Melanoma is the most aggressive form of skin cancer, with fast progression and early dissemination mediated by the melanoma inhibitory activity (MIA) protein. Here, we discovered that dimerization of MIA is required for functional activity through mutagenesis of MIA which showed the correlation between dimerization and functional activity. We subsequently identified the dodecapeptide AR71, which prevents MIA dimerization and thereby acts as a MIA inhibitor. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy demonstrated the binding of AR71 to the MIA dimerization domain, in agreement with in vitro and in vivo data revealing reduced cell migration, reduced formation of metastases and increased immune response after AR71 treatment. We believe AR71 is a lead structure for MIA inhibitors. More generally, inhibiting MIA dimerization is a novel therapeutic concept in melanoma therapy.

Although vast information about the coordination ability of amino acids and peptides to metal ions is available, little use of this has been made in the rational design of selective peptide receptors. We have combined a copper(II) nitrilotriacetato (NTA) complex with an ammonium-ion-sensitive and luminescent benzocrown ether. This compound revealed micromolar affinities and selectivities for glycine- and histidine-containing sequences, which closely resembles those of copper(II) ion peptide binding: the two free coordination sites of the copper(II) NTA complex bind to imidazole and amido nitrogen atoms, replicating the initial coordination steps of non-complexed copper(II) ions. The benzocrown ether recognizes the N-terminal amino moiety intramolecularly, and the significantly increased emission intensity signals the binding event, because only if prior coordination of the peptide has taken place is the intramolecular ammonium ion-benzocrown ether interaction of sufficient strength in water to trigger an emission signal. Intermolecular ammonium ion-benzocrown ether binding is not observed. Isothermal titration calorimetry confirmed the binding constants derived from emission titrations. Thus, as deduced from peptide coordination studies, the combination of a truncated copper(II) coordination sphere and a luminescent benzocrown ether allows for the more rational design of sequence-selective peptide receptors.

Artificial ditopic receptors for the differentiation of phosphorylated peptides varying in i+3 amino acid side chains were synthesized, and their binding affinities and selectivities were determined. The synthetic receptors show the highest binding affinities to phosphorylated peptides under physiological conditions (HEPES, pH 7.5, 154 mM NaCl) reported thus far for artificial systems. The tight and selective binding was achieved by high cooperativity of the two binding moieties in the receptor molecules. All receptors interact with phosphorylated serine by bis(ZnII-cyclen) complex coordination and a second binding site recognizing a carboxylate or imidazole amino acid side chain functionality.

Malignant melanoma is a highly aggressive cancer with a very poor prognosis after the onset of metastasis. We have previously demonstrated that the protein melanoma inhibitory activity (MIA) is involved in the metastasis of and immunosuppression in malignant melanoma. Recently, we further established MIA as a therapeutic target to inhibit metastatic spread in malignant melanoma. We could show that an inhibition of MIA by a synthetic peptide decreased both the number of metastases as well as immunosuppression in a murine model of malignant melanoma. To control recurrence after surgical resection of a primary lesion, it is paramount to have diagnostic tools available that can detect a relapse due to the strong metastatic potential of melanoma. This follow-up is maintained with periodic re-examinations. Due to high cost and the associated radiation exposure, radiology examinations are avoided if possible. The analysis of prognostic markers in patient serum is therefore attractive. In this review, we focus on the quantitative analysis of the MIA protein as a prognostic tool because it has proven to be a useful serum marker for documenting disease progression of malignant melanoma. The MIA quantification assay itself is readily performed using an ELISA kit and common laboratory equipment. Because analysing MIA serum levels in combination with other established markers such as S100B improves their prognostic value, we feel that the quantification of MIA in the serum, among other markers, should be performed as a general standard of care in patients at risk of developing metastatic melanoma.

Fluorescent probes for the detection of protein phosphorylation on SDS-PAGE are presented. The probes were designed using a dinuclear metal-chelate phosphate recognition unit and an environmentally sensitive fluorophore. The specificity of the probes is determined by their binding site selectivity toward phosphate ions and the emission response induced by the change in the electrostatic environment of the fluorophore upon binding to a phosphorylated amino acid residue. The staining is fully reversible due to the noncovalent binding of the probe. Gel bands with less than 100 ng of phosphorylated alpha-casein are detectable with the new probes on a normal UV-table without specialized equipment like a laser-based gel scanner or a cooled camera detector.