Rational design of highly stabilized and selective adrenomedullin analogs.

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids with a disulfide bond and an amidated C-terminus. Due to the vasodilatory and cardioprotective effects, the agonistic activity of the peptide on the adrenomedullin 1 receptor (AM1 R) is of high pharmacological interest. However, the wild-type peptide shows low metabolic stability leading to rapid degradation in the cardiovascular system. Previous work by our group has identified proteolytic cleavage sites and demonstrated stabilization of ADM by lipidation, cyclization, and N-methylation. Nevertheless, these ADM analogs showed reduced activity and subtype selectivity toward the closely related calcitonin gene-related peptide receptor (CGRPR). Here, we report on the rational development of ADM derivatives with increased proteolytic stability and high receptor selectivity. Stabilizing motifs, including lactamization and lipidation, were evaluated regarding AM1 R and CGRPR activation. Furthermore, the central DKDK motif of the peptide was replaced by oligoethylene glycol linkers. The modified peptides were synthesized by Fmoc/t-Bu solid-phase peptide synthesis and receptor activation of AM1 R and CGRPR was measured by cAMP reporter gene assay. Peptide stability was tested in human blood plasma and porcine liver homogenate and analyzed by RP-HPLC and MALDI-ToF mass spectrometry. Combination of the favorable lactam, lipidation, ethylene glycol linker, and previously described disulfide mimetic resulted in highly stabilized analogs with a plasma half-life of more than 144 h. The compounds display excellent AM1 R activity and wild-type-like selectivity toward CGRPR. Additionally, dose-dependent vasodilatory effects of the ADM derivatives lasted for several hours in rodents. Thus, we successfully developed an ADM analog with long-term in vivo activity.

Cyclic Derivatives of the Chemerin C-Terminus as Metabolically Stable Agonists at the Chemokine-like Receptor 1 for Cancer Treatment.

Chemerin is a small chemotactic protein and a modulator of the innate immune system. Its activity is mainly mediated by the chemokine-like receptor 1 (CMKLR1), a receptor expressed by natural killer cells, dendritic cells, and macrophages. Downregulation of chemerin is part of the immune evasion strategy exploited by several cancer types, including melanoma, breast cancer, and hepatocellular carcinoma. Administration of chemerin can potentially counteract these effects, but synthetically accessible, metabolically stable analogs are required. Other tumors display overexpression of CMKLR1, offering a potential entry point for targeted delivery of chemotherapeutics. Here, we present cyclic derivatives of the chemerin C-terminus (chemerin-9), the minimal activation sequence of chemerin. Chemerin-9 derivatives that were cyclized through positions four and nine retained activity while displaying full stability in blood plasma for more than 24 h. Therefore, these peptides could be used as a drug shuttle system to target cancer cells as demonstrated here by methotrexate conjugates.

Development of a ghrelin receptor inverse agonist for positron emission tomography.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Photoinduced receptor confinement drives ligand-independent GPCR signaling.

Cell-cell communication relies on the assembly of receptor-ligand complexes at the plasma membrane. The spatiotemporal receptor organization has a pivotal role in evoking cellular responses. We studied the clustering of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and established a photoinstructive matrix with ultrasmall lock-and-key interaction pairs to control lateral membrane organization of hormone neuropeptide Y2 receptors in living cells by light. Within seconds, receptor clustering was modulated in size, location, and density. After in situ confinement, changes in cellular morphology, motility, and calcium signaling revealed ligand-independent receptor activation. This approach may enhance the exploration of mechanisms in cell signaling and mechanotransduction.

Tryptophan Analogues with Fixed Side-Chain Orientation: Expanding the Scope.

A generalized synthetic strategy is proposed here for the synthesis of asymmetric ß-indoylated amino acids by 8-aminoquinoline (8AQ)-directed C(sp3)-H functionalization of suitably protected precursors. Peptides containing one of the four stereoisomers of (indol-3-yl)-3-phenylalanine at position 2 of the parent peptide KwFwLL-NH2 (w=d-Trp) cover a wide range of activities as ghrelin receptor inverse agonists, among them the most active described until now. This application exemplarily shows how ß-indoylated amino acids can be used for the systematic variation of the position of an indole group in a bioactive peptide.

Peptide-Drug Conjugates and Their Targets in Advanced Cancer Therapies.

Cancer became recently the leading cause of death in industrialized countries. Even though standard treatments achieve significant effects in growth inhibition and tumor elimination, they cause severe side effects as most of the applied drugs exhibit only minor selectivity for the malignant tissue. Hence, specific addressing of tumor cells without affecting healthy tissue is currently a major desire in cancer therapy. Cell surface receptors, which bind peptides are frequently overexpressed on cancer cells and can therefore be considered as promising targets for selective tumor therapy. In this review, the benefits of peptides as tumor homing agents are presented and an overview of the most commonly addressed peptide receptors is given. A special focus was set on the bombesin receptor family and the neuropeptide Y receptor family. In the second part, the specific requirements of peptide-drug conjugates (PDC) and intelligent linker structures as an essential component of PDC are outlined. Furthermore, different drug cargos are presented including classical and recent toxic agents as well as radionuclides for diagnostic and therapeutic approaches. In the last part, boron neutron capture therapy as advanced targeted cancer therapy is introduced and past and recent developments are reviewed.

Adrenomedullin - Current perspective on a peptide hormone with significant therapeutic potential.

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids and plays a pivotal role in the regulation of many physiological processes, particularly those of the cardiovascular and lymphatic system. Like calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin (IMD) and amylin (AMY), it belongs to the CT/CGRP family of peptide hormones, which despite their low little sequence identity share certain characteristic structural features as well as a complex multicomponent receptor system. ADM, IMD and CGRP exert their biological effects by activation of the calcitonin receptor-like receptor (CLR) as a complex with one of three receptor activity-modifying proteins (RAMP), which alter the ligand affinity. Selectivity within the receptor system is largely mediated by the amidated C-terminus of the peptide hormones, which bind to the extracellular domains of the receptors. This enables their N-terminus consisting of a disulfide-bonded ring structure and a helical segment to bind within the transmembrane region and to induce an active receptor confirmation. ADM is expressed in a variety of tissues in the human body and is fundamentally involved in multitude biological processes. Thus, it is of interest as a diagnostic marker and a promising candidate for therapeutic interventions. In order to fully exploit the potential of ADM, it is necessary to improve its pharmacological profile by increasing the metabolic stability and, ideally, creating receptor subtype-selective analogs. While several successful attempts to prolong the half-life of ADM were recently reported, improving or even retaining receptor selectivity remains challenging.

A Selective Carborane-Functionalized Gastrin-Releasing Peptide Receptor Agonist as Boron Delivery Agent for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) allows the selective elimination of malignant tumor cells without affecting healthy tissue. Although this binary radiotherapy approach has been known for decades, BNCT failed to reach the daily clinics to date. One of the reasons is the lack of selective boron delivery agents. Using boron loaded peptide conjugates, which address G protein-coupled receptors overexpressed on tumor cells allow the intracellular accumulation of boron. The gastrin-releasing peptide receptor (GRPR) is a well-known target in cancer diagnosis and can potentially be used for BNCT. Here, we present the successful introduction of multiple bis-deoxygalactosyl-carborane building blocks to the GRPR-selective ligand [d-Phe6, ß-Ala11, Ala13, Nle14]Bn(6-14) (sBB2L) generating peptide conjugates with up to 80 boron atoms per molecule. Receptor activation was retained, metabolic stability was increased, and uptake into PC3 cells was proven without showing any intrinsic cytotoxicity. Furthermore, undesired uptake into liver cells was suppressed by using l-deoxygalactosyl modified carborane building blocks. Due to its high boron loading and excellent GRPR selectivity, this conjugate can be considered as a promising boron delivery agent for BNCT.

Selective Neuropeptide Y Conjugates with Maximized Carborane Loading as Promising Boron Delivery Agents for Boron Neutron Capture Therapy.

G-protein-coupled receptors like the human Y1 receptor (hY1R) are promising targets in cancer therapy due to their high overexpression on cancer cells and their ability to internalize together with the bound ligand. This mechanism was exploited to shuttle boron atoms into cancer cells for the application of boron neutron capture therapy (BNCT), a noninvasive approach to eliminate cancer cells. A maximized number of carboranes was introduced to the hY1R-preferring ligand [F7,P34]-NPY by solid phase peptide synthesis. Branched conjugates loaded with up to 80 boron atoms per peptide molecule exhibited a maintained receptor activation profile, and the selective uptake into hY1R-expressing cells was demonstrated by internalization studies. In order to ensure appropriate solubility in aqueous solution, we proved the need for eight hydroxyl groups per carborane. Thus, we suggest the utilization of bis-deoxygalactosyl-carborane building blocks in solid phase peptide synthesis to produce selective boron delivery agents for BNCT.

Identification and stabilization of a highly selective gastrin-releasing peptide receptor agonist.

The gastrin-releasing peptide receptor (GRPR) is part of the bombesin receptor family and a well-known target in cancer diagnosis and therapy. In the last decade, promising results have been achieved by using peptide-drug conjugates, which allow selective targeting of GRPR expressing tumor cells. Most ligands, however, have been antagonists even though agonists can lead to higher tumor uptake owing to their internalization. So far, only a few studies focused on the identification of small GRPR-selective agonists that are metabolically stable. Here, we developed novel bombesin analogs with high selectivity for the GRPR and improved blood plasma stability. The most promising analog [d-Phe6 , ß-Ala11 , NMe-Ala13 , Nle14 ]Bn(6-14) displays an activity of 0.3nM at the GRPR, a more than 4000-fold selectivity over the other two bombesin receptors and more than 75% stability in human blood plasma after 24 hours. This analog is proposed as a promising drug shuttle for the intracellular delivery of different payloads in targeted tumor therapy approaches.

Side Chain Orientation of Tryptophan Analogues Determines Agonism and Inverse Agonism in Short Ghrelin Peptides.

We describe two synthetic amino acids with inverted side chain stereochemistry, which induce opposite biological activity. Phe4 is an important part of the activation motif of ghrelin, and in short peptide inverse agonists such as KwFwLL-NH2 , the aromatic core is necessary for inactivation of the receptor. To restrict indole/phenyl mobility and simultaneously strengthen the interaction between peptide and receptor, we exchanged the natural monoaryl amino acids for diaryl amino acids derived from tryptophan. By standard solid-phase peptide synthesis, each of them was inserted into ghrelin or in the aromatic core of the inverse agonist. Both ghrelin analogues showed nanomolar activity, indicating sufficient space to accommodate the additional side chain. In contrast, diaryl amino acids in the inverse agonist had considerable influence on receptor signaling. Whereas the introduction of Wsf maintains inverse agonism of the peptide, Wrf shifts the receptor more to active states and can induce agonism depending on its introduction site.

Adrenomedullin disulfide bond mimetics uncover structural requirements for AM1 receptor activation.

Adrenomedullin (ADM) is a vasoactive peptide hormone of 52 amino acids and belongs to the calcitonin peptide superfamily. Its vasodilative effects are mediated by the interaction with the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor (GPCR), associated with the receptor activity modifying protein 2 (RAMP2) and functionally described as AM-1 receptor (AM1 R). A disulfide-bonded ring structure consisting of six amino acids between Cys16 and Cys21 has been shown to be a key motif for receptor activation. However, the specific structural requirements remain to be elucidated. To investigate the influence of ring size and position of additional functional groups that replace the native disulfide bond, we generated ADM analogs containing thioether, thioacetal, alkane, and lactam bonds between amino acids 16 and 21 by Fmoc/t-Bu solid phase peptide synthesis. Activity studies of the ADM disulfide bond mimetics (DSBM) revealed a strong impact of structural parameters. Interestingly, an increased ring size was tolerated but the activity of lactam-based mimetics depended on its position within the bridging structure. Furthermore, we found the thioacetal as well as the thioether-based mimetics to be well accepted with full AM1 R activity. While a reduced selectivity over the calcitonin gene-related peptide receptor (CGRPR) was observed for the thioethers, the thioacetal was able to retain a wild-type-like selectivity profile. The carbon analog in contrast displayed weak antagonistic properties. These results provide insight into the structural requirements for AM1 R activation as well as new possibilities for the development of metabolically stabilized analogs for therapeutic applications of ADM.

Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor.

The peptide ghrelin targets the growth hormone secretagogue receptor 1a (GHSR) to signal changes in cell metabolism and is a sought-after therapeutic target, although no structure is known to date. To investigate the structural basis of ghrelin binding to GHSR, we used solid-state nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, and Rosetta modeling. The use of saturation transfer difference NMR identified key residues in the peptide for receptor binding beyond the known motif. This information combined with assignment of the secondary structure of ghrelin in its receptor-bound state was incorporated into Rosetta using an approach that accounts for flexible binding partners. The NMR data and models revealed an extended binding surface that was confirmed via mutagenesis. Our results agree with a growing evidence of peptides interacting via two sites at G protein-coupled receptors.

A stable meta-carborane enables the generation of boron-rich peptide agonists targeting the ghrelin receptor.

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron-containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein-coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor-directed boron uptake. In this study, we present the generation of short, boron-rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super-agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta-carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron-loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.

The Impact of Adrenomedullin Thr22 on Selectivity within the Calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein System.

Adrenomedullin (ADM) is a peptide hormone of the calcitonin gene-related peptide (CGRP) family. It is involved in the regulation of cardiovascular processes such as angiogenesis, vasodilation, and the reduction of oxidative stress. ADM mediates its effects by activation of the ADM-1 and -2 receptors (AM1 R/AM2 R), but also activates the CGRP receptor (CGRPR) with reduced potency. It binds to the extracellular domains of the receptors with its C-terminal binding motif (residues 41-52). The activation motif, consisting of a disulfide-bonded ring structure (residues 16-21) and an adjacent helix (residues 22-30), binds to the transmembrane region and stabilizes the receptor conformation in the active state. While it was shown that the binding motif of ADM guides AM1 R selectivity, there is little information on the activation motif itself. Here, we demonstrate that Thr22 of ADM contributes to the selectivity. By using solid-phase peptide synthesis and cAMP-based signal transduction, we studied the effects of analogues in the activation motif of ADM on AM1 R and CGRPR activity. Our results indicate that Thr22 terminates the α-helix and orients the ring segment by hydrogen bonding. Using olefin stapling, we showed that the α-helical arrangement of the ring segment leads to decreased AM1 R activity, but does not affect CGRPR activation. These results demonstrate that the conformation of the ring segment of ADM has a strong impact on the selectivity within the receptor system.

Peptide chemistry toolbox - Transforming natural peptides into peptide therapeutics.

The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market.

Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes.

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40-50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.

Development of Fluorinated Non-Peptidic Ghrelin Receptor Ligands for Potential Use in Molecular Imaging.

The ghrelin receptor (GhrR) is a widely investigated target in several diseases. However, the current knowledge of its role and distribution in the brain is limited. Recently, the small and non-peptidic compound (S)-6-(4-bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)-2-methylpyrido[3,2-d]pyrimidin-4(3H)-one ((S)-9) has been described as a GhrR ligand with high binding affinity. Here, we describe the synthesis of fluorinated derivatives, the in vitro evaluation of their potency as partial agonists and selectivity at GhrRs, and their physicochemical properties. These results identified compounds (S)-9, (R)-9, and (S)-16 as suitable parent molecules for 18F-labeled positron emission tomography (PET) radiotracers to enable future investigation of GhrR in the brain.

Adrenomedullin - new perspectives of a potent peptide hormone.

Adrenomedullin (ADM) is a 52-amino acid multifunctional peptide, which belongs to the calcitonin gene-related peptide (CGRP) superfamily of vasoactive peptide hormones. ADM exhibits a significant vasodilatory potential and plays a key role in various regulatory mechanisms, predominantly in the cardiovascular and lymphatic system. It exerts its effects by activation of the calcitonin receptor-like receptor associated with one of the receptor activity-modifying proteins 2 or 3. ADM was first isolated from human phaeochromocytoma in 1993. Numerous studies revealed a widespread distribution in various tissues and organs, which is reflected by its multiple physiological roles in health and disease. Because of its anti-inflammatory, anti-apoptotic and proliferative properties, ADM exhibits potent protective functions under diverse pathological conditions, but it is also critically involved in tumor progression. ADM has therefore raised great interest in therapeutic applications and several clinical trials already revealed promising results. However, because the receptor activation mode has not yet been fully elucidated, a rational design of potent and selective ligands is still challenging. Detailed information on the binding mode of ADM from a recently reported crystal structure as well as efforts to improve its plasma stability and bioavailability may help to overcome these limitations in the future. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Development of Potent and Metabolically Stable APJ Ligands with High Therapeutic Potential.

The apelin ligand receptor system is an important target to develop treatment strategies for cardiovascular diseases. Although apelin exhibits strong inotropic effects, its pharmaceutical application is limited because no agonist with suitable properties is available. On the one hand, peptide ligands are too instable, and on the other hand, small-molecule agonists show only low potency. This study describes the development of apelin (APJ) receptor agonists with not only high activity but also metabolic stability. Several strategies including capping of termini, insertion of unnatural amino acids, cyclization, and lipidation were analyzed. Peptide activity was tested using a Ca2+ -mobilization assay and the degradation of selected analogues was analyzed in rat plasma. The best results were obtained by N-terminal lipidation of a 13-mer apelin derivative. This analogue displayed a half-life of 29 h in rat plasma, compared with 0.025 h for the wild-type peptide. Furthermore, in vivo pharmacokinetics revealed a clearance of 0.049 L h-1 kg-1 and a half-life of 0.36 h. In summary, amino acid substitution and fatty acid modification resulted in a potent and 1000-fold more stable peptide that exhibits high pharmaceutical potential.