Novel analogues of the scorpion venom-derived peptide, opistoporin 1, targeting gram-negative bacteria

Abstract

Antimicrobial peptides (AMPs) are promising candidates and lead compounds for the development of next generation antimicrobials. At the onset of this study, 46 AMP candidates, including 27 novel or with unknown activity, were identified from African species such as scorpions, frogs, ticks, and primates. Antimicrobial screening against a diverse panel of susceptible and resistant Gram-negative and Gram-positive bacterial strains as well as fungi was undertaken. The results revealed that while many of the AMPs exhibited potent antimicrobial activity, significant cytotoxicity towards host cells, a common challenge in AMP therapeutic development, was also identified. To address this, multivariate analyses were employed to correlate 28 physicochemical peptide properties to the antimicrobial efficacy and cytotoxicity of the 46 AMPs, aiming to identify characteristics that drive selective antimicrobial effectiveness while minimising toxicity. Principal component analysis (PCA) and quantitative structure-activity relationships (QSAR) revealed that an increase in lipophilicity, hydrophobicity, or residue side chain surface area results in increased activity as well as toxicity of an AMP. Crucially, the total negative lipophilicity contributed by polar residues (Lp) was identified as the key physicochemical property determining selective antimicrobial activity. The scorpion derived peptides proved particularly effective against Gram-negative bacteria. Among them, Opis16a emerged as a novel peptide with significant potential for therapeutic development. Subsequently, a more detailed assessment of the activity, stability, and mechanism of action of Opis16a, particularly against sensitive strains and drug-resistant Gram-negative clinical isolates, was undertaken. Findings were that Opis16a effectively penetrates the intricate outer membrane of Gram-negative bacteria, leading to rapid bacterial cell death. Mechanism of action studies, conducted with a standard laboratory Escherichia coli ATCC 700928 strain and a multidrug-resistant A. baumannii NICD 15283 clinical strain, revealed that Opis16a binds to lipopolysaccharides, destabilises the outer membrane, depolarises the inner membrane and finally induces lethal permeabilisation of the cytoplasmic membrane. The strong bacterial selectivity and retained activity in serum, further supported the therapeutic potential of Opis16a for topical applications. Consequently, activity was further assessed in the in vivo Galleria mellonella model of A. baumannii burn wound infection. Treatment with Opis16a results in a three-fold improvement in larval survival compared to untreated A. baumannii-infected larvae, with an efficacy similar to polymyxin B. Consequently, Opis16a emerges as a novel membrane-destabilising AMP showing promising in vitro and in vivo efficacy against Gram-negative bacteria, suggesting potential for advancement in topical applications. To further increase the activity of Opis16a through rational design, four novel analogues were designed with increased cationic charge where an Asp residue in position five was replaced with a Lys (Opis16aDtoK) or where Lys residues at various positions in the sequence were replaced with Arg (Opis16aCterKtoR, Opis16aNterKtoR and Opis16aAllKtoR). Molecular dynamics (MD) simulations analyses revealed an increase in hydrogen bonding between the analogues and Gram-negative membrane lipids, along with increased membrane insertion in silico. Computational findings were validated through experimental in vitro screenings against a panel of Gram-negative bacteria, including standard laboratory and drug-resistant clinical strains. The analogues showed improvement in activity up to 16-fold compared with the parent Opis16a peptide, while maintaining strong selectivity for bacteria over HaCat cells (human keratinocyte cell-line). The analogue with the greatest improvement in activity and selectivity, Opis16aCterKtoR, also showed increased membrane permeability and in vivo protection of larvae infected with a multidrug-resistant E. cloacae NICD 16103 clinical isolate. In summary, novel structural characteristics for the design of AMPs were developed, and subsequently Opis16a and Opis16aCterKtoR were discovered as novel AMPs with promising therapeutic potential for the treatment of Gram-negative topical infections.