Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences ...against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively. Plasma and serum are reproducibly obtained from animals and humans allowing consistent for clinical analyses and research applications. However, the spectrum of active or activated proteases appears to vary depending on the activation of proteases and cofactors during coagulation (serum) or inhibition of such enzymes by anticoagulants (plasma), such as EDTA (metallo- and Ca2+-dependent proteases) and heparin (e.g. thrombin, factor Xa). Here, we studied the presumed effects on peptide degradation by taking blood via cardiac puncture of CD-1 mice using a syringe containing a peptide solution. Due to absence of coagulation activators (e.g. glass surfaces and damaged cells), visible blood clotting was prevented allowing to study peptide degradation for one hour. The remaining peptide was quantified and the degradation products were identified using mass spectrometry. When the degradation rates (half-life times) were compared to serum derived freshly from the same animal and commercial serum and plasma samples, peptides of three different families showed indeed considerably different stabilities. Generally, peptides were faster degraded in serum than in plasma, but surprisingly all peptides were more stable in fresh blood and the order of degradation rates among the peptides varied among the six different incubation experiments. This indicates, that proteolytic degradation of peptide-based therapeutics may often be misleading stimulating efforts to stabilize peptides at degradation sites relevant only in vitro, i.e., for serum or plasma stability assays, but of lower importance in vivo.
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.
Display omitted
► Peptides bind in two orientations to the substrate binding site of DnaK. ► Induced-fit effects contribute to substrate promiscuity. ► Hydrophobic interactions with binding pockets −2 to 0 determine specificity. ► The probably isoenergetic reverse binding mode must be considered in determining the specificity profile by computational studies. ► Peptides stimulate ATPase activity of DnaK.
Apidaecins are cationic, proline-rich antimicrobial peptides originally isolated from honeybees and exhibit high Gram-negative activity by inhibiting bacterial protein translation. Pharmacokinetics ...of apidaecin derivative Api137 was studied using single and multiple intravenous or subcutaneous injections as well as continuous subcutaneous infusion and correlated to its efficacy in a lethal murine
Escherichia coli
infection model. Survival rates of infected CD-1 mice were monitored and Api137 and its metabolites were quantified in plasma of uninfected CD-1 mice and Sprague Dawley rats using reversed-phase chromatography coupled online to mass spectrometry. The highest Api137 plasma levels of 23 mg/L were obtained after a single intravenous injection of 20 mg/kg body weight, which declined fast over the next 120 min (half-life time < 30 min). In contrast, continuous subcutaneous infusion of a similar dose over an hour (19.2 mg/kg/h) lead to stable plasma levels of ∼6 mg/L, which was above the minimal inhibitory concentration against
E. coli
ATCC 25922 (4 mg/L). The increased exposure by continuous subcutaneous administration of Api137 at 19.2 mg/kg/h over 48 h improved efficacy in the murine intraperitoneal sepsis model with survival rates of 67% over 5 days compared to 33% after intravenous and subcutaneous administration in different dosing schemes. To the best of our knowledge, continuous subcutaneous infusion using osmotic pumps was successfully utilized for delivery of an antimicrobial peptide for the first time. Additionally, the potential of apidaecin analogs as novel antibiotics is demonstrated even in a scenario where the infection site is clearly separated from the route of administration.
Adiponectin, 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.
We 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.
ADP 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.
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-NH2), 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 μg/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.
Strain‐promoted azide–alkyne cycloadditions (SPAAC) have proven extremely useful for labeling of biomolecules, but typically produce isomeric mixtures. This is not appropriate for the formation of ...bioactive molecules in living cells. Here, the first use of SPAAC for the isomer‐free synthesis of a bioactive molecule is reported both in vitro and inside cultured cells. We developed the symmetrical cyclooctyne SYPCO and used it for the generation of a chemically uniform triazole inhibitor of protein–protein interactions mediated by Bcl‐xL via isomer‐free SPAAC (iSPAAC). Tumor cells treated with the reactants of the iSPAAC reaction contained higher concentrations of triazole, and displayed higher apoptosis levels, than cells treated with pre‐synthesized triazole. We envision iSPAAC as a broadly applicable method for modulating intracellular targets with organic molecules with molecular weights prohibitively large for cellular uptake, via smaller and thus more cell‐permeable components.
Isomer‐free zone: The formation of isomers in strain‐promoted azide–alkyne cycloadditions (SPAAC) has precluded their use for the formation of bioactive molecules in living cells. Here, the first isomer‐free generation of a bioactive molecule by SPAAC is presented. The cycloalkyne SYPCO (symmetrical pyrrolocyclooctyne) facilitated the intracellular synthesis of a rationally designed inhibitor of protein–protein interactions mediated by Bcl‐xL via isomer‐free SPAAC (iSPAAC).
Cryptococcosis and cryptococcal meningitis, caused by
infections, lead to approximately 180,000 deaths per year, primarily in developing countries. Individuals with compromised immune systems, e.g., ...due to HIV infection (AIDS) or chemotherapy, are particularly vulnerable. Conventional treatment options are often limited and can cause severe side effects. Therefore, this study aimed to investigate the antifungal effect of insect-derived proline-rich antimicrobial peptides (PrAMPs) against
. These peptides are known for their low toxicity and their high efficacy in murine infection models, making them a promising alternative for treatment.
A preliminary screening of the minimal inhibitory concentrations (MICs) of 20 AMPs, including the well-known PrAMPs Onc112, Api137, and Chex1Arg20 as well as the cathelicidin CRAMP against the
strains 1841, H99, and KN99α revealed promising results, with MICs as low as 1.6 μmol/L. Subsequent investigations of selected peptides, determining their influence on fungal colony-forming units, confirmed their strong activity. The antifungal activity was affected by factors such as peptide net charge and sequence, with stronger effects at higher net charges probably due to better intracellular uptake confirmed by confocal laser scanning microscopy. Inactive scrambled peptides suggest a specific intracellular target, although scanning electron microscopy showed that PrAMPs also damaged the cell exterior for a low proportion of the cells. Possible pore formation could facilitate entry into the cytosol.
PDF
