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In memory of Rainer RudolphBreast cancer is still one of the most frequently occurring tumors in women. Severe and often therapy-limiting side effects are a major obstacle in chemotherapy. New delivery concepts that reduce systemic side effects are needed to optimize anticancer therapies, and selective targeting concepts are required for early and selective tumor diagnosis. Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, is a C-terminal amidated peptide hormone consisting of 36 amino acid residues.[1, 2] NPY-mediated functions are transmitted by so-called Y receptors, named Y 1 , Y 2 , and Y 5 receptors, which bind NPY with nanomolar affinity. All Y receptors are members of the class A of heptahelix receptors, that signal through heterotrimeric G proteins. [3,4] Reubi et al. have recently described Y-receptor expression in human breast cancer. They have shown that over 90 % of all breast tumors and 100 % of the examined metastases express Y 1 receptors.[5] Interestingly, a shift of the receptor subtype from Y 2 receptors in healthy tissue to Y 1 receptors during neoplasm was found, which is potentially related to reduced differentiation. Based on NPY and the known structure-activity relationships for Y 1 -receptor binding, [6] we designed, synthesized, and characterized two analogues for tumor labeling that vary in the position of the chelator to conjugate 99m Tc. Peptides 1 a and 2 a were synthesized with a N a -histidinyl acetyl (N a His-ac) chelator [7] at the N terminus, whereas peptides 1 b and 2 b were modified at the N e side chain of Lys 4 . The tridentate ligand N a His-ac is able to form stable and biologically active complexes. [8,9] Modification of the resin-bound peptide was performed by an efficient strategy (Scheme 1). In the first step, bromoacetic acid was activated by diisopropylcarbodiimide to form the corresponding anhydride. His(Trt)-OtBu was then added and the NHÀ CH bond was formed by HBr elimination. Cleavage of the peptide yielded His-acetyl peptides either at the N terminus or at the N e side chain of Lys 4 . Rhenium was used as a cold surrogate for 99m Tc and introduced for in vitro studies

Selective NPY analogues are potent tools for tumour targeting. Their Y(1)-receptors are significantly over-expressed in human breast tumours, whereas normal breast tissue only expresses Y(2)-receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y(1)-receptor agonists enhance the tumour to background ratio compared to Y(1)-receptor antagonists, we were interested in the development of Y(1)-receptor selective agonists. We designed 19 peptides containing the C-terminus of NPY (28-36) with several modifications. By using competition receptor binding affinity assays, we identified three NPY analogues with high Y(1)-receptor affinity and selectivity. Metabolic stability studies in human blood plasma of the N-terminally 5(6)-carboxyfluorescein (CF) labelled peptides resulted in half-lives of several hours. Furthermore, the degradation pattern revealed proteolytic degradation of the peptides by amino peptidases. The most promising peptide was further investigated in receptor activation and internalization studies. Signal transduction assays revealed clear agonistic properties, which could be confirmed by microscopy studies that showed clear Y(1)-receptor internalization. For the first time, here we show the design and characterization of a small Y(1)-receptor selective agonist. This agonist might be a useful novel ligand for NPY-mediated tumour diagnostics and therapeutics.

Axonal polyneuropathies are a frequent cause of progressive disability in the elderly. Common etiologies comprise diabetes mellitus, paraproteinaemia, and inflammatory disorders, but often the underlying causes remain elusive. Late-onset axonal Charcot-Marie-Tooth neuropathy (CMT2) is an autosomal-dominantly inherited condition that manifests in the second half of life and is genetically largely unexplained. We assumed age-dependent penetrance of mutations in a so far unknown gene causing late-onset CMT2. We screened 51 index case subjects with late-onset CMT2 for mutations by whole-exome (WES) and Sanger sequencing and subsequently queried WES repositories for further case subjects carrying mutations in the identified candidate gene. We studied nerve pathology and tissue levels and function of the abnormal protein in order to explore consequences of the mutations. Altogether, we observed heterozygous rare loss-of-function and missense mutations in MME encoding the metalloprotease neprilysin in 19 index case subjects diagnosed with axonal polyneuropathies or neurodegenerative conditions involving the peripheral nervous system. MME mutations segregated in an autosomal-dominant fashion with age-related incomplete penetrance and some affected individuals were isolated case subjects. We also found that MME mutations resulted in strongly decreased tissue availability of neprilysin and impaired enzymatic activity. Although neprilysin is known to degrade β-amyloid, we observed no increased amyloid deposition or increased incidence of dementia in individuals with MME mutations. Detection of MME mutations is expected to increase the diagnostic yield in late-onset polyneuropathies, and it will be tempting to explore whether substances that can elevate neprilysin activity could be a rational option for treatment.

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. Gprotein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, autofluorescent proteins as well as the evolving technologies for chemical labeling with peptide-and protein-tags are described and their major applications concerning the GPCR life cycle are presented.