Optimisation and use of cell-based assays and in vivo assay for screening drugs for Alzheimer's Disease

Abstract

Alzheimer's Disease (AD) is the most prevalent form of dementia among elderly people. This disease is a major world health issue as the numbers of patients with this type of dementia will significantly increase in the next few decades. The aetiology of AD is not completely fully understood. The amyloid cascade hypothesis that identifies Beta-amyloid peptide (AB) as the initiator of the AD pathology leads the drug development efforts for AD. Currently only five drugs are approved by the FDA for AD but these drugs just ameliorate the symptoms of the disease for a limited time and do not target the underlaying pathology. Unfortunately, none of the drugs targeting the underlying disease has fully succeeded in clinical trials. In this project, we screened a drug library of 174 compounds that have been previously approved and/or undergone clinical trials and five drugs that have been previously identified to have a potential effect as AD therapies. We used SH-SY5Y cells acutely treated with AB42, and SH-SY5Y695 cells, as models to mimic the neurotoxic effects of AB in AD. First, we evaluated the feasibility and optimised different in vitro assays to be used as primary and secondary screening assays. MTT was selected as one of the primary screening methods as cells treated with AB42 decreased MTT reduction compared to non-treated cells. Mesoscale Discovery (MSD) immunoassays were also selected as another primary screening assay using SH-SY5Y695 cells. This immunoassay allowed us to simultaneously measure the concentration of the following proteins involved in APP processing: sAPP-alpha, sAPPB, AB38, AB40 and AB42. The primary screening using MTT identified SEN304, EGCG and castalagin as hits. MSD immunoassays identified compounds 4 (Acetyl-beta-methylcholine), 8 (arecoline), 133 (Tranylprimine), 169 (tropisetron), castalagin and SEN304 as possible hits. The first part of secondary screening focused on assessing the potency of the hits identified from the MTT primary screening and evaluating the possible advantage of a two-drug combination approach. EGCG was the most potent compound at inhibiting AB toxicity measured by MTT, followed by SEN304, castalagin and vescalagin. The two-drug combination approach did not show any synergetic effect. The second part of the secondary screening focused in assessing whether the hits from both MT and MSD immunoassays could hinder the increase of ROS generated in SH-SY5Y cells treated with AB42. EGCG was the only drug that decreased the ROS caused by AB42. A Drosophila melanogaster model overexpressing AB42 decreased the longevity of fruit flies compared to a control fly model not expressing AB We performed survival assays to evaluate whether EGCG or SEN304 increased the lifespan of the AB42 flies. None of the drugs evaluated increased the life span of flies overexpressing AB42. Overall the assays presented in this thesis allowed us to identify possible hits for the treatment of AD. The results suggest that SEN304, EGCG, vescalagin an castalagin have multiple mechanism of action to inhibit AB toxicity. Mesoscale Discovery immunoassays are a promising drug screening platform as it is possible to evaluate various targets simultaneously.

Date of Award	31 Dec 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Doig (Supervisor) & Andreas Prokop (Supervisor)