Two distinct amphipathic peptide antibiotics with systemic efficacy.

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.

Design of Antimicrobial Peptides: Progress Made with Human Cathelicidin LL-37.

The incorporation of the innate immune system into humans is essential for survival and health due to the rapid replication of invading microbes and the delayed action of the adaptive immune system. Antimicrobial peptides are important components of human innate immunity. Over 100 such peptides have been identified in various human tissues. Human cathelicidin LL-37 is best studied, and there has been a growing interest in designing new peptides based on LL-37. This chapter describes the alternative processing of the human cathelicidin precursor, protease digestion, and lab cutting of LL-37. Both a synthetic peptide library and structure-based design are utilized to identify the active regions. Although challenging, the determination of the 3D structure of LL-37 enabled the identification of the core antimicrobial region. The minimal region of LL-37 can be function-dependent. We discuss the design and potential applications of LL-37 into antibacterial, antibiofilm, antiviral, antifungal, immune modulating, and anticancer peptides. LL-37 has been engineered into 17BIPHE2, a stable, selective, and potent antimicrobial, antibiofilm, and anticancer peptide. Both 17BIPHE2 and SAAP-148 can eliminate the ESKAPE pathogens and show topical in vivo antibiofilm efficacy. Also discussed are other application strategies, including peptide formulation, antimicrobial implants, and peptide-inducing factors such as vitamin D and sunlight. Finally, we summarize what we learned from peptide design based on human LL-37.

The π Configuration of the WWW Motif of a Short Trp-Rich Peptide Is Critical for Targeting Bacterial Membranes, Disrupting Preformed Biofilms, and Killing Methicillin-Resistant Staphylococcus aureus.

Tryptophan-rich peptides, being short and suitable for large-scale chemical synthesis, are attractive candidates for developing a new generation of antimicrobials to combat antibiotic-resistant bacteria (superbugs). Although there are numerous pictures of the membrane-bound structure of a single tryptophan (W), how multiple Trp amino acids assemble themselves and interact with bacterial membranes is poorly understood. This communication presents the three-dimensional structure of an eight-residue Trp-rich peptide (WWWLRKIW-NH2 with 50% W) determined by the improved two-dimensional nuclear magnetic resonance method, which includes the measurements of 13C and 15N chemical shifts at natural abundance. This peptide forms the shortest two-turn helix with a distinct amphipathic feature. A unique structural arrangement is identified for the Trp triplet, WWW, that forms a π configuration with W2 as the horizontal bar and W1/W3 forming the two legs. An arginine scan reveals that the WWW motif is essential for killing methicillin-resistant Staphylococcus aureus USA300 and disrupting preformed bacterial biofilms. This unique π configuration for the WWW motif is stabilized by aromatic-aromatic interactions as evidenced by ring current shifts as well as nuclear Overhauser effects. Because the WWW motif is maintained, a change of I7 to R led to a potent antimicrobial and antibiofilm peptide with 4-fold improvement in cell selectivity. Collectively, this study elucidated the structural basis of antibiofilm activity of the peptide, identified a better peptide candidate via structure-activity relationship studies, and laid the foundation for engineering future antibiotics based on the WWW motif.

Sequence Permutation Generates Peptides with Different Antimicrobial and Antibiofilm Activities.

Antibiotic resistance poses a threat to our society, and 10 million people could die by 2050. To design potent antimicrobials, we made use of the antimicrobial peptide database (APD). Using the database filtering technology, we identified a useful template and converted it into an effective peptide WW291 against methicillin-resistant Staphylococcus aureus (MRSA). Here, we compared the antibacterial activity and cytotoxicity of a family of peptides obtained from sequence permutation of WW291. The resulting eight WW peptides (WW291-WW298) gained different activities against a panel of bacteria. While WW295 inhibited the growth of Escherichia coli, WW298 was highly active against S. aureus USA300 LAC. Consistently with this, WW298 was more effective in permeating or depolarizing the S. aureus membranes, whereas WW295 potently permeated the E. coli membranes. In addition, WW298, but not WW295, inhibited the MRSA attachment and could disrupt its preformed biofilms more effectively than daptomycin. WW298 also protected wax moths Galleria mellonella from MRSA infection causing death. Thus, sequence permutation provides one useful avenue to generating antimicrobial peptides with varying activity spectra. Taken together with amino acid composition modulation, these methods may lead to narrow-spectrum peptides that are more promising to selectively eliminate invading pathogens without damaging commensal microbiota.

A Peptide-Nanoparticle System with Improved Efficacy against Multidrug Resistant Bacteria.

The recent rise of multidrug resistant microbial strains requires development of new and novel therapeutic alternatives. In this study, we present a novel antibacterial system that comprises of modified naturally abundant antimicrobial peptides in conjugation with silver nanoparticles. Further, we propose a simple route to incorporate a cysteine residue either at the N- or C-terminal of the parent peptide. Tagging a cysteine residue at the terminals not only enhances the binding propensity of the resultant peptide with the silver nanoparticle, but also increases its antimicrobial property against several pathogenic bacterial strains including K. pneumoniae. The minimum inhibitory concentration (MIC) values of the cysteine tagged nanoconjugates were obtained in the range of 5-15 µM compared to 50 µM for peptides devoid of the cysteines. The origin and mechanism of such improved activity of the conjugates were investigated using NMR spectroscopy and molecular dynamics (MD) simulations. The application of 13C-isotope labelled media to track the metabolic lifecycle of E. coli cells provided further insights into the system. MD simulations showed that pore formation in membrane bilayer is mediated through a hydrophobic collapse mechanism. The design strategy described herein opens up new-avenues for using biocompatible nanomedicines as a potential alternative to conventional antibiotics.

Host defense antimicrobial peptides as antibiotics: design and application strategies.

This review deals with the design and application strategies of new antibiotics based on naturally occurring antimicrobial peptides (AMPs). The initial candidate can be designed based on three-dimensional structure or selected from a library of peptides from natural or laboratory sources followed by optimization via structure-activity relationship studies. There are also advanced application strategies such as induction of AMP expression from host cells by various factors (e.g., metals, amino acids, vitamin D and sunlight), the use of engineered probiotic bacteria to deliver peptides, the design of prodrug and peptide conjugates to improve specific targeting. In addition, combined uses of newly developed AMPs with existing antimicrobial agents may provide a practical avenue for effective management of antibiotic-resistant bacteria (superbugs), including biofilms. Finally, we highlight AMPs already in use or under clinical trials.