NIK SMI1

Cell experiment:

Human B cells are re-suspended in RPMI with 10% FBS for the proliferation assays and 2.5% FBS for the survival assays. Mouse B cells are plated in Co-star 96-well plates at either 50,000 cells/well for the survival assays or at 150,000 cells/well for the proliferation assays. Compounds (e.g., NIK SMI1) diluted in DMSO (final DMSO assay concentration=0.1%) are added to the cells. The cells are incubated with NIK SMI1 for one hour at 37°C. Stimulus is then added to the plates and survival or proliferation is measured after four days. For the proliferation assays, cells are treated with either Anti-IgM (20 µg/mL) or rhCD40L (10 µg/mL) or anti-mouse CD40 (100 ng/mL). For the BAFF survival assay, cells are treated with human or mouse rBAFF at 10 ng/mL followed by Cell Titer Glo to measure survival on day four[1].

Animal experiment:

Mice[1]Age-matched C57BL/6 mice are used. Only female mice are used in these experiments. The single oral doses of NIK SMI1 are 10, 20, 60, 100, and 200 mg/kg. For PO dosing, animals are manually restrained, then dosed via oral gavage using an appropriately sized gavage needle. Animals are monitored for any signs of aspiration or distress-respiratory abnormalities, lethargy, pale extremities, etc. For sample collection, 3 mice per group are bled a total of 8 times via tail prick using a 27 G needle (lateral tail vein). 10 μL of blood is collected at each timepoint and deposited into a pre-filled costar cluster tube containing 40 μL of 1.7 mg/mL EDTA/water, the tube is capped, votexed for 5 seconds, then stored on dry ice. Samples are transferred to a -80°C freezer for storage[1].

References:

[1]. Blaquiere N, et al. Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase. J Med Chem. 2018 Aug 9;61(15):6801-6813.