Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens

Abstract

According to the World Health Organization (WHO) the development of resistance against antibiotics by microbes is one of the most pressing health concerns. The situation will intensify since only a few pharmacological companies are currently developing novel antimicrobial compounds. Discovery and development of novel antimicrobial compounds with new modes of action are urgently needed. Antimicrobial peptides (AMPs) are known to be able to kill multidrug-resistant bacteria and, therefore, of interest to be developed into antimicrobial drugs. Proteolytic stability and toxicities of these peptides are challenges to overcome, and one strategy frequently used to address stability is cyclization. Here we introduced a disulfide-bond to cyclize a potent and nontoxic 9mer peptide and, in addition, as a proof-of-concept study, grafted this peptide into loop 6 of the cyclotide MCoTI-II. This is the first time an antimicrobial peptide has been successfully grafted onto the cyclotide scaffold. The disulfide-cyclized and grafted cyclotide showed moderate activity in broth and strong activity in 1/5 broth against clinically relevant resistant pathogens. The linear peptide showed superior activity in both conditions. The half-life time in 100% human serum was determined, for the linear peptide, to be 13 min, for the simple disulfide-cyclized peptide, 9 min, and, for the grafted cyclotide 7 h 15 min. The addition of 10% human serum led to a loss of antimicrobial activity for the different organisms, ranging from 1 to >8-fold for the cyclotide. For the disulfide-cyclized version and the linear version, activity also dropped to different degrees, 2 to 18-fold, and 1 to 30-fold respectively. Despite the massive difference in stability, the linear peptide still showed superior antimicrobial activity. The cyclotide and the disulfide-cyclized version demonstrated a slower bactericidal effect than the linear version. All three peptides were stable at high and low pH, and had very low hemolytic and cytotoxic activity.

Rational Designed Hybrid Peptides Show up to a 6-fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides measured by BioSAXS

“…The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold and dependent on the combination of peptides and bacteria. We have observed a very similar pattern while studying lipidation, glycosylation, cyclisation, and grafting into a cyclotide (Grimsey et al, 2020;Koehbach et al, 2021). All these modifications did not lead to a general improvement and were therefore not a "one fits all strategy".…”

“…Two arginine-isoleucine-rich peptides optP1 (KIILRIRWR) and optP7 (KRRVWIIW), were identified from this new prediction strategy, showing broad-spectrum activity. The peptide optP7 was used as a lead and selected for studying lipidation, glycosylation, cyclisation, and grafting into a cyclotide (Grimsey et al, 2020;Koehbach et al, 2021). Hybridization of different AMPs has been investigated to further improve their performance (Shang et al, 2020;Wade et al, 2018Wade et al, , 2019.…”

Rational Designed Hybrid Peptides Show up to a 6-Fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides Measured by BioSAXS

“…The increase in activity compared to the arginineisoleucine-rich peptides was up to 6-fold and dependent on the combination of peptides and bacteria. We have observed a very similar pattern while studying lipidation, glycosylation, cyclisation, and grafting into a cyclotide (Grimsey et al, 2020;Koehbach et al, 2021). All these modifications did not lead to a general improvement and were therefore not a "one fits all strategy".…”

“…Two arginine-isoleucine-rich peptides optP1 (KIILRIRWR) and optP7 (KRRVWIIW), were identified from this new prediction strategy, showing broad-spectrum activity. The peptide optP7 was used as a lead and selected for studying lipidation, glycosylation, cyclisation, and grafting into a cyclotide ( Grimsey et al, 2020 ; Koehbach et al, 2021 ). Hybridization of different AMPs has been investigated to further improve their performance ( Shang et al, 2020 ; Wade et al, 2018 , 2019 ).…”

Host Defense Peptides: Dual Antimicrobial and Immunomodulatory Action

“…Fourthly, because of their charged nature, HDP activity can be compromised by the presence of salt at physiological concentrations [ 88 ]. Finally, although HDPs are touted as giving rise to little bacterial resistance [ 36 , 89 ] (most likely due to their relatively limited use in the clinic), examples of resistance mechanisms have been reported [ 30 , 40 , 90 , 91 ]. Specifically, bacteria can evade HDPs in manners that are analogous to those illustrated in Figure 2 for antibiotics—in other words, resistance arises in broad terms by (i) degradation of the HDPs, (ii) sequestration, in particular in biofilms, (iii) physico-chemical modifications of the molecules encountered on the path of HDP entry into the bacterial cell, or (iv) efflux pumps ( Table 1 ).…”