Abstract
BackgroundInhibitory 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.ResultsA 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.ConclusionsThis 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.
Highlights
Inhibitory molecules in the adult central nervous system, including NogoA, impede neural repair by blocking axon outgrowth
High throughput screen for Shroom3–Rho kinase inhibitors Shroom3 and Rho kinase directly interact through the Shroom3 domain 2 (SD2) domain of Shroom3 and the R2-C1 domain of Rho kinase 2 (ROCK2) (Figure 1a)
Unlabeled R2-C1 effectively competed with biotinylated R2-C1 for binding to immobilized SD2 (Figure 1c), demonstrating that the interaction assayed in the enzyme linked immunosorbent assay (ELISA) is due to specific binding of SD2 with R2-C1
Summary
Inhibitory molecules in the adult central nervous system, including NogoA, impede neural repair by blocking axon outgrowth. PirB mediates Nogo neurite outgrowth inhibition through an intracellular signaling complex organized by Dickson et al BMC Neurosci (2015) 16:34 the scaffold protein plenty of SH3s (POSH; known as Sh3rf1) [14, 16] This signaling complex includes the actin-myosin regulator Shroom, Rho kinase, and leucine zipper kinase (LZK or MAP3K12) [14, 16]. The Shroom interaction domain is located in a central coiled coil region of Rho kinase, directly upstream of the RhoA binding domain [18, 19] Ectopic expression of this domain of Rho kinase disrupts the interaction of endogenous Shroom and Rho kinase and in neurons expression of this domain enhances neurite outgrowth [16, 19, 20]. This observation points to an important role for the Shroom3–Rho kinase protein–protein interaction in negatively regulating neurite outgrowth
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