In vitro activity: LY2811376 demonstrates concentration-dependent inhibition of hBACE1 with an IC50 of 239 and 249 nM against a small synthetic peptide or a larger chimeric protein substrate, respectively. LY2811376 treatment yields a concentration-dependent decrease in Aβ secretion in APP-overexpressing HEK293 cells. LY2811376 inhibits Aβ secretion with EC50 of ~100 nM in primary neuronal cultures of PDAPP transgenic mouse.
Kinase Assay: The stock solution for each FRET peptide substrate is prepared at 30 mM in dimethylsulfoxide (DMSO). The huBACE1:Fc muBACE1:Fc preparation is concentrated through YM10 Centricon. to a final concentration of at least 7 mg/mL. The optimal enzyme concentration for each FRET peptide substrate is determined individually at 30μM FRET peptide substrate in 50 mM ammonium acetate, pH 4.6, 1 mg/mL BSA and 1 mM Triton X-100. The enzymatic efficiency (kcat /Km) of either of the BACE1 orthologs toward individual FRET peptide substrates at 15, 30 and 100μM is determined under the optimal conditions for each substrate. The progress of the reaction is monitored by measuring an increase of the emission signal at 420 nm with excitation wavelength set at 320 nm, using a GEMINI fluorescence plate reader. Amino acid conjugated aminobenzoate is used to convert the emission signal in the relative fluorescence units into the molar concentration of product generated in the reaction mixture. The initial phase of the timedependence curve is fitted with a linear function whose slope is used to calculate the initial rate for huBACE1:Fc toward each peptide substrate. The kcat /Km values are calculated from the linear dependence of the initial rate on the concentration of each peptide.
Cell Assay: The cytotoxicity in the HEK293Swe cell model is assessed using a CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay. | 