Tuning of a Membrane-Perforating Antimicrobial Peptide to Selectively Target Membranes of Different Lipid Composition.

The use of designed antimicrobial peptides as drugs has been impeded by the absence of simple sequence-structure-function relationships and design rules. The likely cause is that many of these peptides permeabilize membranes via highly disordered, heterogeneous mechanisms, forming aggregates without well-defined tertiary or secondary structure. We suggest that the combination of high-throughput library screening with atomistic computer simulations can successfully address this challenge by tuning a previously developed general pore-forming peptide into a selective pore-former for different lipid types. A library of 2916 peptides was designed based on the LDKA template. The library peptides were synthesized and screened using a high-throughput orthogonal vesicle leakage assay. Dyes of different sizes were entrapped inside vesicles with varying lipid composition to simultaneously screen for both pore size and affinity for negatively charged and neutral lipid membranes. From this screen, nine different LDKA variants that have unique activity were selected, sequenced, synthesized, and characterized. Despite the minor sequence changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic lipid bilayers. Long-scale, unbiased atomistic molecular dynamics (MD) simulations directly reveal that rather than rigid, well-defined pores, these peptides can form a large repertoire of functional dynamic and heterogeneous aggregates, strongly affected by single mutations. Predicting the propensity to aggregate and assemble in a given environment from sequence alone holds the key to functional prediction of membrane permeabilization.

The stereochemical effect of SMAP-29 and SMAP-18 on bacterial selectivity, membrane interaction and anti-inflammatory activity.

Sheep myeloid antimicrobial peptide-29 (SMAP-29) is a cathelicidin-related antimicrobial peptide derived from sheep myeloid cells. In order to investigate the effects of L-to-D-amino acid substitution in SMAP-29 on bacterial selectivity, membrane interaction and anti-inflammatory activity, we synthesized its two D-enantiomeric peptides (SMAP-29-E1 and SMAP-29-E2 containing D-Ile and D-allo-Ile, respectively) and two diastereomeric peptides (SMAP-29-D1 and SMAP-29-D2). Additionally, in order to address the effect of L-to-D-amino acid substitution in the N-terminal helical peptide of SMAP-29 (named SMAP-18) on antimicrobial activity, we synthesized its two D-enantiomeric peptides (SMAP-18-E1 and SMAP-18-E2), which are composed of D-amino acids entirely. L-to-D-amino acid substitution in membrane-targeting AMP, SMAP-29 did not affect its antimicrobial activity. However, D-allo-Ile containing-SMAP-29-E2 and SMAP-29-D2 exhibited less hemolytic activity compared to D-Ile containing-SMAP-29-E1 and SMAP-29-D1, respectively. L-to-D-amino acid substitution in intracellular targeting-AMPs, SMAP-18 and buforin-2 improved antimicrobial activity by 2- to eightfold. The improved antimicrobial activity of the D-isomers of SMAP-18 and buforin-2 seems to be due to the stability against proteases inside bacterial cells. Membrane depolarization and dye leakage suggested that the membrane-disruptive mode of SMAP-29-D1 and SMAP-29-D2 is different from that of SMAP-29, SMAP-29-E1, and SMAP-29-E2. L-to-D-amino acid substitution in SMAP-29 improved anti-inflammatory activity in LPS-stimulated RAW 264.7 cells. In summary, we propose here that D-allo-Ile substitution is a more powerful strategy for increasing bacterial selectivity than D-Ile substitution in the design of D-enantiomeric and diastereomeric AMPs. SMAP-29-D1, and SMAP-29-D2 with improved bacterial selectivity and anti-inflammatory activity can serve as promising candidates for the development of anti-inflammatory and antimicrobial agents.

Biocompatible Catanionic Vesicles from Arginine-Based Surfactants: A New Strategy to Tune the Antimicrobial Activity and Cytotoxicity of Vesicular Systems.

Their stability and low cost make catanionic vesicles suitable for application as drug delivery systems. In this work we prepared catanionic vesicles using biocompatible surfactants: two cationic arginine-based surfactants (the monocatenary Nα-lauroyl-arginine methyl ester-LAM and the gemini Nα,Nϖ-bis(Nα-lauroylarginine) α, ϖ-propylendiamide-C3(CA)2) and three anionic amphiphiles (the single chain sodium dodecanoate, sodium myristate, and the double chain 8-SH). The critical aggregation concentration, colloidal stability, size, and charge density of these systems were comprehensively studied for the first time. These catanionic vesicles, which form spontaneously after mixing two aqueous solutions of oppositely charged surfactants, exhibited a monodisperse population of medium-size aggregates and good stability. The antimicrobial and hemolytic activity of the vesicles can be modulated by changing the cationic/anionic surfactant ratio. Vesicles with a positive charge efficiently killed Gram-negative and Gram-positive bacteria as well as yeasts; the antibacterial activity declined with the decrease of the cationic charge density. The catanionic systems also effectively eradicated MRSA (Methicillin-resistant Staphylococcus Aureus) and Pseudomonas aeruginosa biofilms. Interestingly, the incorporation of cholesterol in the catanionic mixtures improved the stability of these colloidal systems and considerably reduced their cytotoxicity without affecting their antimicrobial activity. Additionally, these catanionic vesicles showed good DNA affinity. Their antimicrobial efficiency and low hemolytic activity render these catanionic vesicles very promising candidates for biomedical applications.

The design of a cell-selective fowlicidin-1-derived peptide with both antimicrobial and anti-inflammatory activities.

Fowlicidin-1 (Fowl-1), a cathelicidin expressed in chicken intestine, is known to have both antimicrobial and anti-inflammatory properties. However, its pharmaceutical development has been ultimately compromised by its high host cytotoxicity. In this study, a series of N- and C-terminal-truncated 19-meric Fowl-1 peptides were synthesized. Among these truncated peptides, Fowl-1 (8-26) exhibited broad-spectrum antimicrobial activity without human erythrocyte cytotoxicity while reducing anti-inflammatory activity. Further, Fowl-1 (8-26)-WRK was designed via Thr5→Trp, Ile7→Arg, and Asn11→Lys substitutions in Fowl-1 (8-26) to exhibit more amphipathicity. The results revealed that it exhibited both antimicrobial and anti-inflammatory properties. This study also demonstrated that the inhibitory activity of Fowl-1 (8-26)-WRK against LPS-induced inflammation was mainly due to the binding of LPS to the peptide. Interestingly, compared with human cathelicidin LL-37 and melittin, Fowl-1 (8-26)-WRK showed more potent activity against drug-resistant bacteria. It was also resistant to physiological salts and human serum and acted synergistically in combination with conventional antibiotics, such as chloramphenicol, ciprofloxacin, and oxacillin, suggesting that combined with conventional antibiotics, it is a promising adjuvant. Furthermore, membrane depolarization, SYTOX Green uptake, and flow cytometry revealed that it kills bacteria by damaging their membrane integrity. Therefore, this study suggests that Fowl-1 (8-26)-WRK has considerable potential for future development as an antimicrobial and anti-inflammatory agent for treating antibiotic-resistant infections.

Coordination-Assisted Self-Assembled Polypeptide Nanogels to Selectively Combat Bacterial Infection.

In the present scenario, the invention of bacteria-selective antimicrobial agent comprising negligible toxicity and hemolytic effect is a great challenge. To surmount this challenge, here, a series of polypeptide nanogels (PNGs) have been fabricated by a coordination-assisted self-assembly of a mannose-conjugated antimicrobial polypeptide, poly(arginine-r-valine)-mannose (poly(Arg-r-Val)-M2), with Zn2+ ions. The fabricated PNGs are spherical in shape with a unique structural appearance similar to that of Taxus baccata fruits. PNGs, with a unique structural arrangement and threshold surface charge density, selectively interact with the bacterial membrane and exhibit potent antimicrobial activity, as reflected in their lower minimum inhibitory concentration values (varies from 2 to 16 µg/mL). PNGs show a remarkably high binding constant, 6.02 × 105 M-1 (from isothermal titration calorimetry, ITC), with the bacterial membrane which manifests its potent bactericidal effect. PNGs are nontoxic against mammalian and red blood cells as reflected from their higher cell viability and insignificant hemolytic effect. PNGs are taken up by the bacterial membrane and selectively undergo structural deformation (scrutinized by ITC) followed by an exposure of free poly(Arg-r-Val)-M2 molecules. The free poly(Arg-r-Val)-M2 molecules are enforced to lyse the bacterial membrane (visualized by cryo-transmission electron microscopy) followed by the diffusion of the cytoplasmic component out of the membrane which culminates in the final death of the bacterium.