Mechanistic insights on the antibacterial action of the kyotorphin peptide derivatives revealed by in vitro studies and Galleria mellonella proteomic analysis.

OBJECTIVES: The selected kyotorphin derivatives were tested to improve their antimicrobial and antibiofilm activity. The antimicrobial screening of the KTP derivatives were ascertained in the representative strains of bacteria, including Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa. METHODS: Kyotorphin derivatives, KTP-NH2, KTP-NH2-DL, IbKTP, IbKTP-NH2, MetKTP-DL, MetKTP-LD, were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. Peptides were synthesized by standard solution or solid-phase peptide synthesis and purified using RP-HPLC, which resulted in >95 % purity, and were fully characterized by mass spectrometry and 1H NMR. The minimum inhibitory concentrations (MIC) determined for bacterial strains were between 20 and 419 µM. The direct effect of IbKTP-NH2 on bacterial cells was imaged using scanning electron microscopy. The absence of toxicity, high survival after infection and an increase in the hemocytes count was evaluated by injections of derivatives in Galleria mellonella larvae. Proteomics analyses of G. mellonella hemolymph were performed to investigate the underlying mechanism of antibacterial activity of IbKTP-NH2 at MIC. RESULTS: IbKTP-NH2 induces morphological changes in bacterial cell, many differentially expressed proteins involved in DNA replication, synthesis of cell wall, and virulence were up-regulated after the treatment of G. mellonella with IbKTP-NH2. CONCLUSION: We suggest that this derivative, in addition to its physical activity on the bacterial membranes, can elicit a cellular and humoral immune response, therefore, it could be considered for biomedical applications.

Co-Functionalization of Gold Nanoparticles with C7H2 and HuAL1 Peptides: Enhanced Antimicrobial and Antitumoral Activities.

The functionalization of nanoparticles with therapeutic peptides has been pointed out as a promising strategy to improve the applications of these molecules in the field of health sciences. Peptides are highly bioactive but face several limitations such as low bioavailability due to the difficulty of overcoming the physiological barriers in the body and their degradation by enzymes. In this work, gold nanoparticles (AuNPs) were co-functionalized with two therapeutic peptides simultaneously. The peptides from the complementary determining region of monoclonal antibodies, composed of the amino acid sequences YISCYNGATSYNQKFK (C7H2) and RASQSVSSYLA (HuAL1) were chosen for having exhibited antitumor and antimicrobial activity before. The peptides-conjugated AuNPs were characterized regarding size, morphology, and metal concentration by using TEM, dynamic light scattering, and ICP-OES techniques. Then, peptides-conjugated AuNPs were evaluated regarding the antimicrobial activity against E. coli, P. aeruginosa, and C. albicans. The antitumoral activity was evaluated in vitro by cell viability assays with metastatic melanoma cell line (B16F10-Nex2) and the cytotoxicity was evaluated against human foreskin fibroblast (Hs68) cell line. Finally, in vivo assays were performed by using a syngeneic animal model of metastatic melanoma. Our findings have highlighted the potential application of the dual-peptide AuNPs in order to enhance the antitumor and antimicrobial activity of peptides.

Synthesis, Structure, and Activity of the Antifungal Plant Defensin PvD1.

Available treatments for invasive fungal infections have limitations, including toxicity and the emergence of resistant strains. Therefore, there is an urgent need for alternative solutions. Because of their unique mode of action and high selectivity, plant defensins (PDs) are worthy therapeutic candidates. Chemical synthesis remains a preferred method for the production of many peptide-based therapeutics. Given the relatively long sequence of PDs, as well as their complicated posttranslational modifications, the synthetic route can be considered challenging. Here, we describe a total synthesis of PvD1, the defensin from the common bean Phaseolus vulgaris. Analytical, structural, and functional characterization revealed that both natural and synthetic peptides fold into a canonical CSαß motif stabilized by conserved disulfide bonds. Moreover, synthetic PvD1 retained the biological activity against four different Candida species and showed no toxicity in vivo. Adding the high resistance of synthetic PvD1 to proteolytic degradation, we claim that conditions are now met to consider PDs druggable biologicals.