Development of a Cost-Effective HPLC Method for Measuring BACE1 Activity in the Presence of Peptide Inhibitors

Objectives: Using high-performance liquid chromatography (HPLC) we developed and validated an in vitro assay for the quantitative determination of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) activity, supplementing limited current methodologies to assess the efficacy of BACE1 inhibitor compounds. A hexa-histidine tagged peptide substrate of BACE1 was used as the analyte for the determination of in vitro BACE1 activity; it was validated according to ICH guidelines. Methods: The HPLC analysis was performed on the Agilent 1290 Series Infinity II UHPLC System equipped with a Phenomenex Kinetex EVO C18 (100 × 3 mm) 5 µm column. The method was developed using a gradient program comprising of 10 % aqueous acetonitrile (0.02 M TFA) to 30% aqueous acetonitrile (0.02 M TFA) for 5 minutes at a flow rate of 0.6 ml/min. Results: The method showed linearity over the range of 14.92 to 72 µM with R^2=0.9997. The accuracy of the method in terms of mean recovery ranged between 96.62 to 98.38 %. The %RSD for intra- and inter-day precision were less than 5 %. Two commercial inhibitors, AZD3839 and OM99-2, were used to evaluate the performance of the method at their respective IC50, resulting in inhibition of 53.46 and 50.74 % respectively. The described method addresses the void for a practical and cheap alternative to quantitatively determine the activity of BACE1 compared to current commercially available detection assays. Conclusions: We have successfully developed a HPLC method to measure the inhibitory function of two commercial inhibitors of BACE1, indicating suitability of the method for the identification and characterisation of novel BACE1 inhibitors.