Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening
AuthorMartin, Dale D. O.
Kanuparthi, Prasad S.
Holland, Sabrina M
Sanders, Shaun S.
Einarson, Margret B.
Jacobson, Marlene A.
Thomas, Gareth M.
GroupShriners Hospitals Pediatric Research Center (Temple University)
Moulder Center for Drug Discovery Research (Temple University)
DepartmentAnatomy and Cell Biology
Permanent link to this recordhttp://hdl.handle.net/20.500.12613/8387
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AbstractAfter 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.
CitationMartin, D.D.O., Kanuparthi, P.S., Holland, S.M. et al. Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening. Sci Rep 9, 3632 (2019). https://doi.org/10.1038/s41598-019-39968-8
Citation to related workNature Research
Has partScientific Reports, Vol. 9, No. 3632
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