Small Molecules Dorsomorphin and LDN-193189 Inhibit Myostatin/GDF8 Signaling and Promote Functional Myoblast Differentiation

Daniel Horbelt,Jan H Boergermann,Apirat Chaikuad,Ivan Alfano,Eleanor Williams,Ilya Lukonin,Tobias Timmel,Alex N Bullock,Petra Knaus

doi:10.1074/jbc.m114.604397

Abstract

GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis.

Highlights

For the detection of myogenic marker proteins after short term differentiation, confluent C2C12 cells were switched to differentiation medium (DMEM containing 2% horse serum) in the presence or absence of GDF8 and inhibitors as indicated
Dorsomorphin and LDN-193189 Bind ActRIIA to ALK2—In vitro kinase assays have shown that LDN-193189 potently inhibits the kinase activity of activin-receptor like kinase 4 (ALK4) and ActRIIA with IC50 values of 101 and 210 nM, respectively [26]
Binding measurements determined by isothermal titration calorimetry (ITC) showed that both compounds had KD values in the range 14 –58 nM (Tables 1 and 2)

Summary

Background

We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. Initially identified as Compound C, an inhibitor of AMP-activated protein kinase, dorsomorphin was later recognized for its potential to induce dorsalization in zebrafish embryos and to inhibit BMP Smad- and non-Smad signaling by targeting the BMP type I receptors ALK1, -2, -3, and -6 [27, 28]. By targeting the type II and type I receptors for GDF8, dorsomorphin and LDN-193189 inhibited antimyogenic GDF8 signaling and were efficient promotors of functional myogenesis in vitro in C2C12 cells and primary human skeletal myoblasts

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION