The proteome and phosphoproteome have been determined for BRAF inhibitor resistant and sensitive cells by applying quantitative MS-based proteomics. We identified the intermediate filament protein nestin as one of the highest downregulated proteins in melanoma cells and tumors. The results reported reveal a link between loss of nestin and an invasive phenotype and provide a quantitative view of PI3K/ Akt and integrin pathways involved in resistant and nestin knockout cells.

Blood coagulation factor XIII (FXIII, F13) is considered to be a promising target for anticoagulants with reduced bleeding risk because of its unique position in the coagulation cascade downstream of thrombin. However, until now, no potent drug addressing FXIII has been available, indeed no compound has even entered clinical trials yet. In 2013, we published the co‐crystal structure of FXIII in the active state (FXIIIa°), thereby providing a detailed map of the active site for the rational design of potent FXIIIa blockers. Here we report, for the first time, a structure‐based approach to improving the affinity of FXIIIa inhibitors. FXIII was crystallized in complex with a methyl thiazole moiety to address a novel transient hydrophobic pocket close to the catalytic center. By subsequent structure‐based design to rationalize the introduction of an ethyl ester, the potency of the inhibitor was improved significantly compared to that of the parent lead compound. The occupancy of the hydrophobic pocket described here might turn out to be a key step in the development of a potent reversible and orally available FXIIIa blocker.

Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K+ channel with a mean inward conductance of 55 +/- 6.5 pS (n = 20) and a mean outward conductance of 25 +/- 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl- channel with a mean outward conductance of 30 +/- 4.5 pS (n = 8) and a mean inward conductance of 10 +/- 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca2+ activity on the intracellular side. Accordingly, the Ca2+ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K+ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K+ channel is inhibited by progesterone, while the outwardly rectifying Cl- channel is insensitive to progesterone.

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Immune checkpoint inhibitors are used to restore or augment antitumor immune responses and show great promise in the treatment of melanoma and other types of cancers. However, only a small percentage of patients are fully responsive to immune checkpoint inhibition, mostly due to tumor heterogeneity and primary resistance to therapy. Both of these features are largely driven by the accumulation of patient-specific mutations, pointing to the need for personalized approaches in diagnostics and immunotherapy. Proteogenomics integrates patient-specific genomic and proteomic data to study cancer development, tumor heterogeneity and resistance mechanisms. Using this approach, we characterized the mutational landscape of four clinical melanoma patients. This enabled the quantification of hundreds of sample-specific amino acid variants, among them many that were previously not reported in melanoma. Changes in abundance at the protein and phosphorylation site levels revealed patient-specific over-represented pathways, notably linked to melanoma development (MAPK1 activation) or immunotherapy (NLRP1 inflammasome). Personalized data integration resulted in the prediction of protein drug targets, such as the drugs vandetanib and bosutinib, which were experimentally validated and led to a reduction in the viability of tumor cells. Our study emphasizes the potential of proteogenomic approaches to study personalized mutational landscapes, signaling networks and therapy options.