Discovery of an APP-Selective BACE1 Inhibitor for Alzheimer's Disease

Abstract

Inhibition of amyloid precursor protein (APP) beta-site cleaving enzyme 1 (BACE1; BACE) has been a target for Alzheimers disease (AD) therapeutic development, but has been impaired by off-target effects of clinically evaluated inhibitors, including inhibition of cleavage of non-APP substrates. Here, we report our identification of a BACE inhibitors series that are not only selective for the APP substrate, but also for BACE1 as the targeted enzyme. These APP-selective fluoro aminohydantoin (FAH) inhibitor compounds were identified by screening a compound library for inhibition of BACE cleavage of a maltose binding protein (MBP)-conjugated-APPC125 substrate followed by IC50 determination using the P5-P5 substrate assay. In multiple substrate and enzyme cell-free assays, the lead compound FAH65 displayed substrate selectivity for inhibition of APP cleavage, with little activity against BACE substrates neuregulin 1 (NRG1) or p-selectin glycoprotein ligand -1 (PSGL1). We also demonstrate FAH65 shows little inhibitory activity against the enzyme cathepsin D (Cat D) or BACE2. FAH65 inhibits production of BACE cleavage products soluble APP{beta} (sAPP{beta}) and the {beta} C-terminal fragment ({beta}CTF), as well as amyloid-{beta} (A{beta})1-40 and 1-42, in vitro in cells and in vivo in an animal model of AD. In a murine model of AD, FAH65 improved the discrimination score in the Novel Object Recognition (NOR) memory testing paradigm. The active enantiomer of FAH65, FAH65E(-), was obtained and tested in in vivo pharmacokinetic and pharmacodynamic (PK/PD) analysis, wherein it displayed good brain-penetrance and target engagement. Given its demonstrated selectivity for both enzyme and substrate, along with evidence it can improve cognitive performance in an animal model, FAH65 and its E(-) enantiomer merit continued pre-clinical development towards clinical testing as an APP-selective BACE1 inhibitor. Such a candidate would reduce A{beta} levels and overcome the deleterious effects of the non-selective BACE1 inhibitors that have failed in the clinic and potentially could be used as a maintenance therapy along with or following clearance of A{beta} from the brain with the approved antibody therapy for AD.