Structure–Activity Study, Characterization, and Mechanism of Action of an Antimicrobial Peptoid D2 and Its d- and l-Peptide Analogues

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) constitutes an emerging health problem for companion animals in veterinary medicine. Therefore, discovery of novel antimicrobial agents for treatment of Staphylococcus-associated canine infections is urgently needed to reduce use of human antibiotics in veterinary medicine. In the present work, we characterized the antimicrobial activity of the peptoid D2 against S. pseudintermedius and Pseudomonas aeruginosa, which is another common integumentary pathogen in dogs. Furthermore, we performed a structure–activity relationship study of D2, which included 19 peptide/peptoid analogs. Our best compound D2D, an all d-peptide analogue, showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius (2–4 µg/mL) and P. aeruginosa (4 µg/mL) isolates as well as other selected dog pathogens (2–16 µg/mL). Time–kill assays demonstrated that D2D was able to inhibit MRSP in 30 min at 1× MIC, significantly faster than D2. Our results suggest that at high concentrations D2D is rapidly lysing the bacterial membrane while D2 is inhibiting macromolecular synthesis. We probed the mechanism of action at sub-MIC concentrations of D2, D2D, the l-peptide analog and its retro analog by a macromolecular biosynthesis assay and fluorescence spectroscopy. Our data suggest that at sub-MIC concentrations D2D is membrane inactive and primarily works by cell wall inhibition, while the other compounds mainly act on the bacterial membrane

Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide–oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design

nteractions between DNA and Psoralenamines Studied with Dichroism Techniques

The interaction of a series of water-soluble mono- and bis-psoralenamines with calf thymus DNA has been studied with flow UV linear dichroism (LD), circular dichroism (CD) and equilibrium techniques. The positive charge of a protonated amino group strongly enhances the DNA affinity compared to that of the parent compound, 8-methoxypsoralen. The orientation of the psoralen when bound to DNA, depends on the position of the amino substituent. With amino substituents in the 5-position (on the‘hydrophobic edge’of psoralen) psoralenamines tend to bind with a considerable tilt relative to the average orientation of the DNA base-pairs. The tilt generally increases with an increased psoralen: base-pair ratio, indicating a more random, nonintercalated binding. With the amino substituents in the 8-position the psoralen binds with its plane parallel to that of the DNA bases as expected for intercalation. The DNA CD supports that these psoralenamines induce a considerable perturbation of the DNA structure, and the CD induced in the psoralen chromophore is in qualitative agreement with intercalation. The study also includes a theoretical and an experimental determination of the UV transition moments of the psoralen chromophore