Fig. 1: Drug discovery pipeline, in silico analysis and in vitro validation.
From: Thiorphan reprograms neurons to promote functional recovery after spinal cord injury
a, Drug discovery pipeline consisting of five steps: (1) creation of a transcriptomic dataset; (2) in silico analysis using CMap; (3) medium-throughput in vitro screen; (4) in vivo testing of lead candidate(s); and (5) in vitro monkey and human validation. CST, corticospinal tract. b, In silico analysis shows ranking of compounds in CMap on the basis of connectivity score (left y axis), specificity score (blue, right y axis) and reliability score (green, right y axis) (see text for further detail). c, Neurite extension in composite image of cortical neurons treated with DMSO control and thiorphan (250 μM), Tuj1 labelling. d,e, In vitro screen of top three ‘hits’ from in silico screen, together with predicted negative modulator, adiphenine. Tested in cultures of adult mouse primary cortical neurons for 5 days in vitro, three independent biological replicates were performed on separate days, each using neurons from four adult mice. For each condition, data from all neurons measured across replicates were combined to yield n = 200 neurons per condition. Each neuron was spatially separated and analysed as an independent observation. Total neurite outgrowth per cell (Tuj1 labelling) is shown for each condition. Median values: DMSO (82.9); thiorphan (90.7, 99.8, 132.4, 166.1); triflusal (74.3, 61.7, 94.5, 124.5); milrinone (78.9, 77.4, 68.5, 75.5); adiphenine (74.4, 44.5, 31.7, 3) (d). Maximum neurite length per cell is shown for each condition. Median values: DMSO (93.3); thiorphan (111.1, 105.4, 122.1, 128); triflusal (84.4, 85, 104.9, 114.3); milrinone (100.6, 81.7, 79, 78.6); adiphenine (87.4, 60, 30.6, 6.23) (e). Statistical significance was determined by two-tailed Student’s t-test (**P < 0.01, ***P < 0.001). Error bars ± s.e.m. (d,e). Scale bar, 25 μm (c).
