Targeted inhibition of the Shroom3–Rho kinase protein–protein interaction circumvents Nogo66 to promote axon outgrowth

Heather M Dickson, Amanda Wilbur, Ashley A Reinke, Mathew A Young, Anne B Vojtek

Inhibitory molecules in the adult central nervous system, including NogoA, impede neural repair by blocking axon outgrowth. The actin-myosin regulatory protein Shroom3 directly interacts with Rho kinase and conveys axon outgrowth inhibitory signals from Nogo66, a C-terminal inhibitory domain of NogoA. The purpose of this study was to identify small molecules that block the Shroom3-Rho kinase protein-protein interaction as a means to modulate NogoA signaling and, in the longer term, enhance axon outgrowth during neural repair. A high throughput screen for inhibitors of the Shroom3-Rho kinase protein-protein interaction identified CCG-17444 (Chem ID: 2816053). CCG-17444 inhibits the Shroom3-Rho kinase interaction in vitro with micromolar potency. This compound acts through an irreversible, covalent mechanism of action, targeting Shroom3 Cys1816 to inhibit the Shroom3-Rho kinase protein-protein interaction. Inhibition of the Shroom3-Rho kinase protein-protein interaction with CCG-17444 counteracts the inhibitory action of Nogo66 and enhances neurite outgrowth. This study identifies a small molecule inhibitor of the Shroom3-Rho kinase protein-protein interaction that circumvents the inhibitory action of Nogo66 in neurons. Identification of a small molecule compound that blocks the Shroom3-Rho kinase protein-protein interaction provides a first step towards a potential new strategy for enhancing neural repair.