Fig. 8

Preclinical lead compound identification. The process of lead compound identification includes three phases, single-dose screening, serial dose screening, and potential compound validation. In single-dose screening phase, cells are automatically seeded into each well on the plates. The 9 different compounds (compound B1 to B9 colored from red to pink) would be used to treat the cells. The wells with blue color represented the reference as no compound treatment. After culturing, the detection reagent would then be added. The relative cell viability of each well would be detected. If a compound can suppress the cell viability, these compounds would be regarded as active hits. The active hits, here as B1 and B9, would further be proceeded to the serial dose phase. The B1 (red) and B9 (pink) compounds were treated to the cells using serial dose. The darker circles around the red or pink dots represented the higher compound concentration. The B1 compound showed inhibited the cell viability along with the increased concentration, which would be determined as a true positive. The B1 would then be validated in the potential compound validation phase. The cell lysates from B1 treatment or reference were then collected for the sandwich ELISA. The cells treated with B1 showed lower value than reference, demonstrating B1 as a lead compound