Serpin proteins irreversibly inhibit serine proteases, but only a small part of the serpin reactive-center loop (RCL) is responsible for the initial protein–protein interaction (PPI). To develop peptidic protease inhibitors, kallikrein-related peptidases 7 (KLK7) and 5 (KLK5) were chosen. Firstly, we demonstrated that short peptides derived from RCL sequences can be cleaved by KLK7 in a substrate-like manner. Next, these substrates were grafted onto the protease-binding loop of sunflower trypsin inhibitor-1 (SFTI-1). Peptides based on kallistatin, α₁-antichymotrypsin, and protein C inhibitor (PCI) inhibited KLK7 with K_i=0.4, 0.5, and 0.7 μm, respectively. In contrast, the trypsin-like KLK5 was only blocked by the peptide derived from PCI (K_i=0.6 μm). Thus, serpin function can be mimicked by introducing its PPI site into the rigid structure of the SFTI-1 scaffold. This approach might be applicable not only to KLKs but also to other serine protease members, thus opening up new therapeutic fields.

Peptidic ligands selectively targeting distinct G protein-coupled receptors that are highly expressed in tumor tissue represent a promising approach in drug delivery. Receptor-preferring analogues of neuropeptide Y (NPY) bind and activate the human Y₁ receptor subtype (hY₁ receptor), which is found in 90 % of breast cancer tissue and in all breast-cancer-derived metastases. Herein, novel highly boron-loaded Y₁-receptor-preferring peptide analogues are described as smart shuttle systems for carbaboranes as ¹⁰B-containing moieties. Various positions in the peptide were screened for their susceptibility to carbaborane modification, and the most promising positions were chosen to create a multi-carbaborane peptide containing 30 boron atoms per peptide with excellent activation and internalization patterns at the hY₁ receptor. Boron uptake studies by inductively coupled plasma mass spectrometry revealed successful uptake of the multi-carbaborane peptide into hY₁-receptor-expressing cells, exceeding the required amount of 10⁹ boron atoms per cell. This result demonstrates that the NPY/hY receptor system can act as an effective transport system for boron-containing moieties.

The side effects of chemotherapy can be overcome by linking toxic agents to tumor-targeting peptides with cleavable linkers. Herein, this concept is demonstrated by addressing the human Y₁ receptor (hY₁R), overexpressed in breast tumors, with analogues of the hY₁R-preferring [F⁷,P³⁴]NPY. First, carboxytetramethylrhodamine was connected to [F⁷,P³⁴]NPY by an amide, ester, disulfide, or enzymatic linkage. Live imaging revealed hY₁R-mediated delivery and allowed visualization of time-dependent intracellular release. Next, the fluorophore was replaced by the toxic agent methotrexate (MTX). In addition to linkage through the amide, ester, disulfide bond, or enzymatic cleavage site, a novel disulfide/ester linker was designed and coupled to [F⁷,P³⁴]NPY by solid-phase peptide synthesis. Internalization studies showed hY₁R subtype selective uptake, and cell viability experiments demonstrated hY₁R-mediated toxicity that was clearly dependent on the linkage type. Fast release profiles for fluorophore-[F⁷,P³⁴]NPY analogues correlated with high toxicities of MTX conjugates carrying the same linker types and emphasize the relevance of new structures connecting the toxophore and the carrier.

Pancreatic polypeptide (PP) is a satiety-inducing gut hormone targeting predominantly the Y₄ receptor within the neuropeptide Y multiligand/multireceptor family. Palmitoylated PP-based ligands have already been reported to exert prolonged satiety-inducing effects in animal models. Here, we suggest that other lipidation sites and different fatty acid chain lengths may affect receptor selectivity and metabolic stability. Activity tests revealed significantly enhanced potency of long fatty acid conjugates on all four Y receptors with a preference of position 22 over 30 at Y₁, Y₂ and Y₅ receptors. Improved Y receptor selectivity was observed for two short fatty acid analogues. Moreover, [K³⁰(E-Prop)]hPP₂₋₃₆ (15) displayed enhanced stability in blood plasma and liver homogenates. Thus, short chain lipidation of hPP at key residue 30 is a promising approach for anti-obesity therapy because of maintained selectivity and a sixfold increased plasma half-life.

Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY₁R and hY₅R are orexigenic, while hY₂R and hY₄R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY₂R/hY₄R over hY₁R/hY₅R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.

Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY₁R and hY₅R are orexigenic, while hY₂R and hY₄R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY₂R/hY₄R over hY₁R/hY₅R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.

The prolactin releasing peptide (PrRP) is involved in regulating food intake and body weight homeostasis, but molecular details on the activation of the PrRP receptor remain unclear. C-terminal segments of PrRP with 20 (PrRP20) and 13 (PrRP8-20) amino acids, respectively, have been suggested to be fully active. The data presented herein indicate this is true for the wildtype receptor only; a 5-10-fold loss of activity was found for PrRP8-20 compared to PrRP20 at two extracellular loop mutants of the receptor. To gain insight into the secondary structure of PrRP, we used CD spectroscopy performed in TFE and SDS. Additionally, previously reported NMR data, combined with ROSETTANMR, were employed to determine the structure of amidated PrRP20. The structural ensemble agrees with the spectroscopic data for the full-length peptide, which exists in an equilibrium between α- and 3₁₀-helix. We demonstrate that PrRP8-20's reduced propensity to form an α-helix correlates with its reduced biological activity on mutant receptors. Further, distinct amino acid replacements in PrRP significantly decrease affinity and activity but have no influence on the secondary structure of the peptide. We conclude that formation of a primarily α-helical C-terminal region of PrRP is critical for receptor activation. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 273–281, 2013.

Obesity is one of the main human epidemics today. The increase in fat accumulation, which is associated with obesity, may significantly change the expression of several bioactive molecules known as adipokines. These adipokines interact not only with adipose tissue, but also with metabolically relevant organs such as liver and muscle. Understanding the molecular basics of potential novel targets might help to improve the therapeutic treatment of people who suffer from obesity. Herein we summarize the state of the art of two novel adipokines and their impaired or protective action in obesity: chemerin and vaspin. Their expression patterns, signal transduction activity, and resulting functions within the human body are introduced. We also discuss various possibilities to target these adipokines, which may represent promising new targets for the treatment of obesity by small and synthetic compounds.

Cyclic Arg-Gly-Asp (RGD) peptides show remarkable affinity and specificity to integrin receptors and mediate important physiological effects in tumor angiogenesis. Additionally, they are one of the keyplayers in improving the biocompatibility of biomaterials. The fully biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) is frequently used for biomedical implants and can be applied as nanoparticles for drug delivery. The aim of this work was the generation of a lipidated c[RGDfK] peptide including a second functionality for coating of hydrophobic PLGA. Therefore, we established a general and straightforward strategy for the introduction of two different modifications into the same c[RGDfK] peptide. This allowed the generation of a palmitoylated integrin-binding lipopeptide that shows high affinity to PLGA. Additionally, we coupled 5(6)-carboxyfluorescein to the second site for modification to enable sensitive quantification of the immobilized lipopeptide on PLGA. In conclusion, we present a synthesis protocol that enables the preparation of c[RGDfK] lipopeptides with a strong affinity to PLGA and an additional site for modifications. This will provide the opportunity to introduce a variety of effector molecules site-specifically to the c[RGDfK] lipopeptide, which will enable the introduction of multifunctionality into c[RGDfK]-coated PLGA devices or nanoparticles.

The interactions between regulatory proteins such as interleukin-8 (IL-8) and glycosaminoglycans are of great interest both for the general understanding of regulatory processes in biology and for the development of implant coatings and innovative materials that suppress undesired immune responses and improve wound healing. In previous work, a number of residues of IL-8 that interact strongly with several glycosaminoglycans (GAGs) have been identified. In particular, the negatively charged Glu75 was reported to be involved in interactions with charged GAGs. To improve understanding of the role of this residue, we generated a selectively ¹⁵N-labeled E75K variant of IL-8(1–77) by expressed protein ligation. NMR and fluorescence spectroscopy in combination with molecular modeling were applied to evaluate the particular role of residue 75 in interactions with GAGs. Remarkably, more residues in the variant responded to GAG binding than in the wild-type. For the first time, we identified amino acids 34 to 36 as additional residues in the loop region of IL-8(1–77) that participate in the interactions with GAGs. These findings indicate that the N terminus of the E75K variant is more important as a second binding site for GAGs than that of the wild-type IL-8(1–77).

The chemokine stromal cell-derived factor-1 α (SDF1 α) is strongly involved in organogenesis, as well as inflammation and tissue repair, and acts by attracting different kinds of stem and progenitor cells. Therefore, it constitutes an interesting compound for drug development in regenerative medicine. However, it is prone to inactivation by proteolytic cleavage in human serum. Accordingly, it has to be stabilized against enzymatic degradation for any therapeutic application. We synthesized a palmitoylated SDF1 α analogue by native chemical ligation. Both the N-terminal thioester and the C-terminal palmitoylated fragment were prepared by solid-phase peptide synthesis. The activity of the refolded and pure compound was determined by an inositol phosphate turnover assay and revealed no loss in receptor activation. Additionally, resistance to proteolytic degradation was investigated in porcine liver homogenates and showed a near sevenfold increased half time. This study is a proof of principle approach for the lipidation of SDF1 α and provides a basis for further engineering of the chemokine in order to increase its therapeutic value.

Selectivity is a major issue in closely related multiligand/multireceptor systems. In this study we investigated the RFamide systems of hNPFF₁R and hNPFF₂R that bind the endogenous peptide hormones NPFF, NPAF, NPVF, and NPSF. By use of a systematic approach, we characterized the role of the C-terminal dipeptide with respect to agonistic properties using synthesized [Xaa 7]NPFF and [Xaa 8]NPFF analogues. We were able to identify only slight differences in potency upon changing the position of Arg 7, as all modifications resulted in identical behavior at the NPFF₁R and NPFF₂R. However, the C-terminal Phe 8 was able to be replaced by Trp or His with only a minor loss in potency at the NPFF₂R relative to the NPFF₁R. Analogues with shorter side chains, such as α-amino-4-guanidino butyric acid ([Agb 7]NPFF) or phenylglycine ([Phg 8]NPFF), decreased efficacy for the NPFF₁R to 25–31 % of the maximal response, suggesting that these agonist–receptor complexes are more susceptible to structural modifications. In contrast, mutations to the conserved Asp 6.59 residue in the third extracellular loop of both receptors revealed a higher sensitivity toward the hNPFF₂R receptor than toward hNPFF₁R. These data provide new insight into the subtype-specific agonistic activation of the NPFF₁ and NPFF₂ receptors that are necessary for the development of selective agonists.

Despite a considerable sequence identity of the three mammalian hormones of the neuropeptide Y family, namely neuropeptide Y, peptide YY and pancreatic polypeptide, their structure in solution is described to be different. A so-called pancreatic polypeptide-fold has been identified for pancreatic polypeptide, whereas the structure of the N-terminal segment of neuropeptide Y is unknown. This element is important for the binding of neuropeptide Y to two of its relevant receptors, Y₁ and Y₅, but not to the Y₂ receptor subtype. In this study now, three doubly fluorescent-labeled analogs of neuropeptide Y have been synthesized that still bind to the Y₅ receptor with high affinity to investigate the conformation in solution and, for the first time, to probe the conformational changes upon binding of the ligand to its receptor in cell membrane preparations. The results obtained from the fluorescence resonance energy transfer investigations clearly show considerable differences in transfer efficiency that depend both on the solvent as well as on the peptide concentration. However, the studies do not support a pancreatic polypeptide-like folding of neuropeptide Y in the presence of membranes that express the human Y₅ receptor subtype.

Now that the human genome has been decoded, the demand for novel therapeutic concepts, such as gene and stem cell therapy, is higher than ever before. Although new and better pharmaceutical agents are available, their efficient delivery to the intracellular site of action is still a serious challenge. A possible solution to this problem is the use of cell-penetrating peptides as delivery vectors, including derivatives of human calcitonin (hCT). The aim of this study was to synthesise novel branched hCT-derived peptides for the noncovalent delivery of nucleic acids. The uptake of the resulting oligocationic peptides into various cell lines as well as primary cells was monitored by fluorescence microscopy. To determine the appropriate peptide–plasmid charge ratios for efficient cell transfection, electromobility shift assays were carried out. Finally, flow cytometric and fluorescence microscopic studies of gene expression highlighted two novel hCT-derived peptides as highly effective in the delivery of noncovalently complexed plasmid DNA. Thus, the absence of cytotoxicity paired with highly efficient cell internalisation and transfection rates, in primary cells as well, make both peptides powerful candidates as drug delivery vectors, especially for plasmid DNA, for both in vivo and ex vivo therapeutic applications.