Peptides containing the PCNA interacting motif APIM bind to the β-clamp and inhibit bacterial growth and mutagenesis

Abstract

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

Novel Peptides Targeting the β-Clamp Rapidly Kill Planktonic and Biofilm Staphylococcus epidermidis Both in vitro and in vivo

“…To exclude that this anti-biofilm effect was due to the cell penetrating part of the APIM-peptide, we included the 11-arginine cell penetrating peptide, R11, as a control. However, 60 μM of the R11 peptide gave only ∼1 log decrease in CFU/mL, showing that, similarly to the antibacterial and anti-mutagenic effects ( Nedal et al, 2020 ), the full-length peptide containing APIM is needed for maximal anti-biofilm effect. Preliminary data also shows that coating steel rods with APIM-peptide dissolved in polyethylene glycol (PEG) inhibited biofilm formation (data not shown); thus, APIM-peptides could potentially be exploited for further use in coating of surgical implants.…”

“…The growing problem of antibacterial resistance highlights the need for new antibiotics with a novel mode of action. In this paper, the recently discovered antibacterial effect of APIM-peptides ( Nedal et al, 2020 ) and their potential use in a clinical setting with MRSE infections are explored. The results show that APIM-peptide kill S. epidermidis rapidly in in vitro planktonic cultures as well as in biofilms.…”

“…APIM-peptides (Innovagen, Sweden) used here were TFA-salt (>90% pure). The APIM-peptide consists of the APIM-motif (in bold) connected via a linker to an arginine tail (cell penetrating part), containing 10 or 11 arginine; Ac-MD- RWLVK -WKKKRKI-R11/R10 the C-termini on all peptides were amidated ( Nedal et al, 2020 ). Lyophilized peptide from the manufacturer was dissolved in water.…”

“…These APIM-peptides were recently shown to have antibacterial activities due to binding to the conserved bacterial DNA sliding clamp, the β-clamp, thereby inhibiting DNA replication and mutagenesis. Antibacterial effects were found for several bacterial species, including Escherichia coli , S. epidermidis , and S. aureus ( Nedal et al, 2020 ). Here we explored the possibility of prophylactically using APIM-peptide, in combination with the commonly used antibiotic gentamicin, in bone cement to limit the frequency of PJIs.…”

Status update in the use of cell-penetrating peptides for the delivery of macromolecular therapeutics

“…Hence, the authors showed that their CPPmodified peptide inhibited bacterial DNA replication and bacterial growth in a mouse skin infection model. This is an interesting find that has implications beyond cancer research and is important from the perspective of antimicrobial resistance [26].…”