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Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening
Martin, Dale D. O. Kanuparthi, Prasad S. Sanders, Shaun S. Jeong, Hey-Kyeong Einarson, Margret B. Jacobson, Marlene A. Thomas, Gareth M.
Martin, Dale D. O.
Kanuparthi, Prasad S.
Sanders, Shaun S.
Jeong, Hey-Kyeong
Einarson, Margret B.
Jacobson, Marlene A.
Thomas, Gareth M.
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Journal article
Date
2019-03-06
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Committee member
Department
Anatomy and Cell Biology
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https://doi.org/10.1038/s41598-019-39968-8
Abstract
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.
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Citation
Martin, 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
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Nature Research
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Scientific Reports, Vol. 9, No. 3632
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