Morel, G. | bioRxiv Science

bioRxiv2024

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2

Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound 9 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 16 was designed as a negative control to be used alongside compound 9 in experiments to interrogate CDKL2-mediated biology. A solved co-crystal structure of compound 9 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 9 in cells resulted in inhibition of its activity. When used at relevant concentrations, compound 9 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.

cell biology↗

bioRxiv2023

Modification of gene expression after internalization of Growth Hormone into the cell nucleus

Growth hormone (GH) and many other hormones or growth factors have been shown to be rapidly internalized and translocated into the nucleus. The first event of peptide action is binding to its receptor which initiates both signal transduction pathways and internalization. The latter process involves the nucleus and probably gene transcription. In order to analyze the consequences of internalization of GH on gene expression, we used different populations of CHO cells, transfected with either full length GH receptor, or with defective receptor unable to trigger either signal transduction (deletion of box1) or the internalization of GH (Phe346 mutation in Ala). In addition, functional isolated nuclei were incubated 10 and 30 min with 50 nM GH in order to analyze the direct effect of GH on gene expression without surrounding cytoplasmic structures. The genes involved in signal transduction pathways were not revealed if GH internalization is the only functional activity in the whole cell as well as with isolated nuclei. In intact cell, internalization increased expression of 297 genes and decreased fewer than 10% of those known to be influenced by GH. Variations of expression in purified nuclei showed large variations with time. If cell signaling was not modified, cellular growth and proliferation, nucleic acid metabolism, cellular development, cell cycle and gene expression showed many variations with time. GH internalization shows direct effects on gene expression, different from those stimulated by signal transduction.

cell biology↗