P2‐339: Development of a screening platform for the identification of new Rac1/Rac1b inhibitors active in the prevention of amyloid‐beta production

Using our DATAS™ technology, we have identified genes that are abnormally spliced in brain samples from patients suffering from Alzheimer's disease (AD). Among these genes, the small GTP-binding protein Rac1 is specifically altered, leading to the expression of a constitutively activated Rac1 variant (Rac1b) in the AD brain. Our experiments indicated that oxidative stress and Aβ treatment induced Rac1b expression and that Rac1b can increase Aβ production. Inhibition of β-secretase decreased Rac1b levels concomitantly to Aβ secretion. Furthermore, one of our compounds known to specifically inhibit Rac1 and Rac1b, EHT 1864, prevented reactive oxygen species (ROS) generation and Aβ production in vitro and in vivo. As Rac1b is a particularly attractive novel target for AD, we have developed a screening platform to identify new Rac1/Rac1b inhibitors active in the prevention of Aβ production. Ability of new compounds to inhibit Rac1b activity was assessed using BODIPY-GTP exchange assay and G-LISA™ (Cytoskeleton) experiments. Aβ40/Aβ42 concentrations in supernatant of APP-transfected HEK293 cells were measured by ELISA. Cell toxicity was evaluated using a released lactate deshydrogenase assay (CytoTox 96, Promega) and ROS generation was analysed using the fluorescent probe DCFDA (Molecular probes). Our in vitro approach has been divided into three parts: 1) a medium throughput screening based on our compounds' ability to inhibit Rac1b activity, assessed using BODIPY-GTP exchange assay and G-LISA™ experiments; 2) a cell-based assay using APP-transfected HEK293 cells in which we evaluate the compounds' efficacy in inhibiting Aβ40/Aβ42 production together with their potential toxicity; 3) several cellular assays aimed at exploring the link between Rac1b, oxidative stress and Aβ production, using different cell lines and different ways of modulating Rac1b activity. This screening platform allowed us to identify new Rac1/Rac1b inhibitors which modulate Aβ secretion in vitro, and which will be further evaluated for their capacity to decrease Aβ40/Aβ42 levels in guinea pig brains. Several compounds belonging to two different structural families were identified. They may be good candidates for inhibiting Aβ production and ROS generation in the brain of AD patients and may contribute to the development of novel therapies for Alzheimer's disease.