Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors.

Antibiotic-resistant bacteria are an emerging threat to global public health. New classes of antibiotics and tools for antimicrobial discovery are urgently needed. Type III secretion systems (T3SS), which are required by dozens of Gram-negative bacteria for virulence but largely absent from nonpathogenic bacteria, are promising virulence blocker targets. The ability of mammalian cells to recognize the presence of a functional T3SS and trigger NF-κB activation provides a rapid and sensitive method for identifying chemical inhibitors of T3SS activity. In this study, we generated a HEK293 stable cell line expressing green fluorescent protein (GFP) driven by a promoter containing NF-κB enhancer elements to serve as a readout of T3SS function. We identified a family of synthetic cyclic peptide-peptoid hybrid molecules (peptomers) that exhibited dose-dependent inhibition of T3SS effector secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa without affecting bacterial growth or motility. Among these inhibitors, EpD-3'N, EpD-1,2N, EpD-1,3'N, EpD-1,2,3'N, and EpD-1,2,4'N exhibited strong inhibitory effects on translocation of the Yersinia YopM effector protein into mammalian cells (>40% translocation inhibition at 7.5 µM) and showed no toxicity to mammalian cells at 240 µM. In addition, EpD-3'N and EpD-1,2,4'N reduced the rounding of HeLa cells caused by the activity of Yersinia effector proteins that target the actin cytoskeleton. In summary, we have discovered a family of novel cyclic peptomers that inhibit the injectisome T3SS but not the flagellar T3SS.

Amide-to-ester substitution as a stable alternative to N-methylation for increasing membrane permeability in cyclic peptides.

Naturally occurring peptides with high membrane permeability often have ester bonds on their backbones. However, the impact of amide-to-ester substitutions on the membrane permeability of peptides has not been directly evaluated. Here we report the effect of amide-to-ester substitutions on the membrane permeability and conformational ensemble of cyclic peptides related to membrane permeation. Amide-to-ester substitutions are shown to improve the membrane permeability of dipeptides and a model cyclic hexapeptide. NMR-based conformational analysis and enhanced sampling molecular dynamics simulations suggest that the conformational transition of the cyclic hexapeptide upon membrane permeation is differently influenced by an amide-to-ester substitution and an amide N-methylation. The effect of amide-to-ester substitution on membrane permeability of other cyclic hexapeptides, cyclic octapeptides, and a cyclic nonapeptide is also investigated to examine the scope of the substitution. Appropriate utilization of amide-to-ester substitution based on our results will facilitate the development of membrane-permeable peptides.

Functional hypervariability and gene diversity of cardioactive neuropeptides.

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.

Competitive binding effects on surface-enhanced Raman scattering of peptide molecules.

Surface enhanced Raman scattering (SERS) has been conducted on tryptophan (W), proline (P) and tyrosine (Y) containing peptides that include W-P-Y, Y-P-W, W-P-P-P-Y, Y-P-P-P-W, W-P-P-P-P-P-Y, and Y-P-P-P-P-P-W to gain insight into molecular binding behavior on a metal substrate to eventually apply in protein SERS detection. The peptides are shown to bind through the molecule's carboxylic end, but the strong affinity of the tryptophan residue to the substrate surface, in conjunction with its large polarizability, dominates each molecule's SERS signal with the strong presence of its ring modes in all samples. These results are important for understanding SERS of protein molecules.

HALO384: a halo-based potency prediction algorithm for high-throughput detection of antimicrobial agents.

A high-throughput (HT) agar-based halo assay is described, which allows for rapid screening of chemical libraries for bioactivity in microorganisms such as yeast and bacteria. A pattern recognition algorithm was developed to identify halo-like shapes in plate reader optical density (OD) measurements. The authors find that the total growth inhibition within a detected halo provides an accurate estimate of a compound's potency measured in terms of its EC(50). The new halo recognition method performs significantly better than an earlier method based on single-point OD readings. An assay based on the halo algorithm was used to screen a 21,120-member library of drug-like compounds in Saccharomyces cerevisiae, leading to the identification of novel bioactive scaffolds containing derivatives of varying potencies. The authors also show that the HT halo assay can be performed with the pathogenic bacterium Vibrio cholerae and that liquid culture EC(50) values and halo scores show a good correlation in this organism. These results suggest that the HT halo assay provides a rapid and inexpensive way to screen for bioactivity in multiple microorganisms.