Development of a Small-Molecule Screening Method for Inhibitors of Cellular Response to Myostatin and Activin A

Abstract

Myostatin, a member of the transforming growth factor (TGF)-β family of secreted ligands, is a strong negative regulator of muscle growth. As such, therapeutic inhibitors of myostatin are actively being investigated for their potential in the treatment of muscle-wasting diseases such as muscular dystrophy and sarcopenia. Here, we sought to develop a highthroughput screening (HTS) method for small-molecule inhibitors that target myostatin. We created a HEK293 stable cell line that expresses the (CAGA) 12-luciferase reporter construct and robustly responds to signaling of certain classes of TGF-β family ligands. After optimization and miniaturization of the assay to a 384-well format, we successfully screened a library of compounds for inhibition of myostatin and the closely related activin A. Selection of some of the tested compounds was directed by in silico screening against myostatin, which led to an enrichment of target hits as compared with random selection. Altogether, we present an HTS method that will be useful for screening potential inhibitors of not only myostatin but also many other ligands of the TGF-β family.

Molecular characterization of latent GDF8 reveals mechanisms of activation

“…This observation suggested that perhaps the prodomain remained bound to the mature domain but was not in a fully inhibitory state. To extend these initial observations, we isolated the mammalian-derived latent proGDF8 complex (GDF8 L ) and compared its signaling activity to both the acid-activated state (GDF8 AA ) and to the mature, unbound GDF8 (GDF8 apo ) using a SMAD3-responsive (CAGA) 12 luciferase-reporter HEK293 cell line (21)(22)(23)(24)(25). As expected and consistent with our previous report, GDF8 apo readily signaled with a calculated half-maximal effective concentration (EC 50 ) of 0.72 nM (25), whereas media containing GDF8 L did not readily signal and required nearly 10,000-fold more protein to achieve a similar response compared with GDF8 apo (Fig.…”

“…HEK293-(CAGA) 12 Luciferase-Reporter Assay. Luciferase-reporter assays for activation and inhibition were performed as previously described (21)(22)(23)(24)(25). Briefly, HEK293 (CAGA) 12 cells stably transfected with plasmid containing Firefly luciferase-reporter gene under the control of SMAD3-responsive promoter were seeded in growth media at 20,000 cells per well in a 96well plate.…”

Molecular characterization of latent GDF8 reveals mechanisms of activation

“…This observation suggested that perhaps the prodomain remained bound to the mature domain, but was not in a fully inhibitory state. To extend these initial observations, we isolated the mammalian-derived latent proGDF8 complex (GDF8 L ) and compared its signaling activity to both the acidactivated state (GDF8 AA ) and to the mature, unbound GDF8 (GDF8 apo ) using a SMAD3-responsive (CAGA) 12 luciferase reporter HEK293 cell line (27)(28)(29)(30)(31). As expected and consistent with our previous report, GDF8 apo readily signaled with a calculated half-maximal effective concentration (EC 50 ) of 0.72 nM (31), whereas media containing GDF8 L did not readily signal and required nearly 10,000-fold more protein to achieve a similar response as compared to GDF8 apo (Figure 1a).…”

“…Luciferase reporter assays for activation and inhibition were performed as previously described (27)(28)(29)(30)(31). Briefly, HEK293 (CAGA) 12 h post-transfection using 20 μL per well 1x Passive Lysis Buffer (E1941, Promega, USA), on a plate shaker (800 rpm, 20 min, 20°C).…”

Alternative Binding Modes Identified for Growth and Differentiation Factor-associated Serum Protein (GASP) Family Antagonism of Myostatin

“…Luciferase Reporter Assays-The luciferase reporter assays were performed as previously described (28,29). Briefly, HEK293 CAGA 12 cells were plated in a 96-well plate and grown for ϳ24 h. Subsequently, the growth medium was removed and myostatin at a concentration of 0.62 nM was mixed with the antagonist (GASP-1, GASP-2, or GASP-1 truncations) in serum free medium and applied to the cells for ϳ18 h. The cells were lysed and luminescence was recorded immediately using a Synergy H1 Hybrid plate reader (BioTek).…”