OPC-X screen: Discovery of small molecule inhibitors to overcome extrinsic inhibition of remyelination.

Project Summary/Abstract Multiple Sclerosis (MS) is a chronic inflammatory, neurodegenerative disease involving the destruction of myelin sheaths on neurons resulting in axonal damage leading to permanent functional deficits including paralysis and loss of vision. In MS, the blood-brain barrier is disrupted allowing fibrinogen to enter the brain and fibrinogen is a driver of neuropathology. It prevents remyelination and repair of damaged neurons by blocking multipotent oligodendrocyte progenitor cells (OPCs) from differentiating into myelin producing oligodendrocytes (OL) and directs OPC cell fate to astrocytes. The rationale for our IGNITE (PAR-21-124) grant is to develop a novel high throughput OPC-X screen to identify compounds that in the presence of fibrinogen promote OPC differentiation to mature OLs and decrease the OPC fate-switch to astrocytes to facilitate remyelination of neurons in MS to slow disease progression. We have already developed a medium throughput OPC-X screen in a 96 well format. We made the novel finding that fibrinogen prevents remyelination by activating bone morphogenetic protein (BMP) receptors in OPCs and showed in the assay that small molecule BMP receptor inhibitors (DMH1 and LDN-212854) increase OL production >80% in a dose-dependent manner and suppressed differentiation to astrocytes. To validate the utility of the assay we showed that LDN-212854 was effective in vivo in two EAE animal models of MS; it significantly improved clinical scores, reduced fibrinogen deposition, demyelination, and myelin damage and increased OPC differentiation to OLs. Our in vitro screening assay is unique because while some compounds have been identified that increase myelination in MS by stimulating differentiation of OPCs to OL, they are not designed to overcome the inhibition of remyelination caused by extrinsic factors such as fibrinogen, are ineffective in our assay and may not promote remyelination under conditions that might be most relevant to the disease. This is important because therapies to overcome extrinsic inhibition of remyelination are not widely available and may be critical in the ultimate success in developing more effective treatments of MS. In the R61 component of the IGNITE grant we will optimize our OPC-X screen for compound screening. In the R33 component, we will screen 28,690 compounds that increase OPC differentiation to OL in the presence of fibrinogen. These compounds consist of bio-annotated compounds, comprising FDA approved drugs and compounds selected on the basis of structural diversity, physicochemical properties consistent with brain permeability and drug-like properties, including Lipinsky criteria. Effective “hits” will be further screened in secondary assays we have developed; a safety assay to exclude potential adverse hemorrhagic effects and assays to identify compounds that may regulate BMP receptor activation, cholesterol synthesis or have additional anti-inflammatory effects by blocking fibrin-induced inflammation. These compounds will be further developed in future studies by us to improve drug like properties, with a goal of transitioning to future clinical development to treat MS patients.