Figure 1

Flowchart of the screening protocol used in the study. 1st step. A collection of 700,000 drug-like commercially available compounds was screened in silico. Molecular docking was performed using Surflex-dock version 2.5. After visual inspection, a 1000 compound hit list was selected for experimental testing. 2 ^nd step: The inhibitory activity of the compounds composing the hit list was evaluated in vitro on human TNFα induced apoptosis on the L929 cell line. Top hit compounds displayed an IC₅₀ between 1 and 100 µM. 3 ^rd step: 2D/3D similarity search methods were used to identify analogues of the top hits identified after step 2. Up to 100 analogues were found per top hit with a Tanimoto similarity score >0.6. 4 ^th step: As in step 2, their inhibitory activity was evaluated in vitro on human TNFα induced apoptosis on the L929 cell line. The 10 best compounds after these 4 steps were selected as candidates for in vivo evaluation on a murine model. 5 ^th step: The in vivo evaluation of the candidates was performed in the TNFα-dependent hepatic shock model triggered with LPS/D-Galactosamine via force-feeding. After this step, 1 in vivo active compound was selected. 6 ^th Step: Using 2D/3D similarity search methods, we searched in our large compound collection for new analogues of this best compound identified after step 5. Up to 500 analogues were identified and purchased from the chemical supplier. As in step 2, their inhibitory activity was evaluated in vitro on human and murine TNFα. The 9 best compounds were evaluated in vivo in our murine hepatic shock assay by force-feeding as described in step 5. The best compound identified after the 6^th step is compound 1.