Absence of <i>in vitro</i> innate immunomodulation by insect‐derived short proline‐rich antimicrobial peptides points to direct antibacterial action <i>in vivo</i>

dProline-rich antimicrobial peptides (PrAMPs) from insects and mammals have recently been evaluated for their pharmaceutical potential in treating systemic bacterial infections. Besides the native peptides, several shortened, modified, or even artificial sequences were highly effective in different murine infection models. Most recently, we showed that the 18-residue-long peptide Api88, an optimized version of apidaecin 1b, was efficient in two different animal infection models using the pathogenic Escherichia coli strains ATCC 25922 and Neumann, with a promising safety margin. Here, we show that Api88 is degraded relatively fast upon incubation with mouse serum, by cleavage of the C-terminal leucine residue. To improve its in vitro characteristics, we aimed to improve its serum stability. Replacing the C-terminal amide by the free acid or substituting Arg-17 with Lornithine or L-homoarginine increased the serum stabilities by more than 20-fold (half-life, ϳ4 to 6 h). These analogs were nontoxic to human embryonic kidney (HEK 293), human hepatoma (HepG2), SH-SY5Y, and HeLa cells and nonhemolytic to human erythrocytes. The binding constants of all three analogs with the chaperone DnaK, which is proposed as the bacterial target of PrAMPs, were very similar to that of Api88. Of all the analogs tested, Api137 (Gu-ONNRPVYIPRPRPPHPRL; Gu is N,N,N=,N=-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional alanine and D-amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against an E. coli strain, whereas substitutions of residues 14 to 18 decreased the activity dramatically. Based on the significantly improved resistance to proteolysis, Api137 appears to be a very promising lead compound that should be even more efficient in vivo than Api88.

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“…3C and D). Both findings are consistent with a recent report on PrAMPs, including apidaecin 1b and Api88 (34).…”

Section: Fig 1 Degradation Of Api88 In 25% Aqueous Mouse Serum (Top) supporting

confidence: 93%

Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

Berthold

Czihal

Fritsche

et al. 2013

Antimicrob Agents Chemother

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“…Recently, it was described that oncocin and apidaecin, two short (<20 Amino acids) PR-AMPs also lack any immunomodulatory activity. No chemotactic activity towards DC, no modification of LPS induced immune responses or direct immune stimulating effects on macrophages were observed for these PR-AMPs, in contrast to the murine cathelicidin CRAMP used in the same study [40]. Taken together, our results show that N-terminal PR-39 derived peptides are sufficient for antimicrobial activity and stimulation of TNF-α production by macrophages, but that the full length peptide is required for IL-8 production.…”

Section: Discussionmentioning

confidence: 45%

Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

et al. 2014

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The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics.

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“…Synergistic effects with intrinsic antimicrobial substances produced by the host, e.g., AMPs like CRAMP, and immunomodulatory effects could also explain this discrepancy (Ostorhazi et al, 2013; Otvos and Ostorhazi, 2015; Knappe et al, 2016b). The latter effect was studied for Onc72, but no immunomodulatory effects were observed on unstimulated and lipopolysaccharide (LPS)-stimulated murine dendritic cells or murine macrophages (Fritsche et al, 2012). Only human macrophages and monocytes showed a reduction of LPS-induced TNFα release after treatment with Api88 and Api137 indicating a mild anti-inflammatory effect (Tavano et al, 2011; Keitel et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

In vivo Efficacy and Pharmacokinetics of Optimized Apidaecin Analogs

et al. 2017

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Proline-rich antimicrobial peptides (PrAMPs) represent promising alternative therapeutic options for the treatment of multidrug-resistant bacterial infections. PrAMPs are predominantly active against Gram-negative bacteria by inhibiting protein expression via at least two different modes of action, i.e., blocking the ribosomal exit tunnel of 70S ribosomes (oncocin-type binding) or inhibiting the assembly of the 50S ribosomal subunit (apidaecin-type binding). The in vivo efficacy and favorable biodistribution of oncocins confirmed the therapeutic potential of short PrAMPs for the first time, whereas the in vivo evaluation of apidaecins is still limited despite the promising efficacy of apidaecin-analog Api88 in an intraperitoneal murine infection model. Here, the in vivo efficacy of apidaecin-analog Api137 was studied, which rescued all NMRI mice from a lethal intraperitoneal infection with E. coli ATCC 25922 when administered three times intraperitoneal at doses of 0.6 mg/kg starting 1 h after infection. When Api88 and Api137 were administered intravenous or intraperitoneal at doses of 5 and 20 mg/kg, their plasma levels were similarly low (<3 μg/mL) and four-fold lower than for oncocin-analog Onc72. This contradicted earlier expectation based on the very low serum stability of Api88 with a half-life time of only ~5 min compared to ~6 and ~3 h for Api137 and Onc72, respectively. Pharmacokinetic data relying on a sensitive mass spectrometry method utilizing multiple reaction monitoring and isotope-labeled peptides revealed that Api88 and Api137 were present in blood, urine, and kidney, and liver homogenates at similar levels accompanied by the same major metabolites comprising residues 1–16 and 1–17. The pretended discrepancy was solved, when all peptides were incubated in peritoneal lavage. Api137 was rapidly degraded at the C-terminus, while Api88 was rather stable despite releasing the same degradation products. Onc72 was very stable explaining its higher plasma levels compared to Api88 and Api137 after intraperitoneal administration illuminating its good in vivo efficacy. The data indicate that the degradation of therapeutic peptides should be studied in serum and further body fluids. Moreover, the high efficacy in murine infection models and the fast clearance of Api88 and Api137 within ~60 min after intravenous and ~90 min after intraperitoneal injections indicate that their in vivo efficacy relates to the maximal peptide concentration achieved in blood.

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Absence of in vitro innate immunomodulation by insect‐derived short proline‐rich antimicrobial peptides points to direct antibacterial action in vivo

Cited by 15 publications

References 67 publications

Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

In vivo Efficacy and Pharmacokinetics of Optimized Apidaecin Analogs

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