Figure 4. Validation of primary hits, preliminary structure-activity-relationship (SAR) analysis and their in vitro cytotoxicity.

(a) The activity of all 53 primary hits was validated in PS1-M146L/YC3.6 line. All the hits were capable of reducing the peak response of CCh-induced calcium release to <90% of DMSO-treated controls. (b) The structure-activity-relationship (SAR) map of the screened compounds. The symbols represent compound clusters generated by Benchware DataMiner software. The distance between the clusters correlates with the similarity between their chemical structures. The number of compounds within a cluster is illustrated by the size of the symbol. A cluster with more than 50% active compounds is represented by a star, and marked in blue if the actual number of active compounds is greater than four. The highlighted identified lead structures belong to compound classes Thiazolidine (blue), Phenothiazine (green), Imidazole (turquoise) and Benzhydrilpiperidinamine (brown). Primary hits were also active in HEK293 cells expressing (e) PS1-DeltaE9/YC3.6, (f) PS1-C92S/YC3.6, and (g) PS1-D385N/YC3.6, by attenuating the mutant PS1-induced amplified calcium release. (c) and (d) The hits from the primary screen were classified into 8 categories based on their efficacy in lowering the CCh-evoked calcium release. These categories are separated according to the value of normalized ER calcium response. The noted numbers in each category indicates the number of compounds belonging to that category. (h) Viability of HEK293 cells treated with the primary screen hits was assessed by means of MTT assay after 24 h compound treatment. Values are presented as percentage of viable cells. In (a), (e), (f) and (g), the peak response of DMSO-treated control is set to one. Each color denotes a different lead structure in (a), (b), (e), (f), (g) and (h). The data for analogous molecules belonging to the same lead structure are marked with the same color.