A Designed Analog of an Antimicrobial Peptide, Crabrolin, Exhibits Enhanced Anti-Proliferative and In Vivo Antimicrobial Activity.

Antimicrobial peptides have gradually attracted interest as promising alternatives to conventional agents to control the worldwide health threats posed by antibiotic resistance and cancer. Crabrolin is a tridecapeptide extracted from the venom of the European hornet (Vespa crabro). Its antibacterial and anticancer potentials have been underrated compared to other peptides discovered from natural resources. Herein, a series of analogs were designed based on the template sequence of crabrolin to study its structure-activity relationship and enhance the drug's potential by changing the number, type, and distribution of charged residues. The cationicity-enhanced derivatives were shown to have improved antibacterial and anticancer activities with a lower toxicity. Notably, the double-arginine-modified product, crabrolin-TR, possessed a potent capacity against Pseudomonas aeruginosa (minimum inhibitory concentration (MIC) = 4 µM), which was around thirty times stronger than the parent peptide (MIC = 128 µM). Furthermore, crabrolin-TR showed an in vivo treatment efficacy in a Klebsiella-pneumoniae-infected waxworm model and was non-toxic under its maximum MBC value (MIC = 8 µM), indicating its therapeutic potency and better selectivity. Overall, we rationally designed functional peptides by progressively increasing the number and distribution of charged residues, demonstrating new insights for developing therapeutic molecules from natural resources with enhanced properties, and proposed crabrolin-TR as an appealing antibacterial and anticancer agent candidate for development.

A promising antibiotic candidate, brevinin-1 analogue 5R, against drug-resistant bacteria, with insights into its membrane-targeting mechanism.

In recent decades, antimicrobial peptides (AMPs) have held great promise as novel antibiotic agents. However, they have generally been excluded from clinical use due to certain limitations, such as poor biocompatibility and sensitivity to environmental conditions. In this study, we report a novel brevinin-1 type antimicrobial peptide B1LTe, derived from the skin secretion of Hylarana latouchii. Although the novel peptide B1LTe exhibited remarkable antimicrobial effects, its narrow therapeutic index (TI) can result in adverse drug reactions. Thus, the rational design by systematically scanning and replacing the inherent hydrophobic and cationic residues (Leucine and Lysine) with their D-enantiomeric counterparts was conducted to enhance the application potential of B1LTe. Simultaneously, we also applied lysine-to-arginine substitution within the modification. Among the derivates, 5 R demonstrated the highest selectivity and effectiveness against Methicillin-resistant Streptococcus aureus (MRSA), clinic-isolated Streptococcus pyogenes (S. pyogenes) strain, ranging from their planktonic to biofilm cells, both in vitro and in vivo. Furthermore, the remarkable adaptation of 5 R in saline and 20% serum indicates its potential for clinical application. We employed the in silico approach, which revealed the mechanism of interaction between 5 R and bacterial membranes. In addition, further mechanistic studies of 5 R elucidated the association between the collapsed proton motive force (PMF) and membrane perturbation as peptides aggregate on the bacterial membrane. Overall, our study suggests the D-enantiomeric 5 R can be a promising antibiotic agent against MDR bacteria in further clinical development and highlights the significance of cellular PMF as a potential target for the research of peptides' mode of action.

Progressive Design of a Ranatuerin-2 Peptide from Amolops wuyiensis: Enhancement of Bioactivity and In Vivo Efficacy.

Antimicrobial peptides (AMPs) that exert multiple functions are considered promising candidates to combat the bacterial drug resistance crisis. Nowadays, targeted peptide modification has been widely recognised to improve biological activity and make up for deficiencies in clinical applications such as toxicity. In this study, a helix-loop peptide was isolated and identified from the skin secretion of the Wuyi torrent frog Amolops wuyiensis, namely, ranatuerin-2-AW (R2AW) (GFMDTAKNVAKNVAATLLDKLKCKITGGC). Target modifications were made to R2AW to study the structure-activity relationships and to optimise its bioactivities. Five analogues were progressively designed via residue substitution and truncation and the antibacterial and anticancer activities were evaluated. We found that the serine-substitution and cyclic-domain-deletion products showed similar antibacterial activity to the natural peptide R2AW, implying that the disulphide bridge and Rana box were dispensable for the antibacterial activity of ranatuerin-2 peptides. Notably, the cationicity- and hydrophobicity-enhanced variant, [Lys4,19, Leu20]R2AW(1-22)-NH2, exhibited significantly optimised antibacterial and anticancer activities. Additionally, it killed bacteria by membrane disruption at a highly efficient rate. Moreover, [Lys4,19, Leu20]R2AW(1-22)-NH2 exerted potential in vivo efficacy in a methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Overall, this study demonstrated some rational design ideas for optimising the dual antibacterial and anticancer activities of ranatuerin-2 peptides and it proposes [Lys4,19, Leu20]R2AW(1-22)-NH2 as an appealing candidate for therapeutic development.

Modification Strategy of D-leucine Residue Addition on a Novel Peptide from Odorrana schmackeri, with Enhanced Bioactivity and In Vivo Efficacy.

Brevinins are a well-characterised, frog-skin-derived, antimicrobial peptide (AMP) family, but their applications are limited by high cytotoxicity. In this study, a wild-type des-Leu2 brevinin peptide, named brevinin-1OS (B1OS), was identified from Odorrana schmackeri. To explore the significant role of the leucine residue at the second position, two variants, B1OS-L and B1OS-D-L, were designed by adding L-leucine and D-leucine residues at this site, respectively. The antibacterial and anticancer activities of B1OS-L and B1OS-D-L were around ten times stronger than the parent peptide. The activity of B1OS against the growth of Gram-positive bacteria was markedly enhanced after modification. Moreover, the leucine-modified products exerted in vivo therapeutic potential in an methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Notably, the single substitution of D-leucine significantly increased the killing speed on lung cancer cells, where no viable H838 cells survived after 2 h of treatment with B1OS-D-L at 10 µM with low cytotoxicity on normal cells. Overall, our study suggested that the conserved leucine residue at the second position from the N-terminus is vital for optimising the dual antibacterial and anticancer activities of B1OS and proposed B1OS-D-L as an appealing therapeutic candidate for development.

Generation of truncated derivatives through in silico enzymatic digest of peptide GV30 target MRSA both in vitro and in vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) causing serious hospital-acquired infections and skin infections has become a "superbug" in clinical treatment. Although the clinical treatment of MRSA is continuously improving, due to its unceasing global spread, MRSA has produced much heated discussion and focused study, therefore suggesting an urgent task to find new antibacterial drugs to combat this issue. Antimicrobial peptides (AMPs) are used as the last-resort drugs for treating multidrug-resistant bacterial infections, but their utilisation is still limited due to their low stability and often strong toxicity. Here, we evaluated the structure and the bioactivity of an AMP, GV30, derived from the frog skin secretions of Hylarana guentheri, and designed seven truncated derivatives based on the presence of cleavage sites for trypsin using an online proteomic bioinformatic resource PeptideCutter tool. We investigated the anti-MRSA effect, toxicity and salt- and serum-resistance of these peptides. Interestingly, the structure-activity relationship revealed that removing "Rana box" loop could significantly improve the bactericidal speed on MRSA. Among these derivatives, GV21 (GVIFNALKGVAKTVAAQLLKK-NH2), because of its faster antibacterial effect, lower toxicity, and retains the good antibacterial activity and stability of the parent peptide, is considered to become a new potential antibacterial candidate against MRSA.