Ribosomal Target‐Binding Sites of Antimicrobial Peptides Api137 and Onc112 Are Conserved among Pathogens Indicating New Lead Structures To Develop Novel Broad‐Spectrum Antibiotics

Abstract

Proline‐rich antimicrobial peptides expressed in insects are primarily active against Enterobacteriaceae. Mechanistically, they target the bacterial (70S) ribosome after partially transporter‐based cellular uptake, as revealed for Api137 and Onc112 on Escherichia coli. Following molecular modeling indicating that the Onc112 contact site is conserved among the ribosomes of high‐priority pathogens, the ribosome binding of Api137 and Onc112 was studied. The dissociation constants (Kd) of Onc112 were ∼75 nmol/L for Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, 36 nmol/L for Pseudomonas aeruginosa, and 102 nmol/L for Staphylococcus aureus, thus indicating a very promising lead structure for developing broad‐spectrum antibiotics. Api137 bound weaker with Kd values ranging from 155 nmol/L to 13 μmol/L. For most bacteria, the antibacterial activities were lower than predicted from the Kd values, which was only partially explained by their ability to enter bacterial cells. Other factors limiting the activity expected from the ribosome binding might be off‐target binding.

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria

“…However, increasing Api concentration to 2 mM led to the appearance of a start codon toeprint bands in the zntA and tyrS templates ( Figure 2—figure supplement 3B–C ); the start codon effects were much weaker or completely absent with the other tested genes. The moderate effect of Api on translation initiation is reminiscent of the main mode of action of other PrAMPs ( Gagnon et al, 2016 ; Roy et al, 2015 ; Seefeldt et al, 2016 ; Seefeldt et al, 2015 ) and could be explained by its ability to bind with a reduced affinity to the ribosome even in the absence of RF1/RF2 ( Florin et al, 2017 ; Kolano et al, 2020 ; Krizsan et al, 2014 ). However, it remains to be elucidated whether the mechanism of the weak inhibition of translation initiation by Api is comparable to that of other PrAMPs.…”

“…One of the unexpected aspects of Api action revealed by the in vivo studies is the increased ribosome occupancy of start codons of some genes. These effects could be related to the reported low affinity of Api for the ribosome with the vacant nascent peptide exit tunnel ( Florin et al, 2017 ; Kolano et al, 2020 ; Krizsan et al, 2014 ). Once associated with the ribosome at the start codon, Api may block the first peptide bond formation by displacing the fMet moiety of the initiator tRNA in the P site or may interfere with the first act of translocation.…”

Functional Effects of ARV-1502 Analogs Against Bacterial Hsp70 and Implications for Antimicrobial Activity

“…Dissociation (K d ) and inhibitory constants (K i ) were measured using a previously reported protocol with slight modifications ( Krizsan et al, 2015 ; Kolano et al, 2020 ). Briefly, black 384-well plates (flat bottom, Greiner Bio-One GmbH, Frickenhausen, Germany) were blocked with 0.5% (w/v) casein in phosphate buffered saline (PBS; 10 mmol/L Na 2 HPO 4 , 0.2 mmol/L KH 2 PO 4 , 137 mmol/L NaCl, 2.7 mmol/L KCl, pH 7.4) containing 0.05% (w/v) Tween ® 20 (PBST) at 4°C overnight and washed three times with PBST.…”

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination

“…The moderate effect of Api on translation initiation is reminiscent of the main mode of action of other PrAMPs (Gagnon et al, 2016;Roy et al, 2015;Seefeldt et al, 2016;Seefeldt et al, 2015) and could be explained by its ability to bind with a reduced affinity to the ribosome even in the absence of RF1/RF2 (Florin et al, 2017;Kolano et al, 2020;Krizsan et al, 2014). However, it remains to be elucidated whether the mechanism of the weak inhibition of translation initiation by Api is comparable to that of other PrAMPs.…”