Effect of Amino Acid Substitutions on 70S Ribosomal Binding, Cellular Uptake, and Antimicrobial Activity of Oncocin Onc112

Abstract

Proline‐rich antimicrobial peptides (PrAMPs) are promising candidates for the treatment of infections caused by high‐priority human pathogens. Their mode of action consists of (I) passive diffusion across the outer membrane, (II) active transport through the inner membrane, and (III) inhibition of protein biosynthesis by blocking the exit tunnel of the 70S ribosome. We tested whether in vitro data on ribosomal binding and bacterial uptake could predict the antibacterial activity of PrAMPs against Gram‐negative and Gram‐positive bacteria. Ribosomal binding and bacterial uptake rates were measured for 47 derivatives of PrAMP Onc112 and compared to the minimal inhibitory concentrations (MIC) of each peptide. Ribosomal binding was evaluated for ribosome extracts from four Gram‐negative bacteria. Bacterial uptake was assessed by quantifying each peptide in the supernatants of bacterial cultures. Oncocin analogues with a higher net positive charge appeared to be more active, although their ribosome binding and uptake rates were not necessarily better than for Onc112. The data suggest a complex mode of action influenced by further factors improving or reducing the antibacterial activity, including diffusion through membranes, transport mechanism, secondary targets, off‐target binding, intracellular distribution, and membrane effects. Relying only on in vitro binding and uptake data may not be sufficient for the rational development of more active analogues.

Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity

“…Ribosome binding and cellular uptake are two important considerations for optimizing the antibacterial activity of peptides, but even the combination of both mechanisms does not allow predicting the MIC changes properly indicating a highly complex mode of action [ 23 ]. It is interesting to look for substitutions that change the activity and target binding in the same way.…”

Submonomer Synthesis of Inverse Polyamidoamine (i‐PAMAM) Dendrimer Antibacterials

“…Minimum Inhibitory Concentrations (MIC) were determined by broth dilution on four Gram‐negative ( E. coli W3110, P. aeruginosa PAO1, A. baumannii ATCC19606 and K. pneumoniae NCTC418) and the Gram‐positive methicillin‐resistant S. aureus (MRSA). We tested both in full and in diluted (12.5 %) Mueller Hinton (MH) broth media, a condition known to enhance activity of proline‐rich antimicrobial peptides [39–41] . We tested neutral (pH 7.4) as well as slightly basic (pH 8.5) conditions, which enhance the antimicrobial activity of polymyxin B ( PMB ), peptide dendrimers and i‐PAMAMs [20,42] …”

“…These effects suggest an intracellular mechanism of action as reported for non-membrane disruptive peptoids, [33,43] which partly also require a diluted medium to be active, under which conditions the uptake of peptide-like compounds via transporters is induced, an effect also well known for proline-rich antimicrobial peptides. [39][40][41] This mechanism strongly contrasts with antimicrobial peptide dendrimers [30,31] and PAMAMs [26,27] shown to act as membrane disruptive compounds. Our study therefore highlights the very specific effect of the dendrimer structure, including both the nature of the core and the specific structure of the branches, on the antimicrobial mechanism.…”

“…We tested both in full and in diluted (12.5 %) Mueller Hinton (MH) broth media, a condition known to enhance activity of proline-rich antimicrobial peptides. [39][40][41] We tested neutral (pH 7.4) as well as slightly basic (pH 8.5) conditions, which enhance the antimicrobial activity of polymyxin B (PMB), peptide dendrimers and i-PAMAMs. [20,42] The first-and second-generation dendrimers (1a-b and 2a-b) were all inactive except for 2a, which displayed modest activity against E. coli in 12.5 % MH (MIC = 16-32 μg/mL, Table 2).…”

Evaluation of Potential DnaK Modulating Proline-Rich Antimicrobial Peptides Identified by Computational Screening

“…This aspect was highlighted by a recent publication in which 47 analogues of oncosin were evaluated across several different modes of activity including 70S ribosomal binding, inner and outer membrane transport and antimicrobial activity. The lack of clear correlation between these variables led to the conclusion that several parameters, including additional targets such as DnaK, are in continuous play with respect to ultimate antimicrobial action ( Brakel et al, 2022 ; Kolano et al, 2022 ). Our data confirms this inability to reply solely on one or two in vitro assays for the design and prediction of novel antimicrobial peptide analogues.…”