ABSTRACT Demyelinating diseases, including multiple sclerosis, are characterized by loss of myelin-producing oligodendrocytes in the central nervous system and cause severe disability for millions of patients. Existing therapies for MS exclusively modulate the immune system to prevent additional myelin loss; no regenerative therapies are available that replenish lost oligodendrocytes and repair lost myelin. Multiple researchers have used high-throughput screening of small-molecule libraries to identify drugs that increase the formation of oligodendrocytes from oligodendrocyte progenitor cells (OPCs) in vitro and enhance functional remyelination in vivo. One such molecule, the FDA-approved antihistamine clemastine, was recently shown to enhance optic nerve conduction velocity in MS patients with optic neuritis, providing the first clinical evidence that small molecule treatments can enhance remyelination in the human CNS. While this trial provides proof-of-concept for future remyelinating therapeutics, greater clarity around the optimal pathways and targets controlling oligodendrocyte formation is critical to the success of future translational efforts. Our multi-disciplinary team has leveraged synergistic expertise in glial biology, chemical biology, and organic synthesis to provide compelling preliminary evidence, now published in Nature and Cell Chemical Biology, that almost all promyelinating small molecules identified by HTS enhance oligodendrocyte formation by inhibiting a small number of adjacent enzymes within cholesterol biosynthesis. Inhibition of these enzymes causes accumulation of specific, structurally- related cholesterol precursors (8,9-unsaturated sterols) which are sufficient to enhance the formation of oligodendrocytes from OPCs. Mass spectrometry-based sterol profiling has demonstrated more than three dozen promyelinating small molecules function by this mechanism, including clemastine. This application advances two parallel goals. First, we seek to understand how 8,9-unsaturated sterol accumulation drives oligodendrocyte formation, including defining the optimal sterols and elucidating their cellular target (Aims 1 and 2). Additionally, we aim to optimize the first selective and brain- penetrant EBP inhibitor and to validate that this molecule promotes remyelination in vivo and human myelin formation in vitro (Aim 3). Organic synthesis is a key technique throughout the application, enabling the synthesis of novel 8,9-unsaturated sterols (Aim 1), photoaffinity pulldown reagents (Aim 2), and novel derivatives of our recently-published EBP-inhibiting lead molecules CW9009 and CW9956. Together these studies will accelerate the emerging field of remyelinating therapeutics by developing optimized small molecules for further drug development and identifying novel drug targets that underlie the oligodendrocyte-enhancing effects of 8,9-unsaturated sterols.