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Martin DDO , Kanuparthi PS , Holland SM , Sanders SS , Jeong HK , Einarson MB , Jacobson MA , Thomas GM
Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening
Sci Rep. 2019 Mar 6;9(1) :3632
PMID: 30842471    PMCID: PMC6403299   
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After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can thus serve as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We optimized an HCS assay based on this readout, which showed highly robust performance in a 96-well format. Using this assay we screened a library of 1200 FDA-approved compounds and found that ketoconazole, the compound that most dramatically affected DLK localization in our primary screen, dose-dependently inhibited DLK palmitoylation in follow-up biochemical assays. Moreover, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to trophic deprivation, a well known model of DLK-dependent pro-degenerative signaling. Our HCS platform is thus capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.
2045-2322 Martin, Dale D O Kanuparthi, Prasad S Holland, Sabrina M Sanders, Shaun S Jeong, Hey-Kyeong Einarson, Margret B Jacobson, Marlene A Thomas, Gareth M R50 CA211479/U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI) R01 NS094402/U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS) Journal Article England Sci Rep. 2019 Mar 6;9(1):3632. doi: 10.1038/s41598-019-39968-8.