Daniel Knappe

Design and development of a peptide-based adiponectin receptor agonist for cancer treatment.

BackgroundAdiponectin, a fat tissue-derived adipokine, exhibits beneficial effects against insulin resistance, cardiovascular disease, inflammatory conditions, and cancer. Circulating adiponectin levels are decreased in obese individuals, and this feature correlates with increased risk of developing several metabolic, immunological and neoplastic diseases. Thus, pharmacological replacement of adiponectin might prove clinically beneficial, especially for the obese patient population. At present, adiponectin-based therapeutics are not available, partly due to yet unclear structure/function relationships of the cytokine and difficulties in converting the full size adiponectin protein into a viable drug.ResultsWe aimed to generate adiponectin-based short peptide that can mimic adiponectin action and be suitable for preclinical and clinical development as a cancer therapeutic. Using a panel of 66 overlapping 10 amino acid-long peptides covering the entire adiponectin globular domain (residues 105-254), we identified the 149-166 region as the adiponectin active site. Three-dimensional modeling of the active site and functional screening of additional 330 peptide analogs covering this region resulted in the development of a lead peptidomimetic, ADP 355 (H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2). In several adiponectin receptor-positive cancer cell lines, ADP 355 restricted proliferation in a dose-dependent manner at 100 nM-10 μM concentrations (exceeding the effects of 50 ng/mL globular adiponectin). Furthermore, ADP 355 modulated several key signaling pathways (AMPK, Akt, STAT3, ERK1/2) in an adiponectin-like manner. siRNA knockdown experiments suggested that ADP 355 effects can be transmitted through both adiponectin receptors, with a greater contribution of AdipoR1. In vivo, intraperitoneal administration of 1 mg/kg/day ADP 355 for 28 days suppressed the growth of orthotopic human breast cancer xenografts by ~31%. The peptide displayed excellent stability (at least 30 min) in mouse blood or serum and did not induce gross toxic effects at 5-50 mg/kg bolus doses in normal CBA/J mice.ConclusionsADP 355 is a first-in-class adiponectin receptor agonist. Its biological activity, superior stability in biological fluids as well as acceptable toxicity profile indicate that the peptidomimetic represents a true lead compound for pharmaceutical development to replace low adiponectin levels in cancer and other malignancies.

Insect‐Derived Proline‐Rich Antimicrobial Peptides Kill Bacteria by Inhibiting Bacterial Protein Translation at the 70 S Ribosome

Proline-rich antimicrobial peptides (PrAMPs) have been investigated and optimized by several research groups and companies as promising lead compounds to treat systemic infections caused by Gram-negative bacteria. PrAMPs, such as apidaecins and oncocins, enter the bacteria and kill them apparently through inhibition of specific targets without a lytic effect on the membranes. Both apidaecins and oncocins were shown to bind with nanomolar dissociation constants to the 70S ribosome. In apidaecins, at least the two C-terminal residues (Arg17 and Leu18) interact strongly with the 70S ribosome, whereas residues Lys3, Tyr6, Leu7, and Arg11 are the major interaction sites in oncocins. Oncocins inhibited protein biosynthesis very efficiently in vitro with half maximal inhibitory concentrations (IC50 values) of 150 to 240 nmol L(-1). The chaperone DnaK is most likely not the main target of PrAMPs but it binds them with lower affinity.

Oncocin (VDKPPYLPRPRPPRRIYNR-NH2): a novel antibacterial peptide optimized against gram-negative human pathogens.

Small proline-rich antimicrobial peptides (AMP) have attracted considerable interest, as they target specific intracellular bacterial components and do not act by lytic mechanisms. Here, a novel peptide, termed oncocin (VDKPPYLPRPRPPRRIYNR-NH(2)), is reported that was optimized for the treatment of Gram-negative pathogens. Its minimal inhibitory concentrations in tryptic soy broth medium ranged from 0.125 to 8 microg/mL for 34 different strains and clinical isolates of Enterobacteriaceae and nonfermenters, such as Escherichia coli , Pseudomonas aeruginosa , and Acinetobacter baumannii . Substitutions of two arginine residues by ornithine increased the half-lives in full mouse serum from about 20 min to greater than 180 min and the activity. Both optimized oncocin derivatives were neither toxic to human cell lines nor hemolytic to human erythrocytes. They could freely penetrate lipid membranes and were washed out completely without any sign of lytic activity, as assessed by quartz crystal microbalance. Fluorescence labeled peptides entered the periplasmic space within 20 min at room temperature and homogeneously stained E. coli within 50 min. In conclusion, the optimized oncocin represents a very promising candidate for future in vivo work and may serve as a novel lead compound for an antibacterial drug class.

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.

Rational Design of Oncocin Derivatives with Superior Protease Stabilities and Antibacterial Activities Based on the High-Resolution Structure of the Oncocin-DnaK Complex.

Despite the success story of antibiotics, which began nearly a hundred years ago, bacterial infections are still a major cause of death worldwide. The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global problem of growing importance, with an estimated annual death toll of 50 000 in the EU and 60 000 in the USA. More recently, MDR bacteria have also caused severe community-acquired infections, indicating that we will soon face bacterial strains with the ability to overcome existing antibiotic treatments, and which will therefore represent a severe global threat. Agents responsible for important resistance mechanisms in Gram-negative bacteria include extended spectrum b-lactamases (ESBLs) in Enterobacteriaceae (e.g. , E. coli, K. pneumoniae, and Enterobacter cloacae) or broad-range b-lactamases (e.g. , KPC in Klebsiella pneumoniae or metallo-b-lactamases in Pseudomonas aeruginosa). MDR Acinetobacter baumannii, associated with invasive infections such as pneumonia, meningitis, and bacteremia, has been found to be responsible for outbreaks in intensive care units, including “panresistant” A. baumannii clones susceptible only to polymyxin. To provide effective future treatment options, novel antimicrobial drug classes with novel modes of action are urgently needed. Inducible, gene-encoded antimicrobial peptides (AMPs) represent such a promising alternative, having been selected and optimized by evolution over millions of years. Although AMPs that kill bacteria by lytic effects on the membrane are often toxic to human cells at higher doses, the class of small prolinerich AMPs (PR-AMPs) expressed in mammals and insects has attracted considerable interest. These peptides specifically target intracellular components in Gram-negative bacteria with no indication of any resulting toxic side effects. Despite their favorable antibacterial spectrum against Enterobacteriaceae and nonfermenting species (e.g. , A. baumannii and P. aeruginosa), there are multiple obstacles to be overcome in their further development for therapeutic consideration. We have recently used rational design to optimize the 19-residue-long PR-AMP oncocin (VDKPPYLPRPRPPRRIYNR-NH2) as a potential means of countering the five human pathogens discussed. Substitution of Arg15 and Arg19 by ornithine drastically improved the half-life in mouse serum. Mechanistically, oncocin freely penetrates the bacterial membrane and distributes homogenously within E. coli cells. Here we report its further optimization, based on a positional Ala-scan to deduce residues critical to its antibacterial activity and the crystal structure of an oncocin-DnaK (ligand–target) complex. The new lead compounds were highly resistant against serum (t1=2 8 h) and E. coli proteases (t1=2 >10 h). The mode of action assumed for oncocin and all other PRAMPs has not been worked out in detail, but most likely consists of at least three steps: passive penetration of the bacterial outer membrane, active transport from the periplasmic space into the cytoplasm, and inhibition of DnaK and maybe other targets. 12] A lead optimization strategy therefore has to consider all aspects and cannot focus only on the target binding. We first identified residues crucial for the antibacterial activity of oncocin by determining the minimal inhibitory concentrations (MICs) and inhibition zones for all 19 peptides resulting from a positional Ala-scan (see Figure S1 in the Supporting Information). Substitution of residues 1, 2, 4, 5, 10, and 12–19 reduced the antibacterial activity slightly, whereas substitutions at positions 3, 6–9, and 11 abolished it almost completely. Although the target of oncocin has not yet been identified, the high sequence homology to other insect-derived PR-AMPs, especially pyrrhocoricin, suggests that it might be the bacterial chaperone DnaK. Full-length DnaK was therefore expressed in E. coli and purified in order to study oncocin binding by fluorescence polarization. The binding constants for oncocin and its analogue O2, with a 5(6)-carboxyfluorescein unit at the N terminus, were 1.0 0.2 mm and 4.0 1.0 mm, respectively, whereas all-d oncocin did not bind (Table 1, Figures S2 and S3). These values correspond to binding constants reported for other DnaK-binding sequences, ranging from 0.1 to 10 mm. Cocrystallization of oncocin O2 with the substrate binding domain of DnaK (residues 389 to 607), demonstrated that oncocin residues 4 to 10 (PPYLPR) bound to the peptide binding site of DnaK, whereas the remaining terminal residues of the peptide were flexible (Figures 1 and S4–S8). Interestingly, this sequence stretch matched the residues identified by the Ala-scan as crucial for antibacterial activity. The antibacterial activity of oncocin thus mostly depends on its DnaK binding site, although the activity is also abolished by shortening the sequence, either from the N or the C terminus. Such inactivation, which can occur through the action of proteases either in the bacteria or in blood, should be minimized for systemic applications. Because the peptide O2 is rel[a] D. Knappe, M. Zahn, Prof. Dr. N. Str ter, Prof. Dr. R. Hoffmann Institut f r Bioanalytische Chemie Biotechnologisch-Biomedizinisches Zentrum Fakult t f r Chemie und Mineralogie, Universit t Leipzig Deutscher Platz 5, 04103 Leipzig (Germany) Fax: (+ 49) 341-97-31339 E-mail : hoffmann@chemie.uni-leipzig.de [b] U. Sauer, Dr. G. Schiffer AiCuris GmbH & Co KG Friedrich-Ebert-Strasse 475, Building 302, 42117 Wuppertal (Germany) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000792.

Easy Strategy To Protect Antimicrobial Peptides from Fast Degradation in Serum

ABSTRACT Antimicrobial peptides are promising novel peptide leads, but their low serum stability often limits their further consideration in drug development programs. Here, we describe a generally applicable strategy to stabilize peptides against serum proteases by replacing arginine residues with α-amino-3-guanidino-propionic acid (Agp). Peptide NH2-RRWRIVVIRVRR-CONH2 was nearby totally degraded after 8 h in mouse serum, whereas the variant with Agp substituted was degraded less than 20%. The antimicrobial activity was not affected.

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.

Intracellular Toxicity of Proline-Rich Antimicrobial Peptides Shuttled into Mammalian Cells by the Cell-Penetrating Peptide Penetratin

ABSTRACT The health threat caused by multiresistant bacteria has continuously increased and recently peaked with pathogens resistant to all current drugs. This has triggered intense research efforts to develop novel compounds to overcome the resistance mechanisms. Thus, antimicrobial peptides (AMPs) have been intensively studied, especially the family of proline-rich AMPs (PrAMPs) that was successfully tested very recently in murine infection models. PrAMPs enter bacteria and inhibit chaperone DnaK. Here, we studied the toxicity of intracellular PrAMPs in HeLa and SH-SY5Y cells. As PrAMPs cannot enter most mammalian cells, we coupled the PrAMPs with penetratin (residues 43 to 58 in the antennapedia homeodomain) via a C-terminally added cysteine utilizing a thioether bridge. The resulting construct could transport the covalently linked PrAMP into mammalian cells. Penetratin ligation reduced the MIC for Gram-negative Escherichia coli only slightly (1 to 8 μmol/liter) but increased the activity against the Gram-positive Micrococcus luteus up to 32-fold (MIC ≈ 1 μmol/liter), most likely due to more effective penetration through the bacterial membrane. In contrast to native PrAMPs, the penetratin-PrAMP constructs entered the mammalian cells, aligned around the nucleus, and associated with the Golgi apparatus. At higher concentrations, the constructs reduced the cell viability (50% inhibitory concentration [IC50] ≈ 40 μmol/liter) and changed the morphology of the cells. No toxic effects or morphological changes were observed at concentrations of 10 μmol/liter or below. Thus, the IC50 values were around 5 to 40 times higher than the MIC values. In conclusion, PrAMPs are in general not toxic to mammalian cells, as they do not pass through the membrane. When shuttled into mammalian cells, however, PrAMPs are only slightly cross-reactive to mammalian chaperones or other intracellular mammalian proteins, providing a second layer of safety for in vivo applications, even if they can enter some human cells.

Bactericidal oncocin derivatives with superior serum stabilities.

The proline-rich antimicrobial peptide oncocin is remarkably active in vitro against a number of important Gram-negative bacteria of concern to humans owing to their increasing resistance to antibiotics, i.e. Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae) and non-fermenting species (Acinetobacter baumannii and Pseudomonas aeruginosa). Degradation of oncocin in mouse serum was investigated in this study. Several approaches to stabilise the main cleavage sites (C-terminal to Arg-15 and N-terminal to Arg-19) by substituting either or both arginine (Arg) residues with non-proteinogenic amino acids, i.e. α-amino-3-guanidino-propionic acid, homoarginine, nitro-arginine, N-methyl-arginine, β-homoarginine, D-arginine (D-Arg) or ornithine (Orn), were tested. These modifications were found to increase the half-life of oncocin in full mouse serum. For oncocin with two Orn residues in positions 15 and 19, the half-life in full serum increased from 25 min to 3 h. An increase of >8 h was observed for oncocin with two D-Arg residues at these same positions. The antibacterial activities of these modified sequences were slightly better than the original oncocin sequence. Moreover, the three most stable analogues were found to be bactericidal against E. coli and were not toxic to HeLa cells or haemolytic to human erythrocytes.