Fred H. Rasmussen

ADAM8 as a drug target in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with less than 5% survivors after 5 years. High expression levels of ADAM8, a metalloprotease-disintegrin, are correlated with poor clinical outcome. We show that ADAM8 expression is associated with increased migration and invasiveness of PDAC cells caused by activation of ERK 1/2 and higher MMP activities. For biological function, ADAM8 requires multimerisation and associates with β1-integrin on the cell surface. A peptidomimetic ADAM8 inhibitor, BK-1361, designed by structural modelling of the disintegrin domain, prevents ADAM8 multimerisation. In PDAC cells, BK-1361 affects ADAM8 function leading to reduced invasiveness, and less ERK 1/2 and MMP activation. BK-1361 application in mice decreased tumour burden and metastasis of implanted pancreatic tumour cells and provides improved metrics of clinical symptoms and survival in a KrasG12D-driven mouse model of PDAC. Thus, our data integrate ADAM8 in pancreatic cancer signalling and validate ADAM8 as a target for PDAC therapy.

Fluorescent substrates useful as high-throughput screening tools for ADAM9.

Fluorescence resonance energy transfer substrates were designed and tested as substrates for ADAM9. The donor/quencher pair used were 5-carboxy fluorescein (Fam) and 4-(4-dimethyl-aminophenylazo)benzoyl (Dabcyl) since they have been well studied sensitive fluorescent probes. The peptides based on precursor TNF-alpha, Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Fam)- NH2 and Dabcyl-Leu-Ala-Gln-Ala-HomoPhe-Arg-Ser-Lys(Fam)- NH2, and C-terminal TGF-alpha, Dabcyl-Glu-His-Ala-Asp-Leu-Leu-Ala-Val-Val-Ala-Ala-Lys(Fam)- NH2 cleavage sites were effectively processed by ADAM9 with turnover numbers of 100 +/- 20 x 10(-2) min(-1), 20 +/- 10 x 10(-2) min(-1), and 10 +/- 3 x 10(-2) min(-1). In addition, a peptide based on the 33 kDa cleavage site of the low affinity receptor for IgE, CD23, Dabcyl-Leu-Arg-Ala-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(Fam)- NH2 was processed as well but with less efficiency. A more selective substrate for ADAM9 was found based on the betacellulin cleavage site. However, the valine containing precursor TNF-alpha based substrate was used to measure IC50 values of metalloproteinase inhibitors against ADAM9 since it was processed the most efficiently. The tightest binding inhibitor was the Wyeth Aerst compound, TMI-1, with an IC50 of 2.1 +/- 0.3 nM. In addition, GI254023, previously identified as a selective inhibitor of ADAM10, also inhibited ADAM9 with an IC50 of 280 +/- 110 nM. These results demonstrate that sensitive substrates for ADAM9 can be developed that are useful in high-throughput screening assays for ADAM9.

The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay1

Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98-108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 mM). The results obtained show that the catalytic efficiency of camel chymosin is 3-fold higher than bovine chymosin. The substrate affinity and catalytic activity of bovine and camel chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method.