In vitro activity: FPS-ZM1 inhibits Aβ/RAGE binding in CHO cells with approximately 2-fold greater affinity than its parent molecule, FPS2. FPS-ZM1 inhibits binding of other known RAGE ligands to sRAGE, including S100 calcium-binding protein B and amphoterin. FPS-ZM1 is more effective than FPS2 in reducing Aβ40-induced increases inBACE1 mRNA and protein levels and the generation of sAPPβ, an APP cleavage product of BACE1 indicative of BACE1 activity.

Kinase Assay: Human sRAGE is immobilized (10 μg/mL) overnight at 4°C in 96-well microtiter plates and blocked with 3% bovine serum albumin. 125I-labeled Aβ40, HMGB1, or S100B at 5 nM in the absence and presence of various concentrations of FPS2 or FPS-ZM1 (10 to 1,000 nM) is added to the wells containing immobilized sRAGE and incubated for 1 hour at room temperature in PBS. Wells are washed with cold PBS to remove unbound radiolabeled ligands, and the radioactivity is analyzed.

Cell Assay: To determine whether FPS2 and FPS-ZM1 are toxic to CHO cells, the cells are treated for 72 hours with different concentrations of inhibitors ranging from 10 nM to 10 μM. The cellular toxicity is determined using the WST-8 Assay Kit.

Mice: FPS2 or FPS-ZM1 are administered i.v. (1 mg/kg) via the femoral vein and arterial blood samples (30 μL) collected at 1, 5, 10, 15, and 20 minutes via the cannulated femoral artery. Plasma is separated by centrifugation at 4°C and immediately stored at -80°C until analysis.