">38</a>]. 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.</p

Chemical structures of the 10 peptidomimetic compounds synthesized and tested in this work.

<p>Compound <b>1</b> is the hit from the virtual screening from which derivatives <b>2</b>–<b>10</b> were designed.</p

Potent Heterocyclic Ligands for Human Complement C3a Receptor

The G-protein coupled receptor (C3aR) for human inflammatory protein complement C3a is an important component of immune, inflammatory, and metabolic diseases. A flexible compound (<i>N</i>2-[(2,2-diphenylethoxy)­acetyl]-l-arginine, <b>4</b>), known as a weak C3aR antagonist (IC₅₀ μM), was transformed here into potent agonists (EC₅₀ nM) of human macrophages (Ca²⁺ release in HMDM) by incorporating aromatic heterocycles. Antagonists were also identified. A linear correlation between binding affinity for C3aR and calculated hydrogen-bond interaction energy of the heteroatom indicated that its hydrogen-bonding capacity influenced ligand affinity and function mediated by C3aR. Hydrogen-bond accepting heterocycles (e.g., imidazole) conferred the highest affinity and agonist potency (e.g., <b>21</b>, EC₅₀ 24 nM, Ca²⁺, HMDM) with comparable efficacy and immunostimulatory activity as that of C3a in activating human macrophages (Ca²⁺, IL1β, TNFα, CCL3). These potent and selective modulators of C3aR, inactivated by a C3aR antagonist, are stable C3a surrogates for interrogating roles for C3aR in physiology and disease

Three Homology Models of PAR2 Derived from Different Templates: Application to Antagonist Discovery

Protease activated receptor 2 (PAR2) is an unusual G-protein coupled receptor (GPCR) involved in inflammation and metabolism. It is activated through cleavage of its N-terminus by proteases. The new N-terminus functions as a tethered ligand that folds back and intramolecularly activates PAR2, initiating multiple downstream signaling pathways. The only compounds reported to date to inhibit PAR2 activation are of moderate potency. Three structural models for PAR2 have been constructed based on sequence homology with known crystal structures for bovine rhodopsin, human ORL-1 (also called nociceptin/orphanin FQ receptor), and human PAR1. The three PAR2 model structures were compared and used to predict potential interactions with ligands. Virtual screening for ligands using the Chembridge database, and either ORL-1 or PAR1 derived PAR2 models led to identification of eight new small molecule PAR2 antagonists (IC₅₀ 10–100 μM). Notably, the most potent compound <b>1</b> (IC₅₀ 11 μM) was derived from the less homologous template protein, human ORL-1. The results suggest that virtual screening against multiple homology models of the same GPCR can produce structurally diverse antagonists and that this may be desirable even when some models have less sequence homology with the target protein

Cyclic Penta- and Hexaleucine Peptides without <i>N</i>‑Methylation Are Orally Absorbed

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with <i>N</i>-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no <i>N</i>-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4–17%) approaching that of the heavily <i>N</i>-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for <i>N</i>-methylation or modified amino acids