Aside from having high potency towards ACVR1/ALK2, inhibitor compounds ideally should be highly selective and not target other members of the Transforming Growth Factor beta (TGFb) superfamily. TGFBR1/ALK5 is selected for off-target screening because of its potential role in cardiac functions. Firefly luciferase is transcribed downstream of ACVR1/ALK2 in BRE reporter plasmid and downstream of TBFBR1/ALK5 in CAGA reporter plasmid. Renilla luciferase is constitutively transcribed under a weak promoter as internal control.
C2C12 myoblast cell line responds well to ACVR1/ALK2 ligands (eg: BMP7). However, C2C12 does not respond robustly to TGFBR1/ALK5 ligands (eg: TGFb) and result in incomplete and inconsistent IC50 curves. Several legacy ACVR1/ALK2 inhibitors were tested in these DLAs. HEK293 gave robust DLA TGFBR1/ALK5 IC50 curves. The standard deviation among technical triplicates in HEK293 cells is much smaller than C2C12 cells. However, HEK293 cells did not respond to BMP7 stimulation or Activin A stimulation in the presence of ACVR1/ALK2-R206H. Therefore, DLA using C2C12 cells is still necessary for ACVR1/ALK2 IC50 determination.
As an unpleasant surprise, Activin A appeared to have become less potent and induced a smaller response in ACVR1/ALK2 activation compared to previous experiments (3 folds vs 10+ folds). More verification and troubleshooting are needed to determine whether the ligand had expired or other aspects of the assay had gone south.
For experimental details, please refer to Zenodo.