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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.

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2's transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein- and rhodamine B-conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid-phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L-lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z' factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

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 cCPE

Peptides labeled with short-lived positron-emitting radionuclides are of outstanding interest as probes for molecular imaging by positron emission tomography (PET). Herein, the site-selective incorporation of fluorine-18 into lysine-containing peptides using the prosthetic labeling agent N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) is described. The reaction of [(18)F]SFB with four biologically relevant resin-bound peptides was studied and optimized. For comparison, each peptide was 18F-fluorobenzoylated in solution under different conditions and the product distribution was analyzed confirming the advantages of the solid-phase approach. The method's feasibility for selective radiolabeling either at the N-terminus or at the lysine side chain was demonstrated. Labeling on solid phase with [(18)F]SFB resulted in crude (18)F-fluorobenzoylpeptides whose radiochemical purities were typically greater than 90% and that could be prepared in synthesis times from 65 to 76 min.