Nicole Berthold

Api88 Is a Novel Antibacterial Designer Peptide To Treat Systemic Infections with Multidrug-Resistant Gram-Negative Pathogens

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a K(D) of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin.

Structural Studies on the Forward and Reverse Binding Modes of Peptides to the Chaperone DnaK

Hsp70 chaperones have been implicated in assisting protein folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. For these functions, the chaperones need to exhibit a significant promiscuity in binding to different sequences of hydrophobic peptide stretches. To characterize the structural basis of sequence specificity and flexibility of the Escherichia coli Hsp70 chaperone DnaK, we have analyzed crystal structures of the substrate binding domain of the protein in complex with artificially designed peptides as well as small proline-rich antimicrobial peptides. The latter peptides from mammals and insects were identified to target DnaK after cell penetration. Interestingly, the complex crystal structures reveal two different peptide binding modes. The peptides can bind either in a forward or in a reverse direction to the conventional substrate binding cleft of DnaK in an extended conformation. Superposition of the two binding modes shows a remarkable similarity in the side chain orientations and hydrogen bonding pattern despite the reversed peptide orientation. The DnaK chaperone has evolved to bind peptides in both orientations in the substrate binding cleft with comparable energy without rearrangements of the protein. Optimal hydrophobic interactions with binding pockets -2 to 0 appear to be the main determinant for the orientation and sequence position of peptide binding.

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

ABSTRACT Proline-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 l-ornithine or l-homoarginine increased the serum stabilities by more than 20-fold (half-life, ∼4 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.

Cellular Uptake of Apidaecin 1b and Related Analogs in Gram-negative Bacteria Reveals Novel Antibacterial Mechanism for Proline-rich Antimicrobial Peptides

Proline-rich antimicrobial peptides (PrAMPs) freely penetrate through the outer membrane into the periplasm of Gram-negative bacteria, before they are actively translocated by a permease/transporter-mediated uptake into the cytoplasm where they are reported to inhibit chaperone DnaK. Here we have studied the PrAMP apidaecin 1b, which is produced in honey bees in response to bacterial infections, and optimized apidaecin analogs for their bacterial uptake. The peptides were labeled with 5(6)-carboxyfluorescein and their internalization in Escherichia coli and Klebsiella pneumoniae was visualized by fluorescence microscopy and quantified by flow cytometry for four different time points over an incubation period of 4 h. Apidaecin 1b entered only 40% to 50% of the cells at detectable quantities, whereas designer peptides Api88, Api134 and Api155 entered more than 95% of the bacteria within 30 min at around fourfold higher quantities than the native peptide. Interestingly, a shortened version designated as (1-17)Api88, bound DnaK as efficiently as the 18-residue long Api88 and entered the bacteria at similar kinetics as Api88, but was unable to inhibit the bacterial growth. Similar conflicts with currently proposed mechanisms of PrAMPs were also obtained for some Ala-substituted analogs and reverse apidaecin sequences. Although peptides with C-terminal amides enter the cells much more efficiently than homologous C-terminal acids, this improved cell penetration does not improve the antibacterial activities. These studies suggest that PrAMPs utilize additional modes of action to kill sensitive organisms.

Controlled systemic release of therapeutic peptides from PEGylated prodrugs by serum proteases.

A novel concept to release peptidic drugs systemically by serum proteases from a PEGylated precursor makes it possible to tune release kinetics to fit the medical needs. Drug release depends on the size of the PEG polymer and the sequence and length of the peptide linker. The antimicrobial activities of the prodrugs were even better than those of the free peptides, whereas direct PEGylation abolished the peptide activity.

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

Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well‐known immunomodulatory activities of murine cathelicidin‐related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro‐inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS‐mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections. Copyright

Identification of Api88 Binding Partners in Escherichia coli Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with p-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism.

Structural Identification of DnaK Binding Sites within Bovine and Sheep Bactenecin Bac7

Bacterial resistance against common antibiotics is an increasing health problem. New pharmaceuticals for the treatment of infections caused by resistant pathogens are needed. Small proline-rich antimicrobial peptides (PrAMPs) from insects are known to bind intracellularly to the conventional substrate binding cleft of the E. coli Hsp70 chaperone DnaK. Furthermore, bactenecins from mammals, members of the cathelicidin family, also contain potential DnaK binding sites. Crystal structures of bovine and sheep Bac7 in complex with the DnaK substrate binding domain show that the peptides bind in the forward binding mode with a leucine positioned in the central hydrophobic pocket. In most structures, proline and arginine residues preceding leucine occupy the hydrophobic DnaK binding sites -1 and -2. Within bovine Bac7, four potential DnaK binding sites were identified.