Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling
Fig 2
Identification and validation of splice active compounds by a semi-automated high-throughput screen.
(a) Optimization of the splicing reporter to RBM20 ratio by co-transfection of HEK293 cells. The assay is saturated at a 5-fold excess of RBM20 (N = 8). (b) Assay kinetics with maximum effect after 60 hours of incubation (N = 8). Polypyrimidine Tract Binding Protein 1 (PTBP1) served as negative control not leading to exon exclusion of the splice reporter. (c) Assay suitability for a high-throughput approach—z’ values >0.5 are adequate. (d) Screening strategy to identify splice active compounds. The DLR assay was adapted to the 384-well format followed by the pilot screen with >34,000 compounds at 10 μM. Potentially active compounds were re-evaluated in 9 serial dilutions. Resulting candidates were validated manually in the 96-well format leading to the identification of 7 inhibitors that belong to the group of cardenolides. (e) Number of compounds (cpds) passing the different steps of the screening procedure. *P<0.05, **P<0.01, ***P<0.001 versus CTRL (Dunnett’s post-test). Data are presented as mean ±SD.