Synthesis, 18F-labelling and radiopharmacological characterisation of the C-terminal 30mer of Clostridium perfringens enterotoxin as a potential claudin-targeting peptide.

The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE290-319 appears as a suitable ligand for targeting Cld-4. The synthesis of this 30mer peptide was attempted via several approaches, which has revealed sequential SPPS using three pseudoproline dipeptide building blocks to be the most efficient one. Labelling with fluorine-18 was achieved on solid phase using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents, which was the most advantageous when [18F]SFB was reacted with the resin-bound 30mer containing an N-terminal 6-aminohexanoic spacer. Binding to Cld-4 was demonstrated via surface plasmon resonance using a protein construct containing both extracellular loops of Cld-4. In addition, cell binding experiments were performed for 18F-labelled cCPE290-319 with the Cld-4 expressing tumour cell lines HT-29 and A431 that were complemented by fluorescence microscopy studies using the corresponding fluorescein isothiocyanate-conjugated peptide. The 30mer peptide proved to be sufficiently stable in blood plasma. Studying the in vivo behaviour of 18F-labelled cCPE290-319 in healthy mice and rats by dynamic PET imaging and radiometabolite analyses has revealed that the peptide is subject to substantial liver uptake and rapid metabolic degradation in vivo, which limits its suitability as imaging probe for tumour-associated Cld-4.

The why and how of amino acid analytics in cancer diagnostics and therapy.

Pathological alterations in cell functions are frequently accompanied by metabolic reprogramming including modifications in amino acid metabolism. Amino acid detection is thus integral to the diagnosis of many hereditary metabolic diseases. The development of malignant diseases as metabolic disorders comes along with a complex dysregulation of genetic and epigenetic factors affecting metabolic enzymes. Cancer cells might transiently or permanently become auxotrophic for non-essential or semi-essential amino acids such as asparagine or arginine. Also, transformed cells are often more susceptible to local shortage of essential amino acids such as methionine than normal tissues. This offers new points of attacking unique metabolic features in cancer cells. To better understand these processes, highly sensitive methods for amino acid detection and quantification are required. Our review summarizes the main methodologies for amino acid detection with a particular focus on applications in biomedicine and cancer, provides a historical overview of the methodological pre-requisites in amino acid analytics. We compare classical and modern approaches such as the combination of gas chromatography and liquid chromatography with mass spectrometry (GC-MS/LC-MS). The latter is increasingly applied in clinical routine. We therefore illustrate an LC-MS workflow for analyzing arginine and methionine as well as their precursors and analogs in biological material. Pitfalls during protocol development are discussed, but LC-MS emerges as a reliable and sensitive tool for the detection of amino acids in biological matrices. Quantification is challenging, but of particular interest in cancer research as targeting arginine and methionine turnover in cancer cells represent novel treatment strategies.

Pigment epithelium-derived factor released by Müller glial cells exerts neuroprotective effects on retinal ganglion cells.

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.