Design of Proteolytic-Resistant Antifungal Peptides by Utilizing Minimum d-Amino Acid Ratios.

Antifungal peptides are an appealing alternative to standard antifungal medicines due to their unique mechanism of action and low-level resistance. However, their susceptibility to protease degradation keeps hindering their future development. Herein, a library was established to design peptides with protease resistance and high antifungal activity. The peptides were incorporated with minimal D-amino acids to further improve the protease stability. The most active peptide, IR3, demonstrated good antifungal activity and low toxicity, and its molecular integrity was maintained after protease hydrolysis for 8 h at 2 mg/mL. Furthermore, IR3 could permeate the fungal cell wall, disrupt the cell membrane, produce reactive oxygen species, and induce apoptosis in fungal cells. In vivo experiments confirmed that IR3 could effectively treat fungal keratitis. Collectively, these findings suggest that IR3 is a promising antifungal agent and may be beneficial in the design and development of protease-resistant antifungal peptides.

Self-Assembling Peptide Dendron Nanoparticles with High Stability and a Multimodal Antimicrobial Mechanism of Action.

Self-assembling nanometer-scale structured peptide polymers and peptide dendrimers have shown promise in biomedical applications due to their versatile properties and easy availability. Herein, self-assembling peptide dendron nanoparticles (SPDNs) with potent antimicrobial activity against a range of bacteria were developed based on the nanoscale self-assembly of an arginine-proline repeat branched peptide dendron bearing a hexadecanoic acid chain. The SPDNs are biocompatible, and our most active peptide dendron nanoparticle, C16-3RP, was found to have negligible toxicity after both in vitro and in vivo studies. Furthermore, the C16-3RP nanoparticles showed excellent stability under physiological concentrations of salt ions and against serum and protease degradation, resulting in highly effective treatment in a mouse acute peritonitis model. Comprehensive analyses using a series of biofluorescence, microscopy, and transcriptome sequencing techniques revealed that C16-3RP nanoparticles kill Gram-negative bacteria by increasing bacterial membrane permeability, inducing cytoplasmic membrane depolarization and drastic membrane disruption, inhibiting ribosome biogenesis, and influencing energy generation and other processes. Collectively, C16-3RP nanoparticles show promising biocompatibility and in vivo therapeutic efficacy without apparent resistance development. These advancements may facilitate the development of peptide-based antibiotics in clinical settings.

Design of Heptad Repeat Amphiphiles Based on Database Filtering and Structure-Function Relationships to Combat Drug-Resistant Fungi and Biofilms.

Due to the emergence of reports of multidrug-resistant fungi, infections caused by multidrug-resistant fungi and biofilms are considered to be a global threat to human health due to the lack of effective broad-spectrum drugs. Here, we developed a series heptad repeat sequences based on an antimicrobial peptide database (APD) and structure-function relationships. Among the developed peptides, the target peptide ACR3 exhibited good activity against all fungi and bacteria tested, including fluconazole-resistant Candida albicans (C. albicans) and methicillin-resistant Staphylococcu saureus (S. aureus), while demonstrating relatively low toxicity and good salt tolerance. The peptide ACR3 inhibits the formation of C. albicans biofilms and has a therapeutic effect on mature biofilms in vitro and in vivo. Moreover, we did not observe any resistance of C. albicans and E. coli against the peptide ACR3. A series of assays and microscopy were used to analyze the antimicrobial mechanism. These results showed that the antimicrobial activity of the peptide ACR3 utilizes a multimodal mechanism that degrades the cell wall barrier, alters the cytoplasmic membrane electrical potential, and induces intracellular reactive oxygen species (ROS) production. In general, the peptide ACR3 is a potent antibacterial agent that shows great potential for use in biomedical coatings and healthcare formulas to combat the growing threat of fungal and bacterial infection.

Effect of terminal arrangement of tryptophan on biological activity of symmetric α-helix-forming peptides.

It is traditionally believed that the distribution of tryptophan (Trp) residues is critical for the novo design of antimicrobial peptides (AMPs). However, there is scarce knowledge regarding Trp residues arrangement at the head group level. Thus, a set of α-helical AMPs containing different Trp residue arrangements at the N-/C-terminal of sequence were designed to increase the strategy database and analyze their biological activities. The arrangement of the N-terminal Trp residue significantly improved the bacteriostatic activity of the peptides, but the C-terminal Trp residue arrangement reduced the biocompatibility of them. WL and LW were effective against Gram-negative microbes and had high selectivity for bacteria as compared to human erythrocytes and mammalian cells. They both maintained a relatively desirable activity in the presence of physiological salts and serum. It is observed through electron microscope, flow cytometry and fluorescence spectroscopy that target peptides can penetrate bacterial cell membrane and kill it by damaging cell membrane integrity. Collectively, we determined the structure-activity relationship of Trp residue distributions in a symmetric sequence structure and filled the gap in knowledge related to Trp arrangements at the head group level. The obtained results will be helpful in designing of artificial peptide-based antimicrobials.

Highly Stabilized α-Helical Coiled Coils Kill Gram-Negative Bacteria by Multicomplementary Mechanisms under Acidic Condition.

Although antimicrobial peptides (AMPs) hold tremendous promise in overcoming the threats of multidrug resistance, the main obstacle to successful therapeutic applications is their poor stability. Various synthetic strategies such as unnatural amino acids and chemical modifications have made advances for improving this problem. However, this complicated synthesis often greatly increases the cost of production. Here, we show that a series of novel peptides, designed by combining an α-helical coiled coil model, knowledge of the specificity of proteolysis and major parameters of AMPs, exhibited efficient activity against all tested Gram-negative bacteria under acidic condition and demonstrate low toxicity. Of these α-helical coiled coil peptides, 3IH3 displayed the highest average therapeutic index (GMTI = 294.25) with high stability toward salts, serum, extreme pH, heat, and proteases. Electron microscopy and biological analytical technique analyses showed that 3IH3 killed bacterial cells via a multicomplementary mechanism at pH 6.0, with physical membrane disruption as the dominant bactericidal mechanism. These results suggest that 3IH3 shows great stability as an inexpensive and effective antimicrobial activity agent and has the potential for clinical application in the treatment of infections occurring in body sites with acidic pH.