TAPI-2

Kinase experiment:

Meprin activities are determined using N-benzoyl-L-tyrosyl p-aminobenzoic acid as substrate. The substrate concentration is 40 mM, and the enzyme concentration is always at least 10 times below Ki. Inhibitors are employed in a concentration range from 5 pM to 5 mM, depending on their inhibitory effect. Each inhibitor is tested over a concentration range covering at least ten different concentrations from Ki/5 to 5×Ki. All reactions are carried out at 37°C in 50 mM Tris/HCl, pH 7.5, and 0.5 mM MgCl2[1].

Cell experiment:

TAPI-2 is dissolved in DMSO and diluted with appropriate medium before use. All experiments are performed using 20 μM TAPI-2. Cells are cultured with or without TAPI-2 for 48 hours and then seeded at 3,000 cells per well in 96-well plates. After pretreatment, increasing doses of 5-fluorouracil (5-FU) that are relevant to the recommended clinical dose (up to 2 μg/mL) are added, with or without TAPI-2, for 72 hours. Cell viability is assessed by adding MTT substrate (0.25% in phosphate-buffered saline [PBS]) in growth medium (1:5 dilution) to cells for 1 hour at 37°C. The cells are ished with PBS, and 100 μL of dimethyl sulfoxideis added. Optical density is measured at 570 nM, and relative MTT is presented as a percentage of control[2].

References:

[1]. Kruse MN, et al. Human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors. Biochem J. 2004 Mar 1;378(Pt 2):383-9.
[2]. Wang R, et al. A Disintegrin and Metalloproteinase Domain 17 Regulates Colorectal Cancer Stem Cells and Chemosensitivity Via Notch1 Signaling. Stem Cells Transl Med. 2016 Mar;5(3):331-8.