Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine at Temple University, 3500 N. Broad Street, Philadelphia, PA, 19140, USA.
Fox Chase Cancer Center, Philadelphia, PA, USA.
Sci Rep. 2019 Mar 6;9(1):3632. doi: 10.1038/s41598-019-39968-8.
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.
轴突损伤后，双亮氨酸拉链激酶（DLK）通过其下游靶标 c-Jun N 端激酶（JNK）向神经元细胞体传递逆行促退化信号。我们最近报道，这种信号需要脂肪酸棕榈酸通过棕榈酰化过程对 DLK 进行修饰。因此，抑制 DLK 棕榈酰化的化合物可以减少神经退行性变，但要确定这些抑制剂，需要合适的测定方法。在这里，我们报告说，非神经元细胞中 DLK 的亚细胞定位高度依赖于棕榈酰化，因此可以作为高内涵筛选（HCS）中识别 DLK 棕榈酰化抑制剂的替代读数。我们基于该读数优化了 HCS 测定法，该测定法在 96 孔格式中表现出高度稳健的性能。使用该测定法，我们筛选了 1200 种 FDA 批准的化合物库，发现酮康唑是在我们的初步筛选中最显著影响 DLK 定位的化合物，在后续的生化测定中，酮康唑剂量依赖性地抑制了 DLK 的棕榈酰化。此外，酮康唑显著减弱了营养剥夺下初级感觉神经元中 c-Jun 的磷酸化，这是一种众所周知的依赖于 DLK 的促退化信号模型。因此，我们的 HCS 平台能够识别新的 DLK 棕榈酰化和信号传导抑制剂，这些抑制剂可能具有相当大的治疗潜力。
