Bashore Frances M, Min Sophia M, Chen Xiangrong, Howell Stefanie, Rinderle Caroline H, Morel Gabriel, Silvaroli Josie A, Wells Carrow I, Bunnell Bruce A, Drewry David H, Pabla Navjot S, Ultanir Sila K, Bullock Alex N, Axtman Alison D
Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, UK.
bioRxiv. 2024 May 14:2024.05.12.593776. doi: 10.1101/2024.05.12.593776.
Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound was designed as a negative control to be used alongside compound in experiments to interrogate CDKL2-mediated biology. A solved co-crystal structure of compound bound to CDKL2 highlighted key interactions it makes within its ATP-binding site. Inhibition of downstream phosphorylation of EB2, a CDKL2 substrate, in rat primary neurons provided evidence that engagement of CDKL2 by compound in cells resulted in inhibition of its activity. When used at relevant concentrations, compound does not impact the viability of rat primary neurons or certain breast cancer cells nor elicit consistent changes in the expression of proteins involved in epithelial-mesenchymal transition.
通过对无细胞结合和选择性数据的分析，鉴定出了基于酰基氨基吲唑的CDKL2抑制剂。化合物 基于其对CDKL2酶活性的强效抑制、在细胞中与CDKL2的结合以及出色的全激酶组选择性（尤其是在细胞中使用时），被选为CDKL2化学探针。化合物 被设计为阴性对照，以便在研究CDKL2介导的生物学的实验中与化合物 一起使用。化合物 与CDKL2结合的解析共晶体结构突出了其在ATP结合位点内形成的关键相互作用。在大鼠原代神经元中对CDKL2底物EB2的下游磷酸化的抑制提供了证据，表明化合物 在细胞中与CDKL2的结合导致其活性受到抑制。当以相关浓度使用时，化合物 不会影响大鼠原代神经元或某些乳腺癌细胞的活力，也不会引起上皮-间质转化相关蛋白表达的一致变化。
