Kathrin Bellmann-Sickert

Peptides and peptide conjugates: therapeutics on the upward path

The main benefit of natural peptides, peptide analogs and newly designed peptides as therapeutics, lies in their high selectivity and affinity, which are frequently in the nanomolar range. New drugs targeting protein-protein interactions often require larger interaction sites than small molecules can offer. Thus, many peptidic drugs are already applied in therapy at the current state. The next generation of peptide-based therapeutic agents is currently on its way from basic research to clinical studies and eventually to the pharmaceutical market. Development of more robust and long-lasting drugs owing to well-known and new stabilization strategies is yielding novel and continuously improving peptide drugs. The introduction of smart linkers that exhibit stability towards blood plasma but intracellular lability will lead to target-oriented activity, which might successfully decrease side effects. In this review, peptidic therapeutics on the market, in clinical studies and some of those in basic research are characterized. Stabilization strategies and intelligent linkers are discussed with respect to their use in peptide drug therapy.

Long-Acting Lipidated Analogue of Human Pancreatic Polypeptide Is Slowly Released into Circulation

The main disadvantages of peptide pharmaceuticals are their rapid degradation and excretion, their low hydrophilicity, and low shelf lifes. These bottlenecks can be circumvented by acylation with fatty acids (lipidation) or polyethylene glycol (PEGylation). Here, we describe the modification of a human pancreatic polypeptide analogue specific for the human (h)Y(2) and hY(4) receptor with PEGs of different size and palmitic acid. Receptor specificity was demonstrated by competitive binding studies. Modifications had only a small influence on binding affinities and no influence on secondary structure. Both modifications improved pharmacokinetic properties of the hPP analogue in vivo and in vitro, however, lipidation showed a greater resistance to degradation and excretion than PEGylation. Furthermore, the lipidated peptide is taken up and degraded solely by the liver but not the kidneys. Lipidation resulted in prolonged action of the hPP analogue in respect of reducing food intake in mice after subcutaneous administration. Therefore, the lipidated hPP analogue could constitute a potential new therapeutic agent against obesity.

Type I Arginine Methyltransferases PRMT1 and PRMT-3 Act Distributively

Asymmetric dimethylation of arginine residues is a common posttranslational modification of proteins carried out by type I protein arginine methyltransferases, including PRMT1 and -3. We report that the consecutive transfer of two methyl groups to a single arginine side chain by PRMT1 and -3 occurs in a distributive manner, i.e. with intermittent release of the monomethylated intermediate. The oligomeric state of PRMTs together with the clustering of methylated arginine residues in most proteins carrying this type of modification suggests that multiple methyl transfers to a single polypeptide chain might proceed in a processive manner by cooperation of multiple active sites. However, three different types of experiments provide evidence that the reaction is distributive even with substrates containing multiple methyl-accepting arginines, including one with 13 such residues. PRMT1 also does not prefer substrates already containing one or more singly or doubly methylated arginine residues. Even though the reaction is distributive, the efficiency of methylation of one particular protein strongly depends on the number of methyl-accepting arginine residues it contains.

Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.

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.

Palmitoylated SDF1 α Shows Increased Resistance against Proteolytic Degradation in Liver Homogenates

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.

Position and Length of Fatty Acids Strongly Affect Receptor Selectivity Pattern of Human Pancreatic Polypeptide Analogues

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.

Effects of Peripheral Neurotensin on Appetite Regulation and Its Role in Gastric Bypass Surgery

Neurotensin (NT) is a peptide expressed in the brain and in the gastrointestinal tract. Brain NT inhibits food intake, but the effects of peripheral NT are less investigated. In this study, peripheral NT decreased food intake in both mice and rats, which was abolished by a NT antagonist. Using c-Fos immunohistochemistry, we found that peripheral NT activated brainstem and hypothalamic regions. The anorexigenic effect of NT was preserved in vagotomized mice but lasted shorter than in sham-operated mice. This in combination with a strong increase in c-Fos activation in area postrema after ip administration indicates that NT acts both through the blood circulation and the vagus. To improve the pharmacokinetics of NT, we developed a pegylated NT peptide, which presumably prolonged the half-life, and thus, the effect on feeding was extended compared with native NT. On a molecular level, the pegylated NT peptide increased proopiomelanocortin mRNA in the arcuate nucleus. We also investigated the importance of NT for the decreased food intake after gastric bypass surgery in a rat model of Roux-en-Y gastric bypass (RYGB). NT was increased in plasma and in the gastrointestinal tract in RYGB rats, and pharmacological antagonism of NT increased food intake transiently in RYGB rats. Taken together, our data suggest that NT is a metabolically active hormone, which contributes to the regulation of food intake.

Selective labelling of stromal cell-derived factor 1α with carboxyfluorescein to study receptor internalisation†

SDF1α plays an important role in the regeneration of injured tissue after ischemia or stroke by inducing the migration of progenitor cells. In order to study the function of this therapeutically relevant chemokine site‐specific protein labelling is of great interest. However, modification of SDF1α is complicated because of its complex tertiary structure. Here, we describe the first site‐specific fluorescent modification of SDF1α by EPL. We recombinantly expressed SDF1α (1–49) by intein‐mediated protein expression. The C‐terminal peptide SDF1α (50–68) was synthesised by SPPS and selectively labelled with carboxyfluorescein at Lys56. In a cell migration assay, M‐[K56(CF)]SDF1α showed a clear potency to induce chemotaxis of human T‐cell leukaemia cells. Microscopic analysis on HEK293 cells transfected with the CXCR4 revealed specific binding of the fluorescent ligand. Furthermore, receptor‐induced internalisation of the ligand could be visualised. These results show that site‐specific modification of SDF1α yields in a biologically functional molecule that allows the characterisation of CXCR4 production of cells on a molecular level. Copyright

Peptide-Mediated Specific Immobilization of Catalytically Active Cytochrome P450 BM3 Variant

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants.

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.